DRAFT
FINAL
REPORT
on
COMPARATIVE
EVALUATION
OF
FATHEAD
MINNOW
ASSAYS
FOR
DETECTING
ENDOCRINE­
DISRUPTING
CHEMICALS
EPA
CONTRACT
NUMBER
68­
W­
01­
023
WORK
ASSIGNMENTS
2­
18
and
3­
8
July
2003
Prepared
for
LES
TOUART,
PH.
D.
WORK
ASSIGNMENT
MANAGER
U.
S.
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
DC
BATTELLE
505
King
Avenue
Columbus,
Ohio
43201
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
iii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
CONTENTS
1.0
INTRODUCTION..............................................................................................................................
1
1.1
Purpose.....................................................................................................................................
2
2.0
MATERIALS
AND
METHODS.......................................................................................................
3
2.1
Test
Material
and
Exposure
Regime
........................................................................................
3
2.2
Test
Material
............................................................................................................................
3
2.3
Preparation
and
Sampling
of
Chemical
Exposure
Water.........................................................
3
2.4
Analytical
Procedures
..............................................................................................................
6
2.4.1
Methoxychlor
..............................................................................................................
6
2.4.2
Trenbolone
..................................................................................................................
6
2.4.3
Flutamide.....................................................................................................................
6
2.4.4
Fadrozole.....................................................................................................................
7
2.5
Animals
and
Husbandry...........................................................................................................
7
2.6
Study
Schedule
and
Design......................................................................................................
8
2.7
Description
of
Study
Protocols
................................................................................................
9
2.7.1
Summary
of
the
14­
day
Non­
spawning
Method
.........................................................
9
2.7.2
Summary
of
the
14­
day
EPA
Method
.........................................................................
9
2.7.3
Summary
of
21­
day
EPA
Method.............................................................................
10
2.7.4
Summary
of
Assay
Endpoints
...................................................................................
11
2.8
Statistical
Analyses
................................................................................................................
18
2.9
Quality
Assurance..................................................................................................................
18
2.9.1
Technical
Systems
Audits
.........................................................................................
18
2.9.2
Audits
of
Data
Quality
..............................................................................................
19
2.10
Storage
of
Records
and
Data
Management
...............................................................
20
3.0
RESULTS:
METHOXYCHLOR....................................................................................................
21
3.1
EPA
14­
Day
Assay
for
Methoxychlor
...................................................................................
21
3.1.1
Survival
.....................................................................................................................
21
3.1.2
Vitellogenin...............................................................................................................
21
3.1.3
Appearance
/
Secondary
Sex
Characteristics
............................................................
23
3.1.4
Gonadosomatic
Index................................................................................................
24
3.1.5
Female
Gonad
Histology...........................................................................................
26
3.1.6
Male
Gonad
Histology
..............................................................................................
29
3.1.7
Plasma
Steroid
Concentrations..................................................................................
31
3.1.8
Body
Weight
.............................................................................................................
37
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
iv
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
3.1.9
Fecundity...................................................................................................................
37
3.1.10
Fertilization
Success..................................................................................................
40
3.1.11
Hatchability
and
Larval
Development.......................................................................
41
3.2
EPA
21­
Day
Assay
for
Methoxychlor
...................................................................................
43
3.2.1
Survival
.....................................................................................................................
43
3.2.2
Vitellogenin...............................................................................................................
44
3.2.3
Appearance
/
Secondary
Sex
Characteristics
............................................................
46
3.2.4
Gonadosomatic
Index................................................................................................
46
3.2.5
Female
Gonad
Histology...........................................................................................
48
3.2.6
Male
Gonad
Histology
..............................................................................................
51
3.2.7
Plasma
Steroid
Concentrations..................................................................................
53
3.2.8
Body
Weight
.............................................................................................................
59
3.2.9
Fecundity...................................................................................................................
60
3.2.10
Fertilization
Success..................................................................................................
63
3.2.11
Hatchability
and
Larval
Development.......................................................................
65
3.3
Non­
spawning
Adult
14­
day
Assay
for
Methoxychlor..........................................................
67
3.3.1
Survival
.....................................................................................................................
68
3.3.2
Vitellogenin...............................................................................................................
68
3.3.3
Appearance
/
Secondary
Sex
Characteristics
............................................................
70
3.3.4
Gonadosomatic
Index................................................................................................
71
3.3.5
Female
Gonad
Histology...........................................................................................
73
3.3.6
Male
Gonad
Histology
..............................................................................................
77
3.3.7
Plasma
Steroid
Concentrations..................................................................................
80
3.3.8
Body
Weight
and
Length
..........................................................................................
85
4.0
RESULTS:
TRENBOLONE...........................................................................................................
90
4.1
EPA
14­
Day
Assay
for
Trenbolone
.......................................................................................
90
4.1.1
Survival
.....................................................................................................................
90
4.1.2
Vitellogenin...............................................................................................................
90
4.1.3
Appearance
/
Secondary
Sex
Characteristics
............................................................
92
4.1.4
Gonadosomatic
Index................................................................................................
92
4.1.5
Female
Gonad
Histology...........................................................................................
94
4.1.6
Male
Gonad
Histology
..............................................................................................
97
4.1.7
Plasma
Steroid
Concentrations..................................................................................
99
4.1.8
Fecundity.................................................................................................................
104
4.1.9
Fertilization
Success................................................................................................
106
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
v
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
4.1.10
Hatchability
and
Larval
Development.....................................................................
108
4.1.11
Body
Weight
...........................................................................................................
110
4.2
EPA
21­
Day
Assay
for
Trenbolone
.....................................................................................
110
4.2.2
Vitellogenin.............................................................................................................
110
4.2.3
Appearance
/
Secondary
Sex
Characteristics
..........................................................
112
4.2.4
Gonadosomatic
Index..............................................................................................
112
4.2.5
Female
Gonad
Histology.........................................................................................
114
4.2.6
Male
Gonad
Histology
............................................................................................
117
4.2.7
Plasma
Steroid
Concentrations................................................................................
119
4.2.8
Fecundity.................................................................................................................
124
4.2.9
Fertilization
Success................................................................................................
128
4.2.10
Hatchability
and
Larval
Development.....................................................................
129
4.2.11
Body
Weight
...........................................................................................................
132
4.3
Non­
spawning
Adult
14­
Day
Assay
for
Trenbolone
...........................................................
132
4.3.1
Survival
...................................................................................................................
132
4.3.2
Vitellogenin.............................................................................................................
132
4.3.3
Appearance
/
Secondary
Sex
Characteristics
..........................................................
134
4.3.4
Gonadosomatic
Index..............................................................................................
135
4.3.5
Female
Gonad
Histology.........................................................................................
137
4.3.6
Male
Gonad
Histology
............................................................................................
140
4.3.7
Plasma
Steroid
Concentrations................................................................................
142
4.3.8
Body
Weight
and
Length
........................................................................................
147
5.0
RESULTS:
FLUTAMIDE
............................................................................................................
152
5.1
EPA
14­
Day
Assay
for
Flutamide
.......................................................................................
152
5.1.1
Survival
...................................................................................................................
152
5.1.2
Vitellogenin.............................................................................................................
152
5.1.3
Appearance
/
Secondary
Sex
Characteristics
..........................................................
154
5.1.4
Gonadosomatic
Index..............................................................................................
154
5.1.5
Female
Gonad
Histology.........................................................................................
156
5.1.6
Male
Gonad
Histology
............................................................................................
159
5.1.7
Plasma
Steroid
Concentrations................................................................................
161
5.1.8
Fecundity.................................................................................................................
166
5.1.9
Fertilization
Success................................................................................................
169
5.1.10
Hatchability
and
Larval
Development.....................................................................
170
5.1.11
Body
Weight
...........................................................................................................
172
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
vi
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
5.2
EPA
21­
Day
Assay
for
Flutamide
.......................................................................................
172
5.2.1
Survival
...................................................................................................................
172
5.2.2
Vitellogenin.............................................................................................................
172
5.2.3
Appearance
/
Secondary
Sex
Characteristics
..........................................................
174
5.2.4
Gonadosomatic
Index..............................................................................................
175
5.2.5
Female
Gonad
Histology.........................................................................................
176
5.2.6
Male
Gonad
Histology
............................................................................................
179
5.2.7
Plasma
Steroid
Concentrations................................................................................
181
5.2.8
Fecundity.................................................................................................................
186
5.2.9
Fertilization
Success................................................................................................
190
5.2.10
Hatchability
and
Larval
Development.....................................................................
191
5.2.11
Body
Weight
...........................................................................................................
193
5.3
Non­
spawning
Adult
14­
Day
Assay
for
Flutamide
.............................................................
194
5.3.1
Survival
...................................................................................................................
194
5.3.2
Vitellogenin.............................................................................................................
194
5.3.3
Appearance
/
Secondary
Sex
Characteristics
..........................................................
196
5.3.4
Gonadosomatic
Index..............................................................................................
197
5.3.5
Female
Gonad
Histology.........................................................................................
199
5.3.6
Male
Gonad
Histology
............................................................................................
202
5.3.7
Plasma
Steroid
Concentrations................................................................................
205
5.3.8
Body
Weight
and
Length
........................................................................................
211
6.0
RESULTS:
FADROZOLE............................................................................................................
216
6.1
EPA
14­
Day
Assay
for
Fadrozole........................................................................................
216
6.1.1
Survival
...................................................................................................................
216
6.1.2
Vitellogenin.............................................................................................................
216
6.1.3
Appearance
/
Secondary
Sex
Characteristics
..........................................................
218
6.1.4
Gonadosomatic
Index..............................................................................................
218
6.1.5
Female
Gonad
Histology.........................................................................................
220
6.1.6
Male
Gonad
Histology
............................................................................................
223
6.1.7
Plasma
Steroid
Concentrations................................................................................
226
6.1.8
Fecundity.................................................................................................................
230
6.1.9
Fertilization
Success................................................................................................
233
6.1.10
Hatchability
and
Larval
Development.....................................................................
234
6.1.11
Body
Weight
...........................................................................................................
236
6.2
EPA
21­
Day
Assay
for
Fadrozole........................................................................................
236
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
vii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
6.2.1
Survival
...................................................................................................................
236
6.2.2
Vitellogenin.............................................................................................................
236
6.2.3
Appearance
/
Secondary
Sex
Characteristics
..........................................................
238
6.2.4
Gonadosomatic
Index..............................................................................................
238
6.2.5
Female
Gonad
Histology.........................................................................................
240
6.2.6
Male
Gonad
Histology
............................................................................................
244
6.2.7
Plasma
Steroid
Concentrations................................................................................
247
6.2.8
Fecundity.................................................................................................................
252
6.2.9
Fertilization
Success................................................................................................
256
6.2.10
Hatchability
and
Larval
Development.....................................................................
257
6.2.11
Body
Weight
...........................................................................................................
259
6.3
Non­
spawning
Adult
14­
day
Assay
for
Fadrozole...............................................................
260
6.3.1
Survival
...................................................................................................................
260
6.3.2
Vitellogenin.............................................................................................................
260
6.3.3
Appearance
/
Secondary
Sex
Characteristics
..........................................................
262
6.3.4
Gonadosomatic
Index..............................................................................................
263
6.3.5
Female
Gonad
Histology.........................................................................................
265
6.3.6
Male
Gonad
Histology
............................................................................................
268
6.3.7
Plasma
Steroid
Concentrations................................................................................
272
6.3.8
Body
Weight
and
Length
........................................................................................
277
7.0
DISCUSSION
................................................................................................................................
282
7.1
Fecundity..............................................................................................................................
282
7.2
Histology
and
Gonadosomatic
index
(
GSI)
.........................................................................
283
7.3
Vitellogenin
and
Plasma
Steroid
Levels
..............................................................................
284
7.3.1
Test
Results:
Transferability
and
Inter­
Protocol
Comparison.................................
286
7.3.2
Methoxychlor
(
mode
of
action:
weak
estrogen)......................................................
287
7.3.3
Trenbolone
(
anabolic
steroid)
.................................................................................
288
7.3.4
Flutamide
(
anti­
androgen).......................................................................................
289
7.3.5
Fadrozole
(
aromatase
inhibitor,
indirect
anti­
estrogen)
..........................................
290
7.4
Conclusion
...........................................................................................................................
291
7.5
Protocol
Selection
................................................................................................................
292
7.6
Transferability......................................................................................................................
292
8.0
REFERENCES
...........................................................................................................................
294
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
viii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
FIGURES
Figure
2.1.
Saturator­
column
apparatus......................................................................................................
4
Figure
2.2.
Continuous­
flow
proportional
diluter
system.........................................................................
5
Figure
2.3.
Terracotta
dish
with
nytex
screen.........................................................................................
10
Figure
2.4.
Staff
members
count
eggs
in
support
of
fecundity
measurements
.......................................
11
Figure
2.5.
Flow­
through
embryo­
incubation
chambers
........................................................................
11
Figure
2.6.
Collection
of
the
liver
and
gonads
.......................................................................................
13
Figure
3.
1.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay......................................................................................................
22
Figure
3.2.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay
.............................................................................................................
23
Figure
3.3.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay
..................
24
Figure
3.4.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay
.....................
25
Figure
3.5.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
14­
Day
Methoxychlor
assay
.....................................................
27
Figure
3.6.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay
........................................................................................
28
Figure
3.7.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay.................................................................................................
28
Figure
3.8.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
14­
Day
Methoxychlor
assay
.....................................................
29
Figure
3.9.
Boxplot
of
seminiferous
tubule
diameter
(
mm)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay
.............................................................................................................
30
Figure
3.10.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay
.............................................................................................................
32
Figure
3.
11.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay
.............................................................................................................
33
Figure
3.12.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay......................................................................................................
34
Figure
3.13.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay
.............................................................................................................
35
Figure
3.14.
Boxplot
of
female
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay.
.......................................................................................
36
Figure
3.15.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay.................................................................................................
37
Figure
3.16.
Total
Egg
Production
by
Replicate
per
Treatment
for
the
EPA
14­
Day
Methoxychlor
Assay....................................................................................................................................
38
Figure
3.17.
Boxplot
of
the
Number
of
Eggs
Produced
per
Female
Reproductive
Day
by
Treatment
for
the
EPA
14­
Day
Methoxychlor
Assay
..........................................................
39
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
ix
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
3.18.
Boxplot
of
the
Proportion
of
Eggs
Fertilized
by
Treatment
for
the
EPA
14­
Day
Methoxychlor
Assay
............................................................................................................
40
Figure
3.19.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay......................................................................................................
42
Figure
3.20.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay
.............................................................................................................
43
Figure
3.21.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay......................................................................................................
44
Figure
3.22.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay
.............................................................................................................
45
Figure
3.23.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay
.................
46
Figure
3.24.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay
....................
47
Figure
3.25.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
21­
Day
Methoxychlor
assay
.....................................................
49
Figure
3.26.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
21
Day
Methoxychlor
assay
........................................................................................
50
Figure
3.27.
Boxplot
of
the
proportion
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay.................................................................................................
50
Figure
3.28.
Frequency
Histogram
Showing
the
Quantitative
Developmental
Staging
of
Testes
for
Each
Treatment
of
the
EPA
21­
Day
Methoxychlor
Assay
..................................................
52
Figure
3.29.
Boxplot
of
male
seminiferous
tubule
diameter
(
mm)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay......................................................................................................
53
Figure
3.30.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay
.............................................................................................................
54
Figure
3.31.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay
.............................................................................................................
55
Figure
3.32.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay......................................................................................................
56
Figure
3.33.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay
.............................................................................................................
57
Figure
3.34.
Boxplot
of
female
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay
........................................................................................
58
Figure
3.35.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay.................................................................................................
59
Figure
3.36.
Total
egg
production
per
treatment
during
the
pre­
exposure
and
exposure
periods
of
the
EPA
21­
Day
Methoxychlor
assay
..................................................................................
60
Figure
3.37.
Total
egg
production
by
replicate
per
treatment
during
the
EPA
21­
Day
Methoxychlor
assay.
....................................................................................................................................
61
Figure
3.38.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay
..................................................................................
63
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
x
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
3.39.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay
.............................................................................................................
64
Figure
3.40.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay......................................................................................................
66
Figure
3.41.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay
.............................................................................................................
67
Figure
3.42.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
......................................................................
69
Figure
3.43.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
......................................................................
70
Figure
3.44.
Secondary
sex
characteristics
of
males
used
during
the
methoxychlor
Non­
spawning
Adult
14­
Day
assay.
.............................................................................................................
71
Figure
3.45.
Boxplot
of
female
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
.............................................................................................................
72
Figure
3.46.
Boxplot
of
male
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
.....................................................................................................................................
73
Figure
3.47.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
...........................
75
Figure
3.48.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
..............................................................
76
Figure
3.49.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay.
.....................................................................
77
Figure
3.50.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
...........................
78
Figure
3.51.
Boxplot
of
seminiferous
tubule
diameter
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
......................................................................................
79
Figure
3.52.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
......................................................................................
80
Figure
3.53.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
......................................................................................
81
Figure
3.54.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
......................................................................
82
Figure
3.55.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
......................................................................
83
Figure
3.56.
Boxplot
of
female
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
..............................................................
84
Figure
3.57.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay.
.....................................................................
85
Figure
3.58.
Boxplot
of
female
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
.............................................................................................................
86
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xi
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
3.59.
Boxplot
of
female
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay......................................................................................................
87
Figure
3.60.
Boxplot
of
male
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
.............................................................................................................
88
Figure
3.61.
Boxplot
of
male
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
.............................................................................................................
89
Figure
4.1.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay.
.......................................................................................................
91
Figure
4.2.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay.
...............................................................................................................
92
Figure
4.3.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay
......................
93
Figure
4.4.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay
.........................
94
Figure
4.5.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
14­
Day
Trenbolone
assay..........................................................
95
Figure
4.6.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay
............................................................................................
96
Figure
4.7.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay.....................................................................................................
97
Figure
4.8.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
14­
Day
Trenbolone
assay..........................................................
98
Figure
4.9.
Boxplot
of
male
seminiferous
tubule
diameter
(
mm)
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay..................................................................................................................
99
Figure
4.10.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay................................................................................................................
100
Figure
4.11.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay................................................................................................................
101
Figure
4.
12.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay
........................................................................................................
102
Figure
4.13.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay................................................................................................................
103
Figure
4.14.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay...................................................................................................
104
Figure
4.15.
Total
egg
production
by
replicate
per
treatment
for
the
EPA
14­
Day
Trenbolone
assay.
..................................................................................................................................
105
Figure
4.16.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay
....................................................................................
106
Figure
4.17.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay................................................................................................................
107
Figure
4.18.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
14­
Day
Trenbolone
assayDay
Trenbolone
assay
....................................................................
108
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
4.19.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
14­
Day
Trenbolone
assay................................................................................................................
109
Figure
4.20.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay
........................................................................................................
111
Figure
4.21.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay................................................................................................................
112
Figure
4.22.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay
....................
113
Figure
4.23.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay
.......................
114
Figure
4.24.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
21­
Day
Trenbolone
assay........................................................
115
Figure
4.25.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay
..........................................................................................
116
Figure
4.26.
Boxplot
of
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay
........................................................................................................
116
Figure
4.27.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
21­
Day
Trenbolone
assay........................................................
118
Figure
4.28.
Boxplot
of
male
seminiferous
tubule
diameter
(
mm)
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay................................................................................................................
119
Figure
4.29.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay................................................................................................................
120
Figure
4.30.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay................................................................................................................
121
Figure
4.31.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay
........................................................................................................
122
Figure
4.32.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay................................................................................................................
123
Figure
4.33.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay...................................................................................................
124
Figure
4.34.
Total
egg
production
per
treatment
for
the
EPA
21­
Day
Trenbolone
assay.
The
vertical
line
at
Day
0
denotes
the
start
of
the
exposure
period...........................................
125
Figure
4.35.
Total
egg
production
by
replicate
per
treatment
for
the
EPA
21­
Day
Trenbolone
assay.
..................................................................................................................................
126
Figure
4.36.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay
....................................................................................
127
Figure
4.37.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay................................................................................................................
129
Figure
4.38.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay
........................................................................................................
130
Figure
4.39.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
21­
Day
Trenbolone
assay................................................................................................................
131
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xiii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
4.40.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay
........................................................................
133
Figure
4.41.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay
........................................................................
134
Figure
4.42.
Secondary
sex
characteristics
of
males
used
during
the
flutamide
Non­
spawning
Adult
14­
Day
assay.
...........................................................................................................
135
Figure
4.43.
Boxplot
of
female
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay
...................................................................................................................................
136
Figure
4.44.
Boxplot
of
male
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay
...................................................................................................................................
137
Figure
4.45.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay..............................
138
Figure
4.46.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay
................................................................
139
Figure
4.47.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay
........................................................................
139
Figure
4.
48.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay..............................
141
Figure
4.49.
Boxplot
of
male
seminiferous
tubule
diameter
(
mm)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay
........................................................................
142
Figure
4.50.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.........................................................................................
143
Figure
4.51.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.........................................................................................
144
Figure
4.52.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay
........................................................................
145
Figure
4.53.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay
........................................................................
146
Figure
4.54.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay
........................................................................
147
Figure
4.55.
Boxplot
of
female
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay................................................................................................................
148
Figure
4.56.
Boxplot
of
female
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay
........................................................................................................
149
Figure
4.57.
Boxplot
of
male
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay................................................................................................................
150
Figure
4.58.
Boxplot
of
male
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay................................................................................................................
151
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xiv
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
5.1.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
assay.
.........................................................................................................
153
Figure
5.2.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
assay..................................................................................................................
154
Figure
5.3.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
14­
Day
Flutamide
assay.
.....................
155
Figure
5.5.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
14­
Day
Flutamide
assay..........................................................
157
Figure
5.6.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Flutamide
assay
............................................................................................
158
Figure
5.7.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Flutamide
assay
.....................................................................................................
159
Figure
5.8.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
14­
Day
Flutamide
assay..........................................................
160
Figure
5.9.
Boxplot
of
seminiferous
tubule
diameter
(

m)
by
treatment
for
the
EPA
14­
Day
Flutamide
assay..................................................................................................................
161
Figure
5.10.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
assay..................................................................................................................
162
Figure
5.11.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
assay..................................................................................................................
163
Figure
5.12.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
assay
..........................................................................................................
164
Figure
5.13.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
assay..................................................................................................................
165
Figure
5.14.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
assay
.....................................................................................................
166
Figure
5.15.
Total
egg
production
by
replicate
per
treatment
for
the
EPA
14­
Day
Flutamide
assay.....
167
Figure
5.16.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
14­
Day
Flutamide
assay.......................................................................................
168
Figure
5.17.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
14­
Day
Flutamide
assay..................................................................................................................
169
Figure
5.18.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
14­
Day
Flutamide
assay
..........................................................................................................
171
Figure
5.19.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
14­
Day
Flutamide
assay..................................................................................................................
172
Figure
5.20.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
assay
..........................................................................................................
173
Figure
5.21.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
assay..................................................................................................................
174
Figure
5.22.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
21­
Day
Flutamide
assay
......................
175
Figure
5.23.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
21­
Day
Flutamide
assay..........................
176
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xv
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
5.24.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
21­
Day
Flutamide
assay..........................................................
177
Figure
5.25.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Flutamide
assay
............................................................................................
178
Figure
5.26.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Flutamide
assay
.....................................................................................................
179
Figure
5.27.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
21­
Day
Flutamide
assay..........................................................
180
Figure
5.28.
Boxplot
of
male
seminiferous
tubule
diameter
(

m)
by
treatment
for
the
EPA
21­
Day
Flutamide
assay..................................................................................................................
181
Figure
5.29.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
assay..................................................................................................................
182
Figure
5.30.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
assay..................................................................................................................
183
Figure
5.31.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
assay
..........................................................................................................
184
Figure
5.32.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
assay..................................................................................................................
185
Figure
5.33.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
assay.
....................................................................................................
186
Figure
5.34.
Total
egg
production
per
treatment
for
the
EPA
21­
Day
Flutamide
assay.
The
vertical
line
denotes
the
initiation
of
the
exposure
assay.
...............................................................
187
Figure
5.35.
Total
egg
production
by
replicate
per
treatment
for
the
EPA
21­
Day
Flutamide
assay.....
188
Figure
5.36.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
21­
Day
Flutamide
assay.......................................................................................
189
Figure
5.37.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
21­
Day
Flutamide
assay..................................................................................................................
191
Figure
5.38.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
21­
Day
Flutamide
assay.
.........................................................................................................
192
Figure
5.39.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
21­
Day
Flutamide
assay..................................................................................................................
193
Figure
5.40.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay...........................................................................
195
Figure
5.41.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay...........................................................................
196
Figure
5.42.
Secondary
sex
characteristics
of
males
used
during
the
EPA
Non­
spawning
Adult
14­
Day
Flutamide
assay.
.........................................................................................................
197
Figure
5.43.
Boxplot
of
female
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay.
..................................................................................................................................
198
Figure
5.44.
Boxplot
of
male
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
...................................................................................................................................
199
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xvi
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
5.45.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Flutamide
assay................................
200
Figure
5.46.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
..................................................................
201
Figure
5.47.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay...........................................................................
202
Figure
5.48.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Flutamide
assay................................
203
Figure
5.49.
Boxplot
of
seminiferous
tubule
diameter
(

m)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay...........................................................................................
204
Figure
5.50.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay..........................................................................
206
Figure
5.51.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay...........................................................................................
207
Figure
5.52.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay..........................................................................
208
Figure
5.53.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay..........................................................................
209
Figure
5.54.
Boxplot
of
female
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay..................................................................
210
Figure
5.55.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay..........................................................................
211
Figure
5.56.
Boxplot
of
female
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay..................................................................................................................
212
Figure
5.57.
Boxplot
of
female
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
..........................................................................................................
213
Figure
5.58.
Boxplot
of
male
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay..................................................................................................................
214
Figure
5.59.
Boxplot
of
male
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay..................................................................................................................
215
Figure
6.1.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay
..........................................................................................................
217
Figure
6.2.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay
..................................................................................................................
218
Figure
6.3.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.......................
219
Figure
6.4.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay..........................
220
Figure
6.5.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
14­
Day
Fadrozole
assay
..........................................................
221
Figure
6.6.
Boxplot
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay
..................................................................................................................
222
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xvii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
6.7.
Boxplot
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay
...................................................................................................................................
223
Figure
6.8.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
14­
Day
Fadrozole
assay
..........................................................
224
Figure
6.9.
Boxplot
of
seminiferous
tubule
diameter
(

m)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
.................................................................................................................
225
Figure
6.10.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay
..................................................................................................................
226
Figure
6.11.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
.........................................................................................................
227
Figure
6.12.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay
..................................................................................................................
228
Figure
6.13.
Boxplot
of
female
11­
ketotestosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.............................................................................................
229
Figure
6.14.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay
.....................................................................................................
230
Figure
6.15.
Total
egg
production
by
replicate
per
treatment
for
the
EPA
14­
Day
fadrozole
assay......
231
Figure
6.16.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.......................................................................................
232
Figure
6.17.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay
..................................................................................................................
233
Figure
6.18.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay
..........................................................................................................
235
Figure
6.19.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
.................................................................................................................
236
Figure
6.20.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
..........................................................................................................
237
Figure
6.21.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
.................................................................................................................
238
Figure
6.22.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.......................
239
Figure
6.23.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay..........................
240
Figure
6.24.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
21­
Day
Fadrozole
assay
..........................................................
242
Figure
6.25.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.............................................................................................
243
Figure
6.26.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
.....................................................................................................
244
Figure
6.27.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
21­
Day
Fadrozole
assay
..........................................................
245
Figure
6.28.
Boxplot
of
male
seminiferous
tubule
diameter
(

m)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
..................................................................................................................
246
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xviii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
6.29.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
..................................................................................................................
247
Figure
6.30.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
..........................................................................................................
249
Table
6.28.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.
................................................................................
249
Figure
6.31.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
..................................................................................................................
250
Figure
6.32.
Boxplot
of
female
11­
ketotestosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.............................................................................................
251
Figure
6.33.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
.....................................................................................................
252
Figure
6.34.
Total
egg
production
per
treatment
for
the
15­
Day
pre­
exposure
assay.
..........................
253
Figure
6.35.
Total
egg
production
by
replicate
per
treatment
for
the
EPA
21­
Day
fadrozole
assay......
254
Figure
6.36.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.......................................................................................
255
Figure
6.37.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
..................................................................................................................
257
Figure
6.38.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
..........................................................................................................
258
Figure
6.39.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
..................................................................................................................
259
Figure
6.40.
Boxplot
of
the
body
weights
of
males
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay
...................................................................................................................................
260
Figure
6.41.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay...........................................................................
261
Figure
6.42.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay...........................................................................
262
Figure
6.43.
Secondary
sex
characteristics
of
males
used
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
.................................................................................................................
263
Figure
6.44.
Boxplot
of
female
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
...................................................................................................................................
264
Figure
6.45.
Boxplot
of
male
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
...................................................................................................................................
265
Figure
6.46.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
................................
266
Figure
6.47.
Boxplot
of
atretic
follicles
per
300
follicles
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
.....................................................................................................
267
Figure
6.48.
Boxplot
of
corpora
lutea
per
300
follicles
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
.....................................................................................................
267
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xix
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
6.49.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
................................
269
Figure
6.50.
Boxplot
of
seminiferous
tubule
diameter
(

m)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
...........................................................................................
270
Figure
6.51.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
...........................................................................................
272
Figure
6.52.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay...........................................................................
274
Figure
6.53.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay...........................................................................
275
Figure
6.54.
Boxplot
of
female
11­
ketotestosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay...................................................................
276
Figure
6.55.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay...........................................................................
277
Figure
6.56.
Boxplot
of
female
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
.................................................................................................................
278
Figure
6.57.
Boxplot
of
female
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
..........................................................................................................
279
Figure
6.58.
Boxplot
of
male
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
..................................................................................................................
280
Figure
6.59.
Boxplot
of
male
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
..................................................................................................................
281
TABLES
Table
2.1.
Chemical
concentrations
used
in
the
WA
2­
18
program..........................................................
3
Table
2.2.
Recommended
ranges
of
water­
quality
characteristics
for
testing
fathead
minnows...............
8
Table
2.3.
Testing
schedule
for
P.
promelas
using
four
chemicals
and
three
assays
................................
8
Table
2.4.
Histological
stages
of
fathead
minnow
ovarian
development.
...............................................
14
Table
2.5.
Histological
stages
of
fathead
minnow
testicular
development.
............................................
15
Table
2.6.
Summary
of
the
testing
conditions
for
each
assay
.................................................................
15
Table
3.1.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay...............................................................................
22
Table
3.2.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay...............................................................................
23
Table
3.3.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
14­
Day
Methoxychlor
assay
..........................................................................................
25
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xx
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.4.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
14­
Day
Methoxychlor
assay
..........................................................................................
26
Table
3.5.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
EPA
14­
Day
Methoxychlor
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments
........................................................................................
27
Table
3.6.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
EPA
14­
Day
Methoxychlor
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments
........................................................................................
30
Table
3.7.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
EPA
14­
Day
Methoxychlor
assay
....................................................................................
31
Table
3.8.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay...............................................................................
32
Table
3.9.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.
...................................................................................
33
Table
3.10.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay...............................................................................
34
Table
3.11.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay...............................................................................
35
Table
3.12.
Summary
statistics
and
power
estimates
for
female
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.
...............................................................
36
Table
3.13.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.
...............................................................
37
Table
3.14.
Summary
Statistics
and
Power
Estimates
for
Fecundity
Data
for
the
EPA
14
Day
Methoxychlor
Assay...............................................................................................................
38
Table
3.15.
Summary
Statistics
and
Power
Estimates
for
Fecundity
per
Female
Reproductive
Day
for
the
EPA
14­
Day
Methoxychlor
Assay..............................................................................
39
Table
3.16.
Summary
Statistics
and
Power
Estimates
for
the
Proportion
of
Eggs
Fertilized
for
the
EPA
14­
Day
Methoxychlor
Assay
.........................................................................................
41
Table
3.17.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
14­
Day
Methoxychlor
assay...............................................................................
42
Table
3.18.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
14
Day
Methoxychlor
assay
..........................................................................................
43
Table
3.19.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay...............................................................................
44
Table
3.20.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay...............................................................................
45
Table
3.21.
Summary
Statistics
and
Power
Estimates
for
Female
Gonadosomatic
Index
Data
for
the
EPA
21­
Day
Methoxychlor
Assay
.........................................................................................
47
Table
3.22.
Summary
Statistics
and
Power
Estimates
for
Male
Gonadosomatic
Index
Data
for
the
EPA
21­
Day
Methoxychlor
Assay
.........................................................................................
48
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xxi
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.23
Descriptive
Statistics
of
the
Proportion
of
Ovarian
Cells
in
each
Developmental
Stage
for
Females
from
the
EPA
21­
Day
Methoxychlor
Assay
and
Results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
Comparing
Treatments...........................................................................
49
Table
3.24.
Descriptive
Statistics
of
the
Proportion
of
Testes
Cells
in
Each
Developmental
Stage
for
Males
from
the
EPA
21­
Day
Methoxychlor
Assay
and
Results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
Comparing
Treatments...........................................................................
51
Table
3.25.
Summary
Statistics
and
Power
Estimates
for
Male
Seminiferous
Tubule
Diameter
Data
for
the
EPA
21­
Day
Methoxychlor
Assay..............................................................................
53
Table
3.26
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay...............................................................................
54
Table
3.27.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay.
...................................................................................
54
Table
3.28.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay
with
all
females
having
a
measured
testosterone
value
included.
...................................................................................................
56
Table
3.29.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay...............................................................................
57
Table
3.30.
Summary
statistics
and
power
estimates
for
female
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay.
...............................................................
58
Table
3.31.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay.
...............................................................
59
Table
3.32.
Summary
Statistics
and
Power
Estimates
for
Total
Fecundity
Data
for
the
EPA
21­
Day
Methoxychlor
Assay...............................................................................................................
61
Table
3.33.
Summary
Statistics
and
Power
Estimates
for
Fecundity
per
Female
Reproductive
Day
for
the
EPA
21­
Day
Methoxychlor
Assay..............................................................................
62
Table
3.34.
Summary
Statistics
and
Power
Estimates
for
the
Proportion
of
Eggs
Fertilized
for
the
EPA
21­
Day
Methoxychlor
Assay
.........................................................................................
64
Table
3.35.
Summary
Statistics
and
Power
Estimates
for
the
Proportion
of
Fertile
Eggs
that
Hatched
for
the
EPA
21­
Day
Methoxychlor
Assay
...............................................................
66
Table
3.36.
Summary
Statistics
and
Power
Estimates
for
the
Proportion
of
Normal
Larvae
for
the
EPA
21­
Day
Methoxychlor
Assay
.........................................................................................
67
Table
3.37.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.....................................................
68
Table
3.38.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.....................................................
69
Table
3.39.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
................................................................
72
Table
3.40.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
................................................................
73
Table
3.41.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments...............................................................
75
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xxii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.42.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments...............................................................
78
Table
3.43.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
..........................................................
79
Table
3.44.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.....................................................
80
Table
3.45.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.
.........................................................
81
Table
3.46.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.....................................................
82
Table
3.47.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.....................................................
83
Table
3.48.
Summary
statistics
and
power
estimates
for
female
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.
.....................................
84
Table
3.49.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.
.....................................
85
Table
3.50.
Summary
statistics
and
power
estimates
for
female
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
........................................................................
86
Table
3.51.
Summary
statistics
and
power
estimates
for
female
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
........................................................................
87
Table
3.52.
Summary
statistics
and
power
estimates
for
male
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
........................................................................
88
Table
3.53.
Summary
statistics
and
power
estimates
for
male
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
........................................................................
89
Table
4.1.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Trenbolone
assay...................................................................................
90
Table
4.2.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Trenbolone
assay...................................................................................
91
Table
4.3.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
14­
Day
Trenbolone
assay.
.............................................................................................
93
Table
4.4.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
14­
Day
Trenbolone
assay.
.............................................................................................
94
Table
4.5.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
EPA
14­
Day
Trenbolone
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.
...............................................................................................
95
Table
4.6.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
EPA
14­
Day
Trenbolone
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.
...............................................................................................
98
Table
4.7.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
EPA
14­
Day
Trenbolone
assay.........................................................................................
99
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xxiii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
4.8.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Trenbolone
assay.................................................................................
100
Table
4.9.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Trenbolone
assay.......................................................................................
101
Table
4.10.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Trenbolone
assay.................................................................................
102
Table
4.11.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Trenbolone
assay.................................................................................
103
Table
4.12.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Trenbolone
assay...................................................................
104
Table
4.13.
Summary
statistics
and
power
estimates
for
fecundity
data
for
the
EPA
14­
Day
Trenbolone
assay..................................................................................................................
105
Table
4.14.
Summary
statistics
and
power
estimates
for
fecundity
per
female
reproductive
day
for
the
EPA
14­
Day
Trenbolone
assay.......................................................................................
106
Table
4.15.
Summary
statistics
and
power
estimates
for
the
proportion
of
eggs
fertilized
for
the
EPA
14­
Day
Trenbolone
assay.
...........................................................................................
107
Table
4.16.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
14­
Day
Trenbolone
assay.................................................................................
108
Table
4.17.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
14­
Day
Trenbolone
assay.
...........................................................................................
109
Table
4.18.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Trenbolone
assay.................................................................................
110
Table
4.19.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Trenbolone
assay.................................................................................
111
Table
4.20.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
21­
Day
Trenbolone
assay
............................................................................................
113
Table
4.21.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
21­
Day
Trenbolone
assay
............................................................................................
114
Table
4.22.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
EPA
21­
Day
Trenbolone
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments
..............................................................................................
115
Table
4.23.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
EPA
21­
Day
Trenbolone
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.
.............................................................................................
117
Table
4.24.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
EPA
21­
Day
Trenbolone
assay.......................................................................................
118
Table
4.25.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Trenbolone
assay.................................................................................
119
Table
4.26.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Trenbolone
assay.......................................................................................
120
Table
4.27.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Trenbolone
assay.................................................................................
121
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xxiv
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
4.28.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Trenbolone
assay.................................................................................
122
Table
4.29.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Trenbolone
assay...................................................................
123
Table
4.30.
Summary
statistics
and
power
estimates
for
fecundity
data
for
the
EPA
21­
Day
Trenbolone
assay..................................................................................................................
125
Table
4.31.
Summary
statistics
and
power
estimates
for
fecundity
per
female
reproductive
day
for
the
EPA
21­
Day
Trenbolone
assay.......................................................................................
127
Table
4.32.
Summary
statistics
and
power
estimates
for
the
proportion
of
eggs
fertilized
for
the
EPA
21­
Day
Trenbolone
assay
............................................................................................
128
Table
4.33.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
21­
Day
Trenbolone
assay.................................................................................
130
Table
4.34.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
21­
Day
Trenbolone
assay
............................................................................................
131
Table
4.35.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.......................................................
132
Table
4.36.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.......................................................
133
Table
4.37.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.
.................................................................
135
Table
4.38.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.
.................................................................
136
Table
4.39.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.............................................................
138
Table
4.40.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.............................................................
140
Table
4.41.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.............................................................
141
Table
4.42.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.......................................................
143
Table
4.43.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.............................................................
144
Table
4.44.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.......................................................
145
Table
4.45.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.......................................................
146
Table
4.46.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Trenbolone
assay.........................................
147
Table
4.47.
Summary
statistics
and
power
estimates
for
female
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay...........................................................................
148
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xxv
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
4.48.
Summary
statistics
and
power
estimates
for
female
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay...........................................................................
148
Table
4.49.
Summary
statistics
and
power
estimates
for
male
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay...........................................................................
149
Table
4.50.
Summary
statistics
and
power
estimates
for
male
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Trenbolone
assay...........................................................................
150
Table
5.1.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Flutamide
assay.
..................................................................................
152
Table
5.2.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Flutamide
assay.
..................................................................................
153
Table
5.3.
Table
E14FL
GSI­
1.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
14­
Day
Flutamide
assay.
.......................................
155
Table
5.4.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
14­
Day
Flutamide
assay...............................................................................................
156
Figure
5.4.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
14­
Day
Flutamide
assay............................
156
Table
5.5.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
EPA
14­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.
.............................................................................................
157
Table
5.6.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
EPA
14­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments....................................................................................................
160
Table
5.7.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
EPA
14­
Day
Flutamide
assay.........................................................................................
161
Table
5.8.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Flutamide
assay.
..................................................................................
162
Table
5.9.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Flutamide
assay.........................................................................................
163
Table
5.10.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Flutamide
assay.
..................................................................................
164
Table
5.11.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Flutamide
assay.
..................................................................................
165
Table
5.12.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Flutamide
assay.....................................................................
166
Table
5.13.
Summary
statistics
and
power
estimates
for
fecundity
data
for
the
EPA
14­
Day
Flutamide
assay.
...................................................................................................................
167
Table
5.14.
Summary
statistics
and
power
estimates
for
fecundity
per
female
reproductive
day
for
the
EPA
14­
Day
Flutamide
assay.........................................................................................
168
Table
5.15.
Summary
statistics
and
power
estimates
for
the
proportion
of
eggs
fertilized
for
the
EPA
14­
Day
Flutamide
assay...............................................................................................
169
Table
5.16.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
14­
Day
Flutamide
assay.
..................................................................................
170
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xxvi
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.17.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
14­
Day
Flutamide
assay...............................................................................................
171
Table
5.18.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Flutamide
assay.
..................................................................................
173
Table
5.19.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Flutamide
assay.
..................................................................................
174
Table
5.20.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
21­
Day
Flutamide
assay...............................................................................................
175
Table
5.21.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
21­
Day
Flutamide
assay...............................................................................................
176
Table
5.22.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
EPA
21­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.
.............................................................................................
177
Table
5.23.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
EPA
21­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments....................................................................................................
180
Table
5.24.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
(

m)
data
for
the
EPA
21­
Day
Flutamide
assay.
..........................................................................
181
Table
5.25.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Flutamide
assay.
..................................................................................
182
Table
5.26.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Flutamide
assay.........................................................................................
183
Table
5.27.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Flutamide
assay.
..................................................................................
184
Table
5.28.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Flutamide
assay.
..................................................................................
185
Table
5.29.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Flutamide
assay.....................................................................
186
Table
5.30.
Summary
statistics
and
power
estimates
for
total
fecundity
data
for
the
EPA
21­
Day
Flutamide
assay.
...................................................................................................................
187
Table
5.31.
Summary
statistics
and
power
estimates
for
fecundity
per
female
reproductive
day
for
the
EPA
21­
Day
Flutamide
assay.........................................................................................
189
Table
5.32.
Summary
statistics
and
power
estimates
for
the
proportion
of
eggs
fertilized
for
the
EPA
21­
Day
Flutamide
assay...............................................................................................
190
Table
5.33.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
21­
Day
Flutamide
assay.
..................................................................................
192
Table
5.34.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
21­
Day
Flutamide
assay...............................................................................................
193
Table
5.35.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay.
........................................................
194
Table
5.36.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay.
........................................................
195
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xxvii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.37.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay.....................................................................
197
Table
5.38.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay.....................................................................
198
Table
5.39.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.............................................................
200
Table
5.40.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.............................................................
203
Table
5.41.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay...............................................................
204
Table
5.42.
Histological
observations
for
males
exposed
to
concentrations
of
flutamide
during
the
Non­
spawning
Adult
14­
D
assay.
........................................................................................
205
Table
5.43.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.
.......................................................
206
Table
5.44.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.
.............................................................
207
Table
5.45.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.
.......................................................
208
Table
5.46.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.
.......................................................
209
Table
5.47.
Summary
statistics
and
power
estimates
for
female
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.
.........................................
210
Table
5.48.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.
.........................................
211
Table
5.49.
Summary
statistics
and
power
estimates
for
female
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay.............................................................................
212
Table
5.50.
Summary
statistics
and
power
estimates
for
female
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay.............................................................................
213
Table
5.51.
Summary
statistics
and
power
estimates
for
male
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay.............................................................................
214
Table
5.52.
Summary
statistics
and
power
estimates
for
male
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay.............................................................................
214
Table
6.1.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.
..................................................................................
216
Table
6.2.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.
..................................................................................
217
Table
6.3.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
14­
Day
Fadrozole
assay...............................................................................................
219
Table
6.4.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
14­
Day
Fadrozole
assay...............................................................................................
220
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xxviii
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.5.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
EPA
14­
Day
Fadrozole
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.
.............................................................................................
221
Table
6.6.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
EPA
14­
Day
Fadrozole
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments....................................................................................................
224
Table
6.7.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
EPA
14­
Day
Fadrozole
assay.
........................................................................................
225
Table
6.8.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.
..................................................................................
226
Table
6.9.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.
..................................................................................
227
Table
6.10.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.
..................................................................................
228
Table
6.11.
Summary
statistics
and
power
estimates
for
female
11­
ketotestosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.
....................................................................
229
Table
6.12.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.
....................................................................
230
Table
6.13.
Summary
statistics
and
power
estimates
for
fecundity
data
for
the
EPA
14­
Day
Fadrozole
assay.
...................................................................................................................
231
Table
6.14.
Summary
statistics
and
power
estimates
for
fecundity
per
female
reproductive
day
for
the
EPA
14­
Day
Fadrozole
assay.
........................................................................................
232
Table
6.15.
Summary
statistics
and
power
estimates
for
the
proportion
of
eggs
fertilized
for
the
EPA
14­
Day
Fadrozole
assay...............................................................................................
233
Table
6.16.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
14­
Day
Fadrozole
assay.
..................................................................................
234
Table
6.17.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
14­
Day
Fadrozole
assay...............................................................................................
235
Table
6.18.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.
..................................................................................
237
Table
6.19.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.
..................................................................................
238
Table
6.20.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
21­
Day
Fadrozole
assay...............................................................................................
239
Table
6.21.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
21­
Day
Fadrozole
assay...............................................................................................
240
Table
6.22.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
EPA
21­
Day
Fadrozole
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.
.............................................................................................
241
Table
6.23.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
EPA
21­
Day
Fadrozole
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments....................................................................................................
245
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xxix
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.24.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
EPA
21­
Day
Fadrozole
assay.
........................................................................................
246
Table
6.25.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.
..................................................................................
247
Table
6.26.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.
........................................................................................
248
Table
6.27.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.
..................................................................................
248
Table
6.29.
Summary
statistics
and
power
estimates
for
female
11­
ketotestosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.
....................................................................
250
Table
6.30.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.
....................................................................
251
Table
6.31.
Summary
statistics
and
power
estimates
for
total
fecundity
data
for
the
EPA
21­
Day
Fadrozole
assay.
...................................................................................................................
253
Table
6.32.
Summary
statistics
and
power
estimates
for
fecundity
per
female
reproductive
day
for
the
EPA
21­
Day
Fadrozole
assay.
........................................................................................
255
Table
6.33.
Summary
statistics
and
power
estimates
for
the
proportion
of
eggs
fertilized
for
the
EPA
21­
Day
Fadrozole
assay...............................................................................................
256
Table
6.34.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
21­
Day
Fadrozole
assay.
..................................................................................
258
Table
6.35.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
21­
Day
Fadrozole
assay...............................................................................................
259
Table
6.36.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
........................................................
261
Table
6.37.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
........................................................
262
Table
6.38.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.....................................................................
263
Table
6.39.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.....................................................................
264
Table
6.40.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.............................................................
266
Table
6.41.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.............................................................
268
Table
6.42.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
..............................................................
269
Table
6.43.
Histological
observations
for
males
exposed
to
concentrations
of
fadrozole
during
the
Non­
spawning
Adult
14­
D
assay.
........................................................................................
270
Table
6.44.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
........................................................
272
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
xxx
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.45.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
..............................................................
273
Table
6.46.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
........................................................
273
Table
6.47.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
........................................................
274
Table
6.48.
Summary
statistics
and
power
estimates
for
female
11­
ketotestosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
..........................................
275
Table
6.49.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
..........................................
276
Table
6.50.
Summary
statistics
and
power
estimates
for
female
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.
............................................................................
277
Table
6.51.
Summary
statistics
and
power
estimates
for
female
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.
............................................................................
278
Table
6.52.
Summary
statistics
and
power
estimates
for
male
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.
............................................................................
279
Table
6.53.
Summary
statistics
and
power
estimates
for
male
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.
............................................................................
280
Table
7.1.
Average
Daily
Fecundity
in
Control
Fathead
Minnows
(
mean,
mean
±
SD)
.........................
283
Table
7.2.
Average
Plasma
Markers
in
Control
Female
Fathead
Minnows
(
mean
or
mean
±
SD).........
285
Table
7.3.
Average
Plasma
Markers
in
Control
Male
Fathead
Minnows
(
mean
or
mean
±
SD)
............
285
Table
7.4.
Methoxychlor
Summary
........................................................................................................
287
Table
7.5.
Trenbolone
Summary.............................................................................................................
289
Table
7.6.
Flutamide
Summary...............................................................................................................
290
Table
7.7.
Fadrozole
Summary...............................................................................................................
291
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
1
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
1.0
INTRODUCTION
The
EPA
has
implemented
an
Endocrine
Disruptor
Screening
Program
(
EDSP).
In
1996,
the
Food
Quality
Protection
Act
and
the
Safe
Drinking
Water
Act
were
enacted
by
Congress
to
authorize
the
EPA
to
implement
a
screening
program
to
evaluate
whether
pesticides
and
other
chemicals
found
in
food
or
water
could
affect
endocrine
systems
in
humans.
In
this
program,
comprehensive
toxicological
and
ecotoxicological
screens
and
assays
are
being
developed
to
identify
and
characterize
the
endocrine
effects
of
environmental
contaminants,
industrial
chemicals,
and
pesticides.
A
two­
tiered
approach
is
being
utilized:
Tier
1
employs
a
combination
of
in
vivo
and
in
vitro
screens,
and
Tier
2
involves
in
vivo
testing
using
two­
generation
reproductive
studies.
Validation
of
the
individual
screens
and
assays
is
required,
and
the
Endocrine
Disruptor
Methods
Validation
Subcommittee
(
EDMVS)
will
provide
advice
and
counsel
on
the
validation
assays.

The
Fish
Screening
Assay
was
selected
as
a
component
of
the
Tier
1
screening
by
the
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC)
to
evaluate
the
potential
toxicity
of
chemicals
and
mixtures
on
the
endocrine
system
(
EDSTAC
1998).
The
Tier
1
screening
assays
were
selected
to
obtain
minimum,
yet
sufficient
estimates
of
potential
endocrine
disrupting
activity.
The
Committee
has
several
stated
goals
for
these
assays.
First,
they
should
be
relatively
inexpensive,
quick,
and
technically
easy
to
perform.
Second,
they
should
be
sensitive
and
specific,
capture
multiple
endpoints,
and
be
predictive
across
species,
gender,
and
age.
And
third,
they
should
be
validated
and
standardized
before
they
are
used
routinely
by
testing
laboratories
(
EDSTAC
1998,
Vol.
1,
p
3­
9).
The
purpose
of
using
testing
protocols
within
the
EDSP
is
"
to
characterize
the
nature,
likelihood
of
a
doseresponse
relationship
of
endocrine
disruption
in
humans
and
wildlife"
(
EDSTAC
1998;
EPA
1997).
Subsequently,
the
EPA
has
requested
the
development
of
a
screening
protocol
that
identifies
compounds
having
the
potential
to
affect
selected
endocrine
processes
in
fish.

The
recommended
protocol
to
be
used
as
part
of
a
Tier
1
screening
(
T1S)
battery
includes
a
fish
screen
assay,
which
complements
the
information
from
assays
using
mammals
and
other
ecologically
significant
animal
classes.
Therefore,
the
inclusion
of
the
fish­
screening
assay
in
Tier
1
is
important
because
estrogenic
and
androgenic
controls
in
reproduction
and
development
in
fish
differ
enough
from
those
in
higher
vertebrates
that
mammalian
screening
alone
may
not
identify
potential
endocrine
disrupting
chemicals
in
this
class
of
animals.
It
is
expected
that
the
fish­
screening
assay
will
complement
the
other
screening
assays
such
that
through
its
completion,
the
following
five
criteria
will
be
met.

1.
The
T1S
battery
should
maximize
sensitivity
to
minimize
false
negatives
while
permitting
analysis
of
a
yet
undetermined,
but
acceptable,
level
of
false
positives.
This
criterion
expresses
the
need
to
"
cast
the
screening
net
widely"
to
not
miss
potential
endocrine
disruptors
or
estrogenandrogen
thyroid­
active
materials.

2.
The
T1S
battery
should
include
a
range
of
organisms
representing
known
or
anticipated
differences
in
metabolic
activity.
The
battery
should
include
assays
from
representative
vertebrate
classes
to
reduce
the
likelihood
that
important
pathways
for
metabolic
activation
or
detoxification
of
parent
chemical
substances
or
mixtures
are
not
overlooked.

3.
The
T1S
battery
should
be
designed
to
detect
all
known
modes
of
action
for
the
endocrine
endpoints
of
concern.
All
chemicals
known
to
affect
the
action
of
estrogen,
androgen,
or
thyroid
hormones
should
be
detected.

4.
The
T1S
battery
should
include
a
sufficient
range
of
taxonomic
groups
among
the
test
organisms.
Differences
in
endogenous
ligands,
receptors,
and
response
elements
among
taxa
can
affect
endocrine
activity
of
chemical
substances
or
mixtures.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
2
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
5.
The
T1S
battery
should
incorporate
sufficient
diversity
among
the
endpoints
and
assays
to
reach
conclusions
based
on
"
weight­
of­
evidence"
considerations.
Decisions
based
on
the
battery
results
will
require
weighing
the
data
from
several
assays.

The
Tier
1
screening
must
be
relatively
fast
and
efficient
while
meeting
the
criteria
described
above.
The
screening
includes
a
fish
reproductive
assay,
which
fills
important
needs
in
the
battery
and
complements
the
information
from
assays
using
mammals
and
other
ecologically
significant
animal
classes.
Fish
differ
in
steroid
profiles
from
mammals.
For
example,
11­
ketotestosterone
(
11­
KT),
as
opposed
to
testosterone
(
T),
is
the
most
important
androgen
in
fish,
and
the
estrogen
receptor
(
ER)
in
fish
appears
to
differ
structurally
and
functionally
from
the
mammalian
ER
(
Petit
et
al.,
1995).
In
addition,
steroid
receptors
in
eggs
and
for
hepatic
vitellogenin
(
VTG)
have
no
known
analogous
receptors
in
mammals,
which
would
suggest
sites
of
endocrine
disruption
unique
to
oviparous
animals.
Therefore,
this
assay
is
essential
to
address
these
known
endocrine
differences.

1.1
Purpose
The
purpose
of
this
study
is
to
evaluate
short­
term
screening
assays
designed
to
detect
substances
that
interfere
with
the
estrogen
and
androgen
systems
of
fish.
As
previously
stated,
including
the
fish
screening
assay
in
Tier
1
is
important
because
estrogenic
and
androgenic
controls
on
reproduction
and
development
in
fish
may
differ
enough
from
those
on
higher
vertebrates
that
mammalian
screening
methods
may
not
identify
potential
endocrine­
disrupting
chemicals
in
this
class
of
animals.

The
EDSTAC
recommended
a
fish
gonadal
recrudescence
assay
for
inclusion
in
the
Tier
1
battery
of
assays
(
EDSTAC
1998).
In
subsequent
developmental
work,
a
gonadal
recrudescence
based
approach
with
the
fathead
minnow
did
not
prove
to
be
very
suitable.
Instead,
a
short­
term
reproduction
assay
was
determined
to
be
more
appropriate
for
investigating
EDCs
(
Ankley
et
al.
2001).
Also,
at
this
time
another
approach
using
juvenile
fish
was
suggested
(
Panter
et
al.
2001).
The
Organization
for
Economic
Cooperation
and
Development
established
an
Endocrine
Disrupter
Testing
and
Assessment
Task
Force
which
formed
a
Validation
Management
Group
for
ecotoxicity
tests.
This
group
recommended
that
a
comparison
study
between
the
short­
term
reproduction
assay
(
EPA
2002)
and
a
modified
version
of
the
juvenile
fish
assay
using
non­
spawning
adults
(
OECD
2001)
be
undertaken.

U.
S.
EPA
(
2001)
has
described
a
short­
term
assay
with
the
fathead
minnow
that
considers
reproductive
fitness
as
an
integrated
measure
of
toxicant
effects
and
enables
measurement
of
a
suite
of
histological
and
biochemical
endpoints
that
reflect
effects
associated
with
[
anti­]
estrogens
and
androgens.
The
assay
uses
mature
male
and
female
fish.
During
a
21­
day
chemical
exposure,
survival,
reproductive
behavior,
and
secondary
sexual
characteristics
are
observed;
and
fecundity
and
fertilization
success
are
monitored.
At
selected
times
during
the
assay,
embryo
hatching
and
larval
survivability
are
measured.
At
termination
of
the
assay,
measurements
are
made
of
a
number
of
endpoints
reflective
of
the
status
of
the
reproductive
endocrine
system,
including
the
gonadal­
somatic
index
(
GSI),
gonadal
histology,
and
plasma
concentrations
of
vitellogenin
and
sex
steroids
(
17 ­
estradiol,
testosterone,
11­
ketotestosterone).

The
two
primary
study
objectives
are:
1)
evaluate
the
transferability
and
sensitivity
of
short­
term
reproduction
assays
with
the
fathead
minnow
to
identify
specific
modes
of
action
of
endocrine
disruptors
using
four
model
compounds,
and
2)
conduct
a
side­
by­
side
comparison
of
the
21­
day
fathead
minnow
short­
term
reproduction
assay
(
EPA
2001)
with
two
separate
14­
day
assays:
one
a
shortened
version
of
the
21­
day
assay
with
less
intensive
monitoring
of
reproductive
performance
and
the
other
an
assay
using
pre­
spawning
fathead
minnows
(
OECD
Draft
31
December
2001).
This
latter
assay
is
simplified
by
not
measuring
reproductive
performance
parameters.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
3
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
2.0
MATERIALS
AND
METHODS
2.1
Test
Material
and
Exposure
Regime
The
test
chemical
concentrations
series
used
for
this
project
were
determined
in
consultation
with
representatives
of
EPA
and
are
summarized
in
Table
2.1.
Each
chemical
was
chosen
based
upon
a
suspected
mode
of
action.
Briefly
methoxychlor
is
thought
to
be
a
weak
estrogen
and
is
believed
to
be
metabolized
into
an
active
estrogen
like
metabolite.
Trenbolone
is
an
anabolic
steroid
that
mimics
11­
KT
and
testosterone
to
cause
masculinization
of
females
and
perhaps
enhance
growth.
Flutamide
is
thought
to
be
metabolized
into
the
2­
hydroxylated
form
which
is
a
mammalian
androgen
receptor
antagonist.
If
this
effect
occurs
in
fish,
then
the
normal
effectiveness
of
testosterone
and
11­
KT
would
be
reduced.
Fadrozole
is
an
aromatase
inhibitor.
It
is
also
an
indirect
anti­
estrogen
by
block
synthesis
of
estrogen
(
aromatase
is
the
key
synthetic
step
in
E2
formation).

Table
2.1.
Chemical
concentrations
used
in
the
WA
2­
18
program
Exposure
Concentration
(
µ
g/
L)

Test
Chemical
Low
Mid*
High
Methoxychlor
1
2.5
5
Trenbolone
0.1
0.5
1
Flutamide
6.0
350
650
Fadrozole
5
25
50
*
Mid
concentration
used
for
non­
spawning
method
only.

2.2
Test
Material
Test­
grade
aliquots
of
each
chemical
were
received
from
the
EDSP
chemical
repository
in
Sequim,
Washington.
The
chemicals
were
logged
in
for
analysis
following
the
procedures
for
sample
receipt,
handling,
and
storage:
MSL­
A­
001,
Sample
Log­
In
Procedure,
and
MSL­
A­
002,
Sample
Chain­
of­
Custody.
The
test
materials
were
stored
under
appropriate
conditions
until
transferred
to
the
toxicology
laboratory
for
use
in
the
diluter
system.
A
copy
of
the
chain­
of­
custody
form
accompanied
all
materials.
Prior
to
conducting
the
chemical
exposures,
purity
and
stability
experiments
were
conducted.
A
summary
of
those
results
is
located
in
Appendix
A.

2.3
Preparation
and
Sampling
of
Chemical
Exposure
Water
Methoxychlor
and
flutamide
were
prepared
using
a
saturator
column
(
Figure
2.1)
similar
to
the
method
described
in
Kahl
et
al.
(
1999).
A
complete
description
of
sample
preparation
and
collection
can
be
found
in
chemical
stability
plans
included
as
appendices
to
this
report.
The
saturator­
column
method
was
chosen
because
methoxychlor
and
flutamide
were
found
to
have
low
water
solubility,
and
concentrated
stock
solutions
approaching
the
limit
of
aqueous
solubility
were
found
not
to
be
chemically
stable
over
time.
This
was
documented
by
chemical
stability
studies
performed
by
the
EDSP
chemical
repository.

Specifically,
the
saturator
column
was
used
to
coat
the
chemical
onto
a
large
surface
area
(
i.
e.,
glass
wool)
and
to
expose
the
water
to
the
surfaces
until
an
equilibrium
concentration
was
reached.
Approximately
1
g
of
methoxychlor
and
in
a
later
study,
5
g
of
flutamide,
in
an
aliquot
of
acetone
of
predetermined
volume
were
used
in
the
column,
followed
by
evaporation
of
the
acetone
by
aspirator
vacuum.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
4
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
2.1
Saturator­
column
apparatus
To
prevent
the
need
for
daily
preparation
of
stock
solutions,
large
volumes
were
prepared
with
either
a
32­
gal
or
a
55­
gal
stock
solution
in
a
high­
density
polyethylene
(
HDPE)
drum
lined
with
a
removable
Teflon
bag,
which
provided
an
inert
absorption
surface.
The
stock
solution
was
prepared
by
pumping
water
through
the
column
with
a
fluid
metering
stainless­
steel
pump
at
a
flow
rate
of
about
0.1
L/
min.
The
stock
solution
in
the
drum
was
recirculated
through
the
saturator­
column
system.
The
stock
solution
was
sampled
daily
until
a
stable
concentration
was
reached
(
0.1
mg/
L
methoxychlor
and
20
mg/
L
flutamide),
then
the
drum
was
sampled
twice
per
week
to
determine
stability
and
duration
of
stability
for
each
solution.

Stock­
solution
samples
were
generated
by
use
of
a
saturator
column
as
documented
in
the
stability
and
testing
plans
(
Appendix
X).
Saturator­
column
samples
were
collected
to
verify
test
concentrations
as
new
columns
were
prepared
to
generate
the
large
volumes
of
chemical­
laden
water
required
for
the
study.

Methoxychlor
and
flutamide
sample
concentrations
were
collected
from
the
aquaria
prior
to
beginning
the
test
and
weekly
during
the
test
to
verify
the
concentration
of
the
chemical
solutions.
Samples
from
replicate
diluters
provide
a
measure
of
precision.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
5
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
The
preparation
of
trenbolone
stock
solution
did
not
require
the
use
of
a
saturator
column.
A
stock
solution
of
10
mg/
L
was
initially
prepared
by
the
addition
of
150
mg
trenbolone
into
15
L
deionized
water.
Then,
2
L
of
this
stock
was
added
to
18
L
deionized
water,
which
resulted
in
a
test
solution
of
approximately
1
mg/
L.
Samples
of
the
stock
solution
were
analyzed
prior
to
use.
Test­
solution
samples
were
collected
from
each
test
aquarium
and
analyzed
weekly
throughout
the
duration
of
the
test.

The
preparation
of
the
fadrozole
stock
solution
also
did
not
require
the
use
of
a
saturator.
A
stock
solution
of
40
mg/
L
was
prepared
by
the
addition
of
approximately
800
mg
fadrozole
into
20
L
deionized
water.
Samples
of
the
stock
solution
were
analyzed
prior
to
use.
Test­
solution
samples
were
collected
from
the
aquaria
and
analyzed
weekly
throughout
the
duration
of
the
test.

A
continuous­
flow
proportional
diluter
was
used
to
deliver
chemical
concentrations
to
the
test
aquaria
(
Figure
2.2).
The
diluter
was
a
modified
version
of
the
Mount
and
Brungs
adjusted
to
deliver
three
concentrations
(
including
control)
with
four
replicates
per
concentration
for
the
EPA
methods.
A
second
diluter
was
modified
for
the
non­
spawning
method
to
deliver
four
concentrations
(
including
control)
with
two
replicates
per
concentration.

The
chemical
stock
solution
was
metered
into
the
mixing
cell
of
the
diluter
using
a
fluid
metering
pump.
The
diluter
was
set
to
add
chemical­
laden
water
to
the
test
chamber
every
12
min
and
was
equal
to
six
volume
exchanges
of
water
per
day.
The
diluter
casing
was
covered
in
black
plastic
to
reduce
biological
activity,
such
as
the
development
of
algal
growth,
during
the
test.
Prior
to
introduction
of
the
chemical,
each
diluter
was
calibrated
using
sodium
chloride
as
an
easily
measured
surrogate
test
chemical.
After
completion
of
the
salt
calibrations,
the
chemical
was
added
to
the
mixing
chamber
via
a
fluid
metering
pump,
and
concentrations
in
aquaria
were
checked
prior
to
the
introduction
of
organisms.

Figure
2.2.
Continuous­
flow
proportional
diluter
system
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
6
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
2.4
Analytical
Procedures
After
preparation
of
the
stock
solutions
for
each
of
the
chemicals,
determinations
of
concentrations
were
made
using
the
methods
described
below.
The
concentrations
of
the
chemicals
in
the
exposure
chambers
were
measured
prior
to
adding
fish
to
verify
that
test
concentrations
were
within
30%
of
target
concentrations.
Samples
(
up
to
10
mL)
were
collected
weekly
from
the
test
chambers
and
were
handcarried
and
delivered
to
the
laboratory
on
the
same
day
collected.
Chain­
of­
custody
forms
accompanied
all
samples.
Sample
collection
and
extraction
and
analyses
dates
are
included
in
the
in­
life
chemistry
data.

2.4.1
Methoxychlor
Water
samples
(
approximately
10
mLs)
were
extracted
using
1
mL
hexane
and
analyzed
using
a
gas
chromatograph
with
an
electron
capture
detector
(
GC­
ECD).
Next,
an
internal
standard
(
300
ng
iodonapthalene)
was
added
to
the
water
samples,
and
then
mixed
with
a
vortex
mixer.
The
hexane
layer
was
transferred
to
a
GC
autosampler
vial
and
1
µ
L
injected
onto
the
GC
equipped
with
a
Hewlett­
Packard
30­
m
X
0.25­
mm,
DB­
5
capillary
column.
The
sample
concentration
was
determined
by
comparing
the
peak
heights
in
the
chromatogram
with
the
peak
heights
of
the
calibration
standard
five­
point
curve.
The
temperature
program
was
set
to
start
at
100
º
C,
and
ramped
at
20
º
C/
min
to
a
final
temperature
of
300
º
C.
The
injection
port
temperature
was
set
at
250
º
C
and
the
detector
at
325
º
C.
The
autosampler
was
set
to
inject
1
µ
L
of
the
matrix
dilution
every
sample.

2.4.2
Trenbolone
Quantification
by
GC
with
mass
selective
detection
(
MSD)
was
used
for
the
analysis
of
the
low
concentrations
of
trenbolone
and
control
exposure
water.
Water
samples
(
10
mL)
were
removed
from
the
exposure
tanks
and
stored
in
a
20­
mL
Pyrex
culture
tube,
to
which
the
internal
standard,
17 ­
estradiol,
was
added.
Next,
approximately
1
g
sodium
chloride
(
NaCl)
was
added,
the
sample
was
agitated,
and
2.0
mL
hexane
was
added.
This
mixture
was
blended
with
a
vortex
mixer,
and
the
hexane
layer
removed
and
placed
in
a
new
glass
tube.
This
procedure
was
done
in
triplicate,
and
the
hexane
layers
were
pooled.
The
pooled
hexane
layers
were
then
evaporated
to
dryness
under
helium
(
He)
gas.
Next,
the
evaporated
residue
was
dissolved
in
10
µ
L
of
n­
methyl­
n­
trimethylsilyl­
trifluoroacetamide
(
MSTFA)
with
catalytic
amounts
of
resublimed
iodine
(
1000:
4
v:
w)
added
as
the
derivitizing
agent.
A
1­
µ
L
quantity
was
injected
onto
the
GC.
The
GC­
MSD
was
operated
in
the
selected
ion­
monitoring
mode
with
the
molecular
ions
m/
z
442
(
trenbolone
/
285
(
internal
standard,
17 ­
estradiol))
used
for
quantitative
purposes.
The
observed
retention
time
for
trenbolone
is
approximately
18.29
minutes
and
for
the
internal
standard,
14.55
minutes.
A
five­
point
calibration
curve
was
prepared
and
used
for
quantification
of
the
trenbolone
in
the
water
samples.

Analysis
of
the
mid­
and
high­
trenbolone­
concentration
exposure
water
was
conducted
by
highperformance
liquid
chromatograph
(
HPLC)
analysis
using
the
fluorescence
detector.
Analyses
of
the
trenbolone
stock
solution
were
done
by
HPLC
analysis
using
the
ultraviolet/
visible
(
UV/
VIS)
detector
at
347
nm.

2.4.3
Flutamide
The
water
samples
(
approximately
10
mLs)
were
mixed
with
50:
50
v:
v
ACN
and
analyzed
using
an
HPLC
with
a
UV/
VIS
detector
at
the
220­
nm
wavelength.
A
60:
40
ACN:
water
(
v:
v)
mix
was
used
for
the
eluent
at
1.5
mL/
min.
Separation
was
attained
using
a
Supelco
polynuclear
aromatic
hydrocarbon
(
PAH)
(
25­
cm
X
4.8­
mm,
C­
18
column).
For
samples
analyzed
using
the
HPLC
system,
data
were
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
7
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
stored
in
MSL5,
Room
219
on
the
computer
with
a
property
number
of
WV04738.
Multipiers
of
40
(
1­
mL
total
sample
volume
made
from
0.025­
mL
of
sample
and
0.975
mL
ACN:
water)
and
2
(
0.5­
mL
sample
with
0.5­
mL
ACN)
were
used
in
the
Varian
Star
software.

2.4.4
Fadrozole
The
samples
were
analyzed
using
an
HPLC
with
a
UV/
VIS
detector
at
the
236­
nm
wavelength
with
a
five­
point
calibration
curve.
A
1.0­
mL
quantity
of
each
sample
was
injected
for
separation
and
analysis.
The
eluent
was
a
25­
mM
sodium
phosphate
buffer
solution
and
acetonitrile
(
lot
#
Y02820)
at
a
30:
70
v:
v
proportion.
Separation
was
attained
using
a
Pherominex
Synergy
4u
Hydrus
RP
80A
250­
mm
X
4.6­
mm
column.
For
samples
analyzed
using
the
HPLC
system,
data
were
stored
in
MSL5,
Room
219,
on
the
computer.

2.5
Animals
and
Husbandry
The
requirements
for
age
and
size
of
the
test
species
stated
that
the
minnows
must
be
sexually
dimorphic,
should
be
first­
time
spawning
conditions,
approximately
120
days
old,
and
the
minimum
size
must
be
2.5
g
for
males
and
1.5
g
for
females.
These
requirements
were
met
by
purchasing
organisms
from
two
suppliers:
Environmental
Consulting
and
Testing
(
EC&
T),
Superior,
Wisconsin,
and
ABC
Laboratories,
Columbia,
Missouri.
Documentation
of
chain­
of­
custody,
the
condition
of
the
animals
when
shipped
and
upon
receipt,
and
environmental
parameters
(
temperature)
at
the
time
of
shipping
for
comparison
with
conditions
encountered
at
the
time
of
receipt,
and
verification
of
the
taxonomy
of
the
organisms
(
genus,
species)
and
disease­
free
status
were
submitted
with
each
batch
of
organisms.
The
vendor
wrote
an
informal
note
stating
that
there
was
no
reported
incidence
of
disease
during
the
care
and
maintenance
of
the
minnows.

Approximately
1200,
4­
month­
old
P.
promelas
were
obtained
from
EC&
T
and
used
in
the
methoxychlor
experiments.
Because
of
questions
regarding
overall
fish
health
that
arose
after
the
methoxychlor
test,
it
was
decided
that
younger
P.
promelas
should
be
purchased
and
held
for
2
to
3
months
prior
to
the
test.
Most
aquatic
suppliers
of
P.
promelas
do
not
rear
minnows
to
120
days.
Therefore,
it
is
possible
that
any
problem
with
the
health
of
the
fish
became
apparent
only
during
their
later
more
mature
stages.

Approximately
2400,
30­
to
60­
day
old
P.
promelas
were
purchased
from
ABC
Laboratories
and
cultured
to
the
stage
of
reproductive
differentiation
and
sexual
maturation
following
the
guidance
described
in
Guidelines
for
the
Culture
of
Fathead
Minnows
(
Pimephales
promelas)
for
Use
in
Toxicity
Tests
(
EPA
1987)
and
SOP
Number
EDSP.
E­
001­
01.
This
group
of
P.
promelas
was
used
in
experiments
conducted
with
trenbolone,
flutamide,
and
fadrozole.

Water
conditions
for
both
supplies
of
P.
promelas
were
maintained
at
24
°
C
to
26
°
C.
A
flow­
through
system
design
provided
adequate
volume
replacement
for
organism
needs
while
maintaining
the
required
constant
temperature.
A
continuous,
gentle
aeration
from
an
oil­
free
air
supply
was
provided
to
the
tanks.
The
minnows
were
housed
in
30­
gal
tanks
upon
arrival
and
until
sexually
differentiated,
whereupon
they
were
separated
by
sex
and
transferred
to
clean,
10­
gal
aquaria
until
needed
for
testing.
To
establish
breeding
pairs,
four
females
and
two
males
were
transferred
at
the
time
of
assay
to
5­
gal
containers,
which
contained
spawning
tiles
made
of
terracotta.
Table
2.2
provides
the
water­
quality
characteristics
for
culturing
and
testing
the
fathead
minnow:
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
8
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
2.2.
Recommended
ranges
of
water­
quality
characteristics
for
testing
fathead
minnows
Water
Characteristic
Preferred
Range
Temperature
(
oC)
24
°
C
­
26
°
C
Dissolved
Oxygen
(
mg/
L)
>
4.9
mg/
L
(>
60%
saturation)
pH
6.5
­
9.0
pH
units
Total
Alkalinity
(
mg/
L
as
CaCo3)
>
20
mg/
L
Total
Organic
Carbon
(
mg/
L)
 
5
mg/
L
Unionized
Ammonia
 
35
mg/
L
2.6
Study
Schedule
and
Design
The
design
of
this
study
was
to
evaluate
the
sensitivity
of
short­
term
reproduction
and
nonreproductive
assays
with
the
fathead
minnow
(
P.
promelas)
to
identify
specific
modes
of
action
of
endocrine
disruptors
using
four
model
compounds,
and
to
conduct
a
side­
by­
side
comparison
of
the
21­
day
fathead
minnow,
short­
term
reproduction
assay
(
EPA
2001)
with
two
separate
14­
day
assays:
a
shortened
version
of
the
21­
day
assay
and
an
assay
that
does
not
measure
reproductive
performance
(
males
and
females
are
kept
in
separate
chambers
and
do
not
spawn)
(
OECD
2001).
Two
different
proportional
flow
diluters
were
used
for
a
given
chemical.
One
diluter
was
prepared
to
deliver
three
concentrations
(
low,
medium,
and
high)
and
a
control,
and
was
used
for
the
non­
spawning
method.
The
other
was
prepared
to
deliver
two
concentrations
and
a
control,
and
was
used
for
both
the
EPA
14­
and
21­
day
assays.
Two
different
stock
solutions
were
prepared
and
monitored
for
each
diluter.
The
testing
schedule
is
presented
in
Table
2.3.

Table
2.3.
Testing
schedule
for
P.
promelas
using
four
chemicals
and
three
assays
Chemical
Task
Date
Methoxychlor
EPA
14
Pre­
exp.
9/
29/
2002
EPA
21
Pre­
exp.
9/
29/
2002
EPA
14­
day
test
10/
14/
2002
EPA
21­
day
test
10/
14/
2002
Non­
spawning
10/
14/
2002
Trenbolone
EPA
14
Pre­
exp.
2/
4/
2003
EPA
21
Pre­
exp.
1/
20/
2003
EPA
14­
day
test
2/
11/
2003
EPA
21­
day
test
2/
3/
2003
Non­
spawning
2/
10/
2003
Flutamide
EPA
14
Pre­
exp.
2/
18/
2003
EPA
21
Pre­
exp.
2/
11/
2003
EPA
14­
day
test
2/
25/
2003
EPA
21­
day
test
2/
25/
2003
Non­
spawning
3/
10/
2003
Fadrozole
EPA
14
Pre­
exp.
3/
17/
2003
EPA
21
Pre­
exp.
3/
10/
2003
EPA
14
test
3/
27/
2003
EPA
21
day
test
3/
25/
2003
Non­
spawning
4/
1/
2003
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
9
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
For
the
two
EPA
assays,
the
P.
promelas
were
first
held
under
a
pre­
exposure
phase
with
no
chemical
present
for
7
to
14
days
to
establish
a
record
of
spawning
success
and
to
measure
viability
of
embryos.
The
assay
units
(
5­
gal
aquaria
with
4
females
and
2
males)
were
then
chosen
for
a
14­
or
21­
day
chemical
exposure.
There
was
no
pre­
exposure
phase
for
the
non­
spawning
method
prior
to
introduction
of
the
chemical
into
the
test
system.

2.7
Description
of
Study
Protocols
The
experimental
design
included
the
comparison
of
three
fish­
screening
protocols
in
side­
by­
side
experiments
using
four
different
chemicals.

2.7.1
Summary
of
the
14­
day
Non­
spawning
Method
The
14­
day
non­
spawning
assay
was
based
on
a
description
in
the
OECD
draft
proposal
 
31
December
2001
(
OECD
2001).
The
assay
was
intended
to
detect
endocrine
disruptor
effects
of
specific
chemicals
with
a
mode
of
action
that
is
either
(
anti)
androgenic
or
(
anti)
estrogenic.

P.
promelas
chosen
for
this
protocol
were
pre­
spawning
adults,
which
were
separated
by
sex.
The
assay
was
initiated
with
healthy,
sexually
dimorphic
adult
fish
(
males
and
females
contained
in
separate
chambers
to
prevent
induction
of
spawning).
The
chambers
included
two
replicate
tanks
(
one
for
each
gender)
per
treatment
with
four
treatments:
a
dilution
water
control,
and
low,
medium,
and
high
concentrations.
The
non­
spawning
assay
was
conducted
at
the
same
time
as
the
EPA
14­
and
21­
day
assays;
however,
a
different
chemical
stock
solution,
proportional
diluter,
and
water
table
were
used
for
this
method.
The
stock
solutions
and
water
chemistry
parameters
were
monitored
throughout
testing,
and
observations
conducted
daily
of
test
aquaria.

The
measurement
endpoints
included
survival,
morphological,
and
histological
parameters.
At
the
conclusion
of
the
test,
blood
samples
were
collected
from
the
adults
for
determination
of
sex
steroids
and
vitellogenin
(
VTG),
and
the
gonads
were
sampled
for
measurement
of
the
GSI
and
histological
analyses.
In
addition,
fork
length
measurements
were
taken
and
general
gross
morphological
observations
were
made.

2.7.2
Summary
of
the
14­
day
EPA
Method
The
14­
day
assay
began
with
at
least
a
7­
day
pre­
exposure
phase.
This
phase
was
conducted
using
5­
gal
aquaria
containing
three
terracotta
nesting
tiles.
Breeding
pairs
for
each
aquarium
included
four
females
and
two
males
that
were
randomly
assigned
to
an
aquarium
at
each
treatment
concentration.
Additional
exposure
chambers
were
set
up
to
account
for
non­
spawning
or
otherwise
compromised
breeding
pairs
in
some
chambers
during
this
time.

The
pre­
exposure
phase
was
conducted
under
conditions
(
temperature,
photoperiod,
feeding,
and
flow)
identical
to
those
used
during
the
chemical
exposure.
The
P.
promelas
were
fed
frozen
Artemia
up
to
three
times
daily
ad
libitum
and
were
monitored
daily
for
alterations
in
secondary
sex
characteristics
(
breeding
tubercles
in
males;
ovipositor
in
females),
reproductive
behavior,
and
spawning
activity.
No
quantitative
measures
of
fecundity
were
made
during
the
pre­
exposure
phase.
However,
larval
hatchability
was
assessed
using
a
subset
of
normal
eggs
(
approximately
50)
incubated
at
test
conditions
for
3
to
6
days
until
eggs
hatched.

The
data
collected
from
the
pre­
exposure
phase
were
examined
using
criteria
as
recommended
in
EPA
guidelines
(
page
37,
EPA
2001):
regular
spawning
occurs
in
each
test
chamber
every
3
to
4
days,
and
a
greater
than
90%
hatching
rate
of
larvae
is
observed
after
7
days.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
10
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Breeding
pairs
that
passed
the
requirements
from
the
preexposure
phase
were
then
transferred
to
the14­
day
chemical
exposure
phase.
Test
treatments
included
four
replicate
containers,
a
dilution­
water
control,
and
a
low
and
high
concentration
that
matched
the
low
and
high
concentrations
measured
for
the
non­
spawning
assay.
The
14­
day
assay
measured
the
reproductive
performance
of
groups
of
fathead
minnows
as
the
primary
indicator
for
endocrine
disruption.
The
fish's
appearance,
behavior,
and
fecundity
were
assessed
daily,
and
routine
water
chemistry
measurements
were
taken.
Terracotta
tiles
and
nytex
screen
were
used
as
housing
for
fish
during
spawning.
The
screened
terracotta
dishes
allowed
enumeration
of
eggs
that
did
not
adhere
to
the
underside
of
the
tile.
(
Figure
2.3)
Larval
hatching
as
described
above
was
conducted
one
time
during
the
14­
day
chemical
exposure
phase.
At
termination
of
the
exposure,
blood
samples
were
removed
from
adults
and
analyzed
for
sex
steroids
and
VTG.
The
gonads
were
also
removed
for
GSI
determination
and
later
histological
analyses.
In
addition,
fork
length
measurements
and
general
gross
morphological
conditions
were
noted
(
appearance
of
adults).

2.7.3
Summary
of
21­
day
EPA
Method
The
21­
day
EPA
assay
method
paralleled
the
14­
day
method
with
the
exception
that
larval
hatching
was
conducted
once
during
the
pre­
exposure
phase
and
three
times
during
chemical
exposure.
Under
this
scenario,
the
pre­
exposure
phase
continued
for
14­
days
(
rather
than
7)
and
quantitative
counts
of
fecundity
were
performed
daily.
The
data
generated
from
the
pre­
exposure
phase
were
used
to
determine
which
spawning
pairs
were
suitable
for
the
21­
day
chemical
exposure.
The
aquaria
chosen
for
chemical
exposure
were
then
transferred
to
the
proportional
diluter
table
in
the
same
system/
tanks
as
was
used
for
the
pre­
exposure
phase.

The
21­
day
chemical
exposure
was
conducted
using
the
same
chemical
stock
solution,
proportional
diluter,
and
water
table
as
the
14­
day
EPA
assay.
This
side­
by­
side
comparison
using
the
same
chemical
concentrations
and
source
of
heated
dilution
water
allowed
for
more
robust
statistical
comparisons.

During
the
21­
day
chemical­
exposure
period,
the
fish's
appearance,
behavior,
and
fecundity
were
assessed
daily.
Viability
of
resultant
embryos
(
e.
g.,
hatching
success)
was
measured
on
Days
7,
14,
and
21
by
incubating
approximately
50
eggs
in
dilution
water
for
3
to
6
days
until
the
majority
of
eggs
hatched.

At
conclusion
of
the
assay,
blood
samples
were
collected
from
the
adults
for
determination
of
sex
steroids
and
VTG,
and
the
gonads
were
sampled
for
measurement
of
the
GSI
and
for
histological
analyses.
In
addition,
fork
length
measurements
and
general
gross
morphological
conditions
were
noted
(
appearance
of
adults).
Figure
2.3
Terracotta
dish
with
nytex
screen
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
11
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
2.7.4
Summary
of
Assay
Endpoints
Survival:
Daily
assessment
of
survival
was
made
to
provide
a
basis
for
expression
and
interpretation
of
reproductive
output:
that
is,
number
of
eggs
per
female
per
day.

Behavior
of
Adults:
Abnormal
behavior
relative
to
controls,
such
as
hyperventilation,
loss
of
equilibrium,
and
feeding
abstinence,
was
noted
during
the
daily
observations.
Alterations
in
reproductive
behavior,
particularly
loss
of
territorial
aggressiveness
by
males,
were
noted.

Fecundity/
Fertilization
Success:
Egg
production
was
determined
daily.
The
terracotta
spawning
substrates
were
removed
from
the
tanks,
and
the
eggs
were
allowed
to
harden
prior
to
enumeration
of
eggs.
After
hardening,
the
eggs
were
carefully
rolled
off
the
tile
with
a
gentle
circular
motion
of
a
gloved
index
finger
and
visually
inspected
under
appropriate
magnification.
If
no
embryos
were
present,
the
substrate
was
left
in
the
aquarium.
If
spawning
occurred
that
morning,
embryos
typically
underwent
late
cleavage,
and
determination
of
the
fertility
rate
(
number
of
embryos/
number
of
eggs
x
100)
was
easily
achieved.
Infertile
eggs
were
opaque
or
clear
with
a
white
dot
where
the
yolk
precipitated;
viable
embryos
remained
clear
for
36
to
48
hours
until
reaching
the
eyed
stage.
Fecundity
was
expressed
on
the
basis
of
surviving
females
per
reproductive
(
test)
day
per
replicate.
This
approach
was
used
for
both
the
14­
and
21­
day
assays
(
Figure
2.4).

Hatchability
and
Larvae
Appearance:
This
endpoint
was
assessed
as
follows:
14­
day
assay:
once
during
preexposure
(
Day
7)
and
once
during
exposure
(
Day
7);
21­
day
assay:
once
during
pre­
exposure
(
Day
7)
and
three
times
during
exposure
(
Days
7,
14,
and
21).

Approximately
50
normal
healthy
looking
embryos
were
transferred
to
incubation
chambers
(
1­
L
flowthrough
jars
with
gentle
flow
and
aeration)
and
held
at
25
°
C
(
Figure
2.5).
The
larvae
typically
hatched
in
3
to
6
days.
Daily
observations
were
conducted
and
the
number
of
embryos,
newly
hatched
larvae,
and
any
dead
embryos
or
larvae
were
scored.
The
data
from
the
hatching
test
were
used
to
determine
the
percentage
of
eggs
that
hatched
and
number
of
normal
appearing
larvae.

Figure
2.5.
Flow­
through
embryo­
incubation
chambers
Figure
2.4
Staff
members
count
eggs
in
support
of
fecundity
measurements.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
12
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Appearance
of
Adults:
The
external
appearance
of
the
adults
was
assessed
as
part
of
the
daily
observations,
and
any
unusual
changes
were
noted.
External
features
of
particular
importance
included
body
color
(
light
or
dark),
coloration
patterns
(
presence
of
vertical
bands),
body
shape
(
head
and
pectoral
region),
and
specialized
secondary
sex
characteristics
(
dorsal
nape
pad,
nuptial
tubercles
in
males;
ovipositor
in
females).
The
daily
observation
forms
are
included
as
appendices
to
this
report.

Blood
Sampling:
At
the
conclusion
of
the
exposure,
the
fish
were
anesthetized
by
transfer
to
an
oxygenated
solution
of
tricane
sulfate
(
MS­
222)
(
250
mg/
L
buffered
with
200
mg
NaHCO3/
L).
Blood
was
collected
from
the
caudal
vein
with
a
heparinized
microhematocrit
capillary
tubule.
Depending
on
the
size
of
the
fathead
minnow,
which
usually
is
gender­
dependent,
blood
volumes
generally
ranged
from
30
µ
L
to
80
µ
L.
Plasma
was
separated
from
the
blood
via
centrifugation
(
2
min)
and
stored
with
protease
inhibitors
at
­
80
º
C
until
analyzed
for
VTG
and
sex
steroids.

Vitellogenin:
The
measurement
of
VTG
plasma
samples
was
performed
using
an
enzyme­
linked
immunoabsorbant
test
(
ELISA).
Vitellogenin
levels
were
quantitated
in
plasma
samples
from
each
individual
fish,
for
all
treatments,
using
research
quality
test
kits
(
product
number
V01003401)
procured
from
Biosense
Laboratories
(
AS
 
HIB­
Thormohlengsgt.
55
N­
5008,
Bergen,
Norway).
The
analyses
were
conducted
on
a
Bio­
Tek
Synergy
HT
microtiter
plate
reader
interfaced
to
a
Dell
computer,
employing
the
Bio­
Tek
KC4
test
analysis
software.

Plasma
samples
were
frozen
in
5­
µ
L
aliquots
and
stored
in
600­
µ
L
microcentrifuge
tubes
at
­
80
°
C
until
the
day
of
analysis.
For
analyses,
samples
were
removed
from
­
80
°
C
storage
and
placed
on
ice.
The
samples
were
rehydrated
with
495
µ
L
cold
phosphate
buffered
saline
(
PBS)
containing
1%
bovine
serum
albumin
(
BSA).
Appropriate
dilutions
of
the
samples,
beyond
the
initial
1:
100
dilution,
were
carried
out
prior
to
the
analysis.
Dilutions
ranged
from
1:
100
to
1:
108,
determined
by
the
sex
of
the
fish
and
specific
chemical
exposure
defined
in
the
study.
The
test
requires
approximately
6
to
8
hours
to
complete,
depending
on
the
number
of
samples
run
and
the
number
of
dilutions
of
each
sample
required.
A
typical
daily
run
would
include
82
samples
divided
between
two
96­
well
microtiter
plates.

The
test
has
a
calibration
range
of
0.24
ng/
mL
to
250
ng/
mL,
with
a
viable
quantitation
range
of
approximately
0.6
ng/
mL
through
125
ng/
mL.
For
data
analyses,
a
value
of
zero
(
0)
was
assigned
to
samples
in
which
vitellogenin
was
not
detected.
Data
were
natural­
log
transformed
(
ln
(
concentration
+
1))
before
analyses
were
conducted.

The
test
required
three
1­
hour
incubations,
each
with
a
different
antibody­
based
reagent
required
to
capture
the
sample
analyte
and
create
the
detectable
sandwich.
The
sandwich
was
made
up
of
the
capture
antibody,
the
analyte,
the
detecting
antibody,
and
the
secondary
antibody
labeled
with
the
horseradish
peroxidase
(
HRP)
enzyme.
In
the
final
step,
the
microtiter
plate
wells
were
thoroughly
washed
with
a
wash
buffer,
removing
all
test
components,
save
the
analyte
captured
within
the
bound
sandwich.
An
HRP
substrate
was
then
added
to
the
wells,
and
following
a
30­
min
incubation,
the
absorbance
of
each
well
was
read
at
492
nm.
Absorbance
levels
increased
with
increased
calibrator
or
sample
concentrations.

Sex
Steroid:
Plasma
concentrations
of
 ­
estradiol,
testosterone,
and
11­
ketotesosterone
were
measured
using
competitive
enzyme
immunoassays
(
EIAS)
commercially
available
for
each
steroid
of
interest.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
13
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Gonad
Size
and
Morphology:
After
blood
was
sampled,
fish
were
weighed,
and
the
gonads
were
removed
and
weighed
to
the
nearest
0.1
mg
to
determine
the
GSI
(
GSI
=
100
X
gonad
wt/
body
wt)
(
Figure
2.4).
Typical
GSI
values
for
reproductively
active
fathead
minnows
range
from
8%
to
13%
for
females
and
from
1%
to
2%
for
males.
Many
chemicals
that
reduce
fecundity
also
will
reduce
the
GSI
in
one
or
both
sexes.
After
removal
of
the
gonads,
the
remainder
of
the
carcass
of
the
fish
was
discarded.

Histology:
Routine
histological
procedures
were
used
to
assess
the
condition
of
testes
and
ovaries
from
the
fish.
Gonads
were
placed
in
fixative
(
10%
buffered
formalin)
and
embedded
in
paraffin.
Serial
sections
4­
to
5­
µ
m
thick
were
cut
along
the
long
axis
of
the
gonad.
At
least
two
serial
sections
were
collected
from
at
least
three
steps
equally
spaced
between
the
leading
edge
of
the
tissue
and
the
midline
of
the
gonad,
for
a
total
of
six
tissue
sections
per
sample
(
Figure
2.6).
Sections
were
stained
with
hematoxylin
and
eosin,
and
were
submitted
to
and
evaluated
by
a
board
certified
histologist
without
prior
knowledge
of
the
treatment
regime
associated
with
specific
samples.
A
summary
of
the
methods
used
by
the
histologist
follows.

The
histology
method
was
previously
described
in
Ankley
et
al.
(
2001);
methods,
original
references,
and
photomicrographs
of
normal
developmental
stages
of
the
reproductive
tract
of
both
female
and
male
fathead
minnows
were
included.
The
methods
were
developed
for
the
systematic
assessment
of
fathead
minnow
reproductive
tracts
and
reference
to
pathological
changes
that
may
occur
upon
exposures
to
endocrine
disruptor
chemicals
(
EDCs).
These
methods
were
followed.
The
paraffinembedding
technique
was
selected
over
the
glycol
methacrylate­
embedding
technique
for
these
analyses.

The
following
is
an
outline
summary
of
the
procedures
that
were
used
to
evaluate
the
histological
sections.
Multiple
measurements
from
each
individual
were
taken
from
a
variety
of
locations
on
the
tissue
sections.

Females:
1.
The
ovary
was
staged,
that
is,
given
a
number
from
1a,
1b,
2,
3,
4,
or
5,
based
on
the
most
advanced
stage
present
(
Table
2.4).
The
explanation
of
the
stage
numbering
was
provided
in
Ankley
et
al.
(
2001).
This
staging
was
done
in
six
locations
on
each
of
the
three
histological
slides.

2.
Oogonia
and
oocytes
were
typed,
and
100
cells
from
each
of
three
sections
were
rated
according
to
developmental
stage
(
stages
noted
above,
including
atretic
follicles
and
corpora
lutea).

3.
Abnormalities
in
the
ovary
were
noted.

Males:
1.
Testes
were
staged,
that
is,
given
a
number
from
1,
2a,
2b,
3a,
3
b,
4,
or
5,
based
on
the
most
advanced
stage
present
(
Table
2.5).
This
staging
was
done
on
four
locations
on
each
of
the
three
slides.
Figure
2.6.
Collection
of
the
liver
and
gonads
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
14
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
2.
Based
on
stages
noted
above,
100
spermatic
cells
were
typed
from
each
of
the
three
slides.
This
typing
is
done
by
counting
the
cells
along
a
straight
line
on
an
ocular
grid
in
a
predetermined
pattern.
More
than
one
line
on
the
grid
is
used
if
required
to
obtain
the
100
cells.

3.
The
testicular
lumen
diameter
(
µ
m)
was
measured
from
six
tubules
on
each
of
three
slides.

4.
Other
changes
were
noted,
including
changes
to
the
interstitial
tissues,
such
as
proliferation
of
Sertoli
or
Leydig
cells.
In
addition,
abnormal
patterns
of
development
were
noted,
such
as
premature
shedding
of
spermatocytes
into
the
tubule
lumen
or
foci
of
necrotic
spermatocytes.
The
presence
of
any
ovatestes
or
patterns
of
testicular
atrophy
were
noted.

Quality
Assurance:

Over
300
randomly
picked
slides
from
the
first
data
set
were
examined
in
detail
for
conformance
with
the
descriptions
provided
in
Ankley
et
al.
(
2001)
prior
to
beginning
the
systematic
examination
of
the
histology
slides.
The
raw
data
from
systematic
slide
evaluation
were
recorded
on
hand­
written
sheets.
These
data
were
transferred
to
a
Microsoft
Excel
spreadsheet.
A
second
100%­
accuracy
check
of
data
transfer
was
conducted
after
the
initial
transfer
of
data
to
the
spreadsheets.
In
addition,
spot
checks
of
comments
and
abnormal
conditions
were
conducted
by
re­
examining
the
slides
for
conformance
with
the
original
comments.
In
addition,
a
final
examination
of
the
spreadsheet
for
accuracy
was
made
by
the
histology
principal
investigator.

Table
2.4.
Histological
stages
of
fathead
minnow
ovarian
development.

Stage
Characteristics
1.
Primary
Growth
Oogonia
and
primary
oocytes
1a.
Oocytes
in
nests;
small
cytoplasmic
volume
1b.
Oocytes
larger,
out
of
nests,
surrounded
by
follicle
cells;
many
pleiomorphic
nucleoli
bordering
the
nuclear
envelope
2.
Cortical
Alveolus
Appearance
of
cortical
alveoli
and
possibly
small
lipid
droplets
3.
Early
Vitellogenic
Appearance
of
yolk
bodies;
initially
few
and
small,
ultimately
many
and
variably­
sized;
centrallylocated
germinal
vesicle
is
round
to
oval
with
several
peripheral
nucleoli
4.
Late
Vitellogenic
Germinal
vesicle
loses
nucleoli,
moves
towards
the
periphery
and
breaks
down;
yolk
bodies
frequently
fill
the
entire
center
of
the
oocyte
and
a
germinal
vesicle
may
not
be
evident
5.
Mature/
Spawning
Oocytes
Germinal
vesicle
breakdown
complete;
yolk
bodies
fuse
and
may
become
larger
than
cortical
alveoli
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
15
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
2.5.
Histological
stages
of
fathead
minnow
testicular
development.

Stage
Characteristics
1.
Resting
Germ
Cells
No
development
2.
Spermatogonia
2a.
Primary
Spermatogonia:
Large
cells
near
edges
of
tubule;
have
a
lightly
staining
nucleus
with
a
prominent
nucleolus
2b.
Secondary
Spermatogonia:
Clusters
of
medium­
sized
cells
with
a
round,
lightly
basophilic
nucleus;
cluster
or
cyst
is
the
result
of
several
mitotic
divisions
of
primary
spermatocyte
3.
Spermatocytes
3a.
Primary
Spermatocytes:
Smaller
cells
with
smaller,
more
basophilic
nuclei
than
Spermatogonia;
will
undergo
meiosis
I
to
produce
secondary
spermatocytes
3b.
Secondary
Spermatocytes:
Small
cells
with
smaller,
more
basophilic
nuclei
than
primary
spermatocytes;
will
undergo
meiosis
II
to
produce
spermatids
4.
Spermatids
and
some
spermatozoa
in
lumen
of
seminiferous
tubules;
small
tubule
lumen
Spermatids
have
a
small,
intensely
basophilic
nucleus;
mature
into
spermatozoa
5.
Abundant
sperm
in
an
expanded
lumen
Table
2.6
provides
a
detailed
summary
of
each
assay
and
the
associated
data
collected
during
testing.

Table
2.6.
Summary
of
the
testing
conditions
for
each
assay
Parameters
Non­
spawning
14­
day
EPA
14­
day
EPA
21­
day
Age
of
organisms
Prebreeding
adult
(
5­
6
months)
fathead
minnows
Reproductive
adult
fathead
minnows
(
120
day
minimum)
Reproductive
adult
fathead
minnows
(
120
day
minimum)
Holding
Conditions:
Temp:
25
°
C
±
1
°
C
D.
O.(
a)
>
4.9
mg/
L
Light:
16
h
light:
8
h
dark
with
400
 
500
lux
Fed:
live
brine
shrimp
and
ground
salmon
starter
(
automatic
feeder)
until
able
to
eat
frozen
brine
shrimp.
Brine
shrimp
was
given
up
to
three
times
daily
Temp:
25
°
C
±
1
°
C
D.
O.
>
4.9
mg/
L
Light:
16
h
light:
8
h
dark
with
400
 
500
lux
Fed:
live
brine
shrimp
and
ground
salmon
starter
(
automatic
feeder)
until
able
to
eat
frozen
brine
shrimp.
Brine
shrimp
was
given
up
to
three
times
daily
Temp:
25
°
C
±
1
°
C
D.
O.
>
4.9
mg/
L
Light:
16
h
light:
8
h
dark
with
400
 
500
lux
Fed:
live
brine
shrimp
and
ground
salmon
starter
(
automatic
feeder)
until
able
to
eat
frozen
brine
shrimp.
Brine
shrimp
was
given
up
to
three
times
daily
Assay
Conditions
Flow­
through
continuous
dispersal
of
chemical
concentrations
using
a
proportional
diluter
Flow­
through
continuous
dispersal
of
chemical
concentrations
using
a
proportional
diluter
Flow­
through
continuous
dispersal
of
chemical
concentrations
using
a
proportional
diluter
Duration:
14­
day
14­
day
21­
day
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
16
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Parameters
Non­
spawning
14­
day
EPA
14­
day
EPA
21­
day
Dilution
water
Clean
artesian
well
water
monitored
yearly
for
drinking
water
standards
Clean
artesian
well
water
monitored
yearly
for
drinking
water
standards
Clean
artesian
well
water
monitored
yearly
for
drinking
water
standards
material
EDC­
chemical
EDC­
chemical
EDC­
chemical
chamber
size
18
L
18
L
(
40
x
20
x
20
cm)
18
L
(
40
x
20
x
20
cm)
Volume:
10
L
10
L
10
L
#
Exchanges/
day
6
volumes
6
volumes
6
volumes
#
Of
conc./
chemical
3
2
2
#
Replicates:
2
4
4
Weight
of
each
fish
Adult
female:
1.5
±
10%
Adult
male:
2.5
±
10%
Not
specified
Not
specified
#
Fish/
replicate
10
(
must
be
all
one
sex)
4
females
and
2
males
per
test
aquarium
with
nesting
tiles
4
females
and
2
males
per
test
aquarium
with
nesting
tiles
Feeding
regime
Frozen
brine
shrimp,
two
to
three
times
per
day.
Typically
up
to
1
mL
per
tank.
Frozen
brine
shrimp,
two
to
three
times
per
day.
Typically
up
to
1
mL
per
tank.
Frozen
brine
shrimp,
two
to
three
times
per
day.
Typically
up
to
1
mL
per
tank.
#
Controls
Dilution
water
Dilution
water
Dilution
water
#
Replicates/
control
2
4
4
#
Fish/
control
10
adult
females
10
adult
males
(
in
separate
vessels)
=
20
4
adult
females
and
2
adult
males
per
replicate
=
24
4
adult
females
and
2
adult
males
per
replicate
=
24
Conditions:
Photoperiod:
16
h
light:
8
h
dark
16
h
light:
8
h
dark
16
h
light:
8
h
dark
Temperature:
25
°
C
±
1
°
C­
monitored
continuously
in
one
chamber
and
daily
in
one
test
replicate
per
concentration.
Monitoring
conducted
using
a
min­
max
thermometer
with
readings
recorded
every
24
hours
25
°
C
±
1
°
C­
monitored
continuously
in
one
chamber
and
daily
in
one
test
replicate
per
concentration.
Monitoring
conducted
using
a
min­
max
thermometer
with
readings
recorded
every
24
hours
25
°
C
±
1
°
C­
monitored
continuously
in
one
chamber
and
daily
in
one
test
replicate
per
concentration.
Monitoring
conducted
using
a
min­
max
thermometer
with
readings
recorded
every
24
hours
Light
intensity
540­
1080
lux,
monitored
at
the
start
and
end
of
assay
540­
1080
lux,
monitored
at
the
start
and
end
of
assay
540­
1080
lux,
monitored
at
the
start
and
end
of
assay
Aeration:
D.
O.
>
4.9
mg/
L
D.
O.
>
4.9
mg/
L
D.
O.
>
4.9
mg/
L
pH
±
0.5
pH
units
Not
Specified
Not
Specified
Alkalinity:
>
20
mg/
L
CaCO3
>
20
mg/
L
CaCO3
>
20
mg/
L
CaCO3
Hardness:
>
140
mg/
L
CaCO3
>
140
mg/
L
CaCO3
>
140
mg/
L
CaCO3
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
17
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Parameters
Non­
spawning
14­
day
EPA
14­
day
EPA
21­
day
Total
ammonia
<
0.5
mg/
L
<
0.5
mg/
L
<
0.5
mg/
L
Monitoring:
WQ
Frequency:
Start
and
end
of
assay:
Temperature,
D.
O,
pH,
and
total
ammonia
in
all
replicates
Daily:
Temperature,
pH
and
D.
O.
one
replicate
of
each
treatment
Weekly
total
ammonia:
20%
of
samples
Once
during
assay
out
of
the
water
head
tank
 
hardness
and
alkalinity
Start
and
end
of
assay:
Temperature,
D.
O,
pH,
and
total
ammonia
in
all
replicates
Daily:
Temperature,
pH
and
D.
O.
one
replicate
of
each
treatment
Weekly
total
ammonia:
20%
of
samples
Once
during
assay
out
of
the
water
head
tank
 
hardness
and
alkalinity
Start
and
end
of
assay:
Temperature,
D.
O,
pH,
and
total
ammonia
in
all
replicates
Daily:
Temperature,
pH
and
D.
O.
one
replicate
of
each
treatment
Weekly
total
ammonia:
20%
of
samples
Once
during
assay
out
of
the
water
head
tank
 
hardness
and
alkalinity
Corrective
Actions:
Temperature:
adjust
controller
Total
ammonia:
increase
water
flow
and
clean
tanks
Photoperiod:
adjust
controller
Light
intensity:
adjust
bulbs
over
tables
pH:
no
action
Alkalinity:
no
action
Hardness:
no
action
Temperature:
adjust
controller
Total
ammonia:
increase
water
flow
and
clean
tanks
Photoperiod:
adjust
controller
Light
intensity:
adjust
bulbs
over
tables
pH:
no
action
Alkalinity:
no
action
Hardness:
no
action
Temperature:
adjust
controller
Total
ammonia:
increase
water
flow
and
clean
tanks
Photoperiod:
adjust
controller
Light
intensity:
adjust
bulbs
over
tables
pH:
no
action
Alkalinity:
no
action
Hardness:
no
action
Biological
endpoints:
Survival
Behavior
Secondary
sexual
characteristics
Gross
morphology
(
GSI)
and
Gonadal
history
plasma
VTG
and
sex
steroids
(
 ­
estradiol,
testosterone,
11­
KT(
b))
concentrations
Adult
survival,
reproductive
behavior,
fecundity,
fertility,
embryo
hatch,
secondary
sexual
characteristics,
Gross
morphology
(
GSI)
and
gonadal
history,

plasma
VTG
and
sex
steroids
(
 
­
estradiol,
testosterone,
11­
KT)
concentrations
Adult
survival,
reproductive
behavior,
fecundity,
fertility,
embryo
hatch,
secondary
sexual
characteristics,
Gross
morphology
(
GSI)
and
gonadal
history,

plasma
VTG
and
sex
steroids
(
 
­
estradiol,
testosterone,
11­
KT)
concentrations
Validity
Criteria:
D.
O.
 
60%
saturation;
Mean
temp.
25
°
C
±
2
°
C
should
be
maintained
and
corrective
action
taken
if
water
quality
is
outside
of
these
limits
Also,
90%
survival
in
the
controls
D.
O.
 
60%
saturation;
Mean
temp.
25
°
C
±
2
°
C
should
be
maintained
and
corrective
action
taken
if
water
quality
is
outside
of
these
limits
Also,
90%
survival
in
the
controls
D.
O.
 
60%
saturation;
Mean
temp.
25
°
C
±
2
°
C
should
be
maintained
and
corrective
action
taken
if
water
quality
is
outside
of
these
limits
Also,
90%
survival
in
the
controls
a)
D.
O.
dissolved
oxygen.
b)
KT
ketotestosterone.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
18
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
2.8
Statistical
Analyses
The
screening
assays
as
described
were
designed
to
detect
potential
EDCs
with
high
power
(
minimum
of
80%)
and
not
to
produce
a
precise
estimate
of
toxicity.
The
statistical
considerations
were
restricted
to
the
demands
of
the
screening
test.
The
amount
of
information
obtained
from
the
screening
test
was
limited
to
detecting
effects
on
reproductive
traits
when
both
genders
are
exposed
or
in
determining
whether
or
not
gender­
specific
differences
are
detected
when
gender­
selective
exposure
was
used.

Descriptive
statistics,
including
the
mean,
standard
deviation,
minimum,
maximum,
and
quartiles,
were
used
to
characterize
each
endpoint
measured
in
the
three
tests.
Statistical
significance
for
each
endpoint
and
chemical
was
evaluated
based
on
the
difference
in
the
mean
characteristics
between
the
treated
and
control
groups
using
analysis
of
variance,
Tukey's
multiple
comparisons
test,
and
the
nonparametric
Kruskal­
Wallis
test.
Chemical­
dosing
regimes
were
considered
classifications
of
fixed
effects
(
i.
e.,
control,
low
dose,
mid
dose,
and
high
dose).
Box
plots
were
used
to
visually
characterize
the
effect
of
each
treatment.

Power
analysis
assuming
a
Type
I
error
rate
of
 
=
0.05
was
used
to
compare
the
sensitivity
of
selected
endpoints.
The
minimum
detectable
difference
assuming
80%
power
for
the
given
sample
size
and
standard
deviation
achieved
during
testing
was
calculated.
The
achieved
power,
given
the
observed
maximum
difference
between
means,
was
also
calculated.
Finally,
the
required
sample
size
needed
to
achieve
80%
power
was
calculated,
given
the
achieved
maximum
mean
difference.

Consistency
between
the
screening
test
decisions
across
endpoints
and
chemicals
were
evaluated
using
Kendall's
coefficient
of
concordance
(
Daniel
1978).
The
smallest
difference
between
means
detected
at
a
power
of
80%
and
 
=
0.05
for
a
given
endpoint
and
protocol
provided
a
quantitative
measure
of
sensitivity.
Recommendations
as
to
potential
changes
to
the
number
of
replicates
to
achieve
a
given
level
of
power
are
provided.

Appropriate
data
transformations
were
applied
to
maintain
homogeneity
of
the
within­
class
variances
(
i.
e.,
data
expressed
as
a
percentage
may
be
arcsine­
square­
root
or
light
transformed,
counts
may
be
square­
root
or
log
transformed,
and
continuous
data
may
be
transformed
to
the
natural
logarithm)
(
Snedecor
and
Cochran
1980).
A
rank
transformation
or
nonparametric
statistics
were
used
when
the
common
data
transformation
was
not
successful
in
controlling
heterogeneity
(
Daniel
1978).

Analysis
may
have
been
conducted
both
with
and
without
suspected
outliers
(
Chapman
et
al.
1996).
Potential
outliers
may
have
been
identified
by
values
that
exceed
the
median
plus
three
times
the
interquartile
range
(
i.
e.,
the
difference
between
the
75th
and
25th
percentiles).
If
an
explanation
could
not
be
made
for
the
divergence
of
data,
then
both
analyses
were
presented,
assuming
that
the
results
differed.
If
there
were
no
changes
to
the
results,
then
the
analysis
including
the
outliers
was
presented.
If
differences
occurred,
then
the
implications
of
removing
the
outliers
were
carefully
documented.
If
an
explanation
could
be
made
for
the
existence
of
outliers,
the
analysis
excluding
outliers
may
have
been
sufficient.

2.9
Quality
Assurance
2.9.1
Technical
Systems
Audits
The
Battelle,
Sequim
QA
Unit
performed
assessments
on
activities
and
operations
affecting
data
quality,
the
raw
data
and
final
report.
Any
findings
were
reported
to
the
WA
Project
Manager
and
management
to
ensure
that
the
requirements
in
relevant
SOPs,
WA
protocol,
QAPP,
and
the
QMP
were
met.
The
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
19
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
assessments
for
this
study
included
technical
systems
audits
(
TSAs)
and
audits
of
data
quality
(
ADQs)
that
included
reviews
of
project
notebooks,
data
base
entry
verifications
from
raw
data
sheets,
and
reviews
of
statistical
analyses
performed.

TSAs
were
performed
at
the
start
of
the
study,
and
for
critical
elements
during
the
study
such
as:

°
personnel
training
files
for
documentation
that
EDSP
SOPs,
the
work
plan
and
the
WA
QAPP
have
been
read
and
understood
by
WA
personnel
before
any
activities
begin
°
calibration
status
of
project
instrumentation
°
dosing
and
sample
collection
of
dosing
solutions,
body
and
feed
weights,
and
clinical
observations
°
chemical
analysis
of
test
chemicals
°
and
termination
of
each
experiment
During
TSA
activities,
the
Battelle,
Sequim
QA
Unit
recorded
observations
to
be
used
later
in
preparing
the
audit
report.
The
Battelle,
Sequim
QA
Unit
observed
completion
of
permitting
requirements,
implementation
of
procedures,
data
recording
and
record
keeping,
and
equipment
maintenance
and
calibration
procedures
and/
or
documentation,
noting
whether
or
not
the
activities
adhered
to
the
work
plan,
and
the
QAPP,
applicable
SOPs,
and
the
QMP.
Any
findings
were
communicated
to
the
technical
personnel
at
the
completion
of
the
WA
activity
unless
an
error
could
compromise
the
WA
(
e.
g.,
misdosing
an
animal).
If
necessary,
the
EDSP
QA
team
members
immediately
notified
the
WA
Leader/
Study
Director
by
telephone
and/
or
e­
mail
of
any
adverse
findings
that
could
impact
the
conduct
of
the
WA.
This
direct
communication
was
also
documented
in
the
audit
report.

2.9.2
Audits
of
Data
Quality
Audits
of
data
quality
(
ADQs)
focused
on
the
accuracy
of
data
collection,
recording,
traceability,
and
calculations
to
ensure
that
the
reported
results
were
documented,
traceable,
and
of
high
quality
that
accurately
reflected
the
raw
data;
that
the
report
accurately
described
the
materials
and
methods
used
in
the
WA;
and
that
conclusions
were
supported
by
the
data.
The
assessment
criteria
for
ADQs
were
that
data
collection,
analysis,
and
reporting
met
the
requirements
of
the
applicable
facility
and
program
SOPs,
the
work
plan,
QAPP,
and
the
EDSP
QMP,
and
that
deviations
were
documented
as
per
the
requirements
of
the
procedure.
Deviation
reports
relative
to
the
work
assignment
were
submitted
to
the
WA
Leader
and
included
in
the
project
records.

Direct
and
frequent
communication
between
the
Project
Manager,
Laboratory
staff,
and
the
QA
Unit
Manager
was
designed
to
provide
for
sufficient
time
to
perform
an
ADQ
so
that
the
submission
date
of
the
audited
final
report
met
those
specified
in
the
work
plan.

All
data
and
records
for
review
were
submitted
to
the
QA
Unit
Manager
or
delegate,
who
reviewed
the
data
packages
for
completeness
and,
if
incomplete,
requested
that
the
additional
records
needed
for
review
be
submitted.
The
EDSP
QA
team
members
reviewed
a
minimum
of
10%
of
the
raw
data,
depending
on
the
level
of
prior
technical
review,
the
tabulated
data,
and
WA
records
of
performance
and
methods,
to
ensure
compliance
with
planning
documents
mentioned
previously.
All
tables
and
graphs
were
reviewed
for
completeness
and
accuracy
of
titles,
headers,
and
footnotes.
The
EDSP
QA
team
members
checked
all
tabulated
data
designated
as
statistically
significant.
Findings
were
reported
and
corrective
actions
undertaken
as
described
earlier.
The
EDSP
QA
team
members
reviewed
the
report
using
the
audited
data
and
corrected
tables
to
ensure
that
the
reported
results
were
of
high
quality
and
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
20
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
accurately
reflected
the
raw
data,
and
that
the
report
accurately
described
the
materials
and
methods
used
in
the
WA.
Findings
were
reported
and
corrective
actions
undertaken
as
described
earlier.

The
eTSA
and
ADQ
were
conducted
throughout
the
duration
of
this
WA.
Neither
of
these
activities
resulted
in
any
major
findings
nor
any
stop
work
associated
with
the
conduct
of
the
experiments.

2.10
Storage
of
Records
and
Data
Management
The
data
for
this
study
were
collected
on
preprinted
data
collection
forms.
The
data
forms
included,
as
appropriate,
the
following
items:
study
code,
protocol
number,
tank
number,
treatment
(
Rx)
code,
and
others.
The
forms
had
preprinted
dates
for
collection
of
data
when
possible.
Otherwise,
the
dates
for
data
collection
were
hand
printed
on
the
forms
as
needed
prior
to
or
on
the
day
of
collection
of
the
data.
All
data
forms
were
initialed
and
dated
by
the
person
collecting
the
data,
and
all
forms
received
documented
technical
review
and
signature
approval.
Corrections
to
data
entries
were
made
by
drawing
a
single
line
through
the
error
and
recording
the
correct
entry,
initials,
date,
and
error
code
that
explained
the
reason
for
the
correction.

The
datasheets
were
clearly
divided
by
chemical
and
protocol
and
placed
in
a
workbook.
These
workbooks
were
kept
next
to
the
tanks
until
data
entry
into
the
database.
The
data
were
entered
into
a
Microsoft
Access
database.
The
database
forms
corresponded
to
the
datasheets
in
the
workbooks.
Data
entry
included
transferring
information
on
the
written
form
to
the
database
form.
These
database
forms
and
tables
associated
with
the
forms
had
data
integrity
such
that
deletions
were
not
allowed
by
the
data
entry
personnel.
Also,
there
was
a
quality
control
(
QC)
process
during
the
data
entry
to
identify
and
correct
any
obvious
discrepancies
in
the
data.

The
original
raw
data
collected
on
the
data
forms
remain
in
the
wet
lab
project
file
until
there
is
a
signed
final
report,
at
which
time
they
are
inventoried
and
archived
on
CD­
ROMs
for
at
least
2
years
(
longer
if
required
by
study
protocol
or
government
regulations),
unless
the
sponsor
requests
that
they
be
transferred
to
an
archive
location
other
than
at
Battelle.

All
specimens
and
records
remain
the
responsibility
of
Battelle
PNWD
and
are
retained
in
the
Battelle
archive
for
the
length
of
time
stipulated
in
the
contract,
which
is
typically
5
years.
The
archive
is
located
at
Battelle's
facility
in
Sequim,
Washington,
and
is
maintained
according
to
a
policy
of
limited
access.
The
Battelle
sample
custodian
is
responsible
for
archiving
and
retrieving
work
assignment
materials.
An
archive
inventory
is
maintained
and
storage
capability
is
provided
for
the
expedient
retrieval
of
materials.
Specimens
and
samples
are
disposed
of
only
after
assessing
that
they
no
longer
afford
evaluation.
