DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
216
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
6.0
RESULTS:
FADROZOLE
6.1
EPA
14­
Day
Assay
for
Fadrozole
The
EPA
14­
Day
Fadrozole
assay
was
conducted
from
March
17,
2003,
to
March
27,
2003
(
pre­
exposure
assay),
and
from
March
27,
2003,
to
April
10,
2003
(
exposure
assay).

6.1.1
Survival
All
males
and
females
in
all
treatments
survived
the
EPA
14­
Day
Fadrozole
assay.

6.1.2
Vitellogenin
Vitellogenin
concentrations
in
Control
treatment
females
used
during
the
EPA
14­
Day
fadrozole
assay
ranged
from
3,426,500
ng/
mL
to
6,920,500
ng/
mL
(
Figure
6.1).
Among
females
exposed
to
the
two
fadrozole
concentrations,
vitellogenin
concentrations
ranged
from
0
ng/
mL
(
not
detected)
to
2,798,000
ng/
mL.
Significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
6.1)
were
detected
(
Kruskal­
Wallis,
H
=
41.80,
p
=
<
0.001,
df
=
2).
Vitellogenin
concentrations
in
Controltreatment
females
were
significantly
greater
than
those
in
females
exposed
to
the
Low
and
High
fadrozole
concentrations.
Additionally,
vitellogenin
concentrations
in
females
from
the
Low
concentration
were
greater
than
those
in
females
from
the
High
concentration.
The
achieved
power
for
this
endpoint
was
100%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
2
(
Table
6.1).

Table
6.1.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
16
5,364,000
1,059,237
20%
100%
2
low
16
1,148,553
703,249
61%
high
16
1,693
1,986
117%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
16.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
217
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
6000000
4000000
2000000
0
treatment
Fad­
VTG
Figure
6.1.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Vitellogenin
concentrations
in
Control
treatment
males
used
during
the
EPA
14­
Day
fadrozole
assay
ranged
from
0
ng/
mL
(
not
detected)
to
7,123
ng/
mL
(
Figure
6.2).
Among
most
males
exposed
to
the
two
fadrozole
concentrations,
vitellogenin
concentrations
ranged
from
0
ng/
mL
(
not
detected)
to
5,604
ng/
mL.
One
male
exposed
to
the
Low
fadrozole
concentration
treatment
had
a
vitellogenin
concentration
of
15,191
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
6.2)
were
detected
(
Kruskal­
Wallis,
H
=
0.67,
p
=
0.716,
df
=
2).
The
achieved
power
for
this
endpoint
was
6%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
528
(
Table
6.2).

Table
6.2.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
2,150
3,088
144%
6%
528
low
8
2,856
5,111
179%
high
8
1,056
1,914
181%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
218
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
15000
10000
5000
0
treatment
Fad­
VTG
Figure
6.2.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

6.1.3
Appearance
/
Secondary
Sex
Characteristics
All
of
the
females
used
during
the
EPA
14­
Day
Fadrozole
assay
exhibited
typical
female
morphology
(
no
fat
pad,
no
tubercles,
no
vertical
banding,
ovipositor
present).

Most
of
the
males
used
during
the
EPA
14­
Day
Fadrozole
assay
exhibited
typical
male
morphology
(
fat
pads,
tubercles,
vertical
banding,
no
ovipositor
present).
One
male
from
the
Control
treatment
lacked
a
fat
pad
and
vertical
banding.

6.1.4
Gonadosomatic
Index
The
range
of
GSI
values
calculated
for
females
in
the
all
treatments
varied
about
two­
fold
(
Figure
6.3),
and
the
overall
variability
within
the
treatment
was
moderate
(
CVs
=
22%
 
24%;
Table
E14FAD
GSI­
1).
The
highest
female
GSI
values
were
about
21
 
23
(
two
fish
each
in
the
Low
and
High
concentrations).
Significant
differences
in
mean
GSI
values
(
Table
6.3)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
13.54,
p
=
0.001,
df
=
2).
The
mean
GSI
values
for
females
exposed
to
the
Low
and
High
concentrations
were
significantly
greater
than
GSI
values
for
females
from
the
Control
treatment.
The
achieved
power
for
this
endpoint
was
92%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
12
(
Table
6.3).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
219
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.3.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
16
11.0
2.4
22%
92%
12
low
16
15.1
3.5
23%
high
16
14.8
3.5
24%
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
16.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

Low
High
Control
23
18
13
8
treatment
GSI
Figure
6.3.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

The
range
of
most
GSI
values
calculated
for
males
during
the
EPA
14­
Day
Fadrozole
assay,
was
small,
ranging
from
0.7
to
1.6
(
Figure
6.4),
which
approximates
the
typical
range
for
reproductively­
active
male
fathead
minnows.
However,
five
fish
had
GSI
values
 
2.0
(
at
least
one
fish
from
each
treatment).
The
highest
male
GSI
value
was
3.6
for
a
small
fish
(
2.5
g
body
weight)
exposed
to
the
Low
fadrozole
concentration
that
had
a
relatively
large
gonad
(
0.09
g).
There
were
no
significant
differences
in
mean
GSI
values
(
Table
6.4)
among
treatments
(
Kruskal­
Wallis,
H
=
4.12,
p
=
0.128,
df
=
2).
The
achieved
power
for
this
endpoint
was
24%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
32
(
Table
6.4).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
220
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.4.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
1.23
0.42
34%
24%
32
low
8
1.45
0.87
60%
high
8
1.66
0.45
27%
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

Low
High
Control
3.5
2.5
1.5
0.5
treatment
GSI
Figure
6.4.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

6.1.5
Female
Gonad
Histology
Histological
analyses
were
conducted
on
the
ovaries
of
48
females
exposed
to
fadrozole
during
the
EPA
14­
Day
Assay.

General
Ovary
Staging
 
Statistical
analysis
of
the
mean
ovarian
staging
from
12
microscope
fields
per
female
from
the
EPA
14­
Day
fadrozole
assay
revealed
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
8.78,
p
=
0.012,
df
=
2).
The
mean
ovarian
stage
of
females
from
the
High
concentration
was
significantly
less
than
those
of
females
from
the
Control
treatment
and
the
Low
concentration.

Quantitative
Ovarian
Staging
 
One
hundred
cells
in
each
of
three
sections
per
female
were
examined
to
quantitatively
determine
the
developmental
stage
of
the
ovaries.
Ova
from
females
from
the
Control
treatment
and
Low
concentration
ranged
from
Stage
1a
to
Stage
5
(
see
Methods
for
a
description
of
the
stages),
whereas
ovaries
in
females
from
the
High­
concentration
treatment
showed
Stage
1a
to
Stage
4
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
221
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
development
(
Figure
6.5).
Variability
within
treatments
for
each
stage
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
400%
(
Table
6.5).
Statistical
analyses
revealed
significant
difference
among
treatments
in
the
proportion
of
cells
in
three
of
the
five
developmental
stages
(
Table
6.5).
The
proportion
of
cells
in
developmental
Stage
1a
was
greater
in
females
from
the
Control
treatment
than
in
females
from
the
High
and
Low
concentrations.
Also,
the
proportion
of
cells
in
developmental
Stage
1b
was
greater
in
females
from
the
Control
treatment
than
in
females
from
the
High
concentration.
The
proportion
of
cells
in
developmental
Stage
3
was
greater
in
females
from
the
High
concentration
than
in
females
from
the
Control
treatment
and
the
Low
concentration.
The
proportion
of
cells
in
developmental
Stage
4
was
less
in
females
from
the
High
concentration
than
in
females
from
the
Control
treatment
and
the
Low
concentration.

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.

Control
(
n
=
16)
Low
(
n
=
16)
High
(
n
=
16)
Kruskal­
Wallis
Stage
Mean
Stdev
CV
Mean
Stdev
CV
Value
Stdev
CV
H
p
1a
0.085
0.028
33%
0.058
0.019
32%
0.050
0.025
50%
14.19
0.001*
1b
0.277
0.067
24%
0.220
0.057
26%
0.205
0.067
33%
8.83
0.012**
2
0.200
0.048
24%
0.198
0.032
16%
0.189
0.073
39%
1.21
0.545
3
0.192
0.046
24%
0.228
0.060
26%
0.351
0.100
28%
20.70
<
0.001***
4
0.206
0.107
52%
0.272
0.080
29%
0.103
0.065
63%
20.79
<
0.001***
5
0.015
0.045
308%
0.000
0.002
400%
0
0
 
2.18
0.337
*
p
<
0.01
**
p
<
0.05
***
p
<
0.001
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
222
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
0.00
0.10
0.20
0.30
0.40
0.50
0.60
1a
1b
2
3
4
5
Ovarian
Stage
Proportion
Control
Low
High
Figure
6.5.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
14­
Day
Fadrozole
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.

Atretic
Follicles
 
The
mean
proportion
of
atretic
follicles
per
300
follicles
(
counted
per
fish)
ranged
from
0.009
follicles
for
females
in
the
Control
treatment
to
0.102
follicles
for
females
in
the
High
concentration
(
Figure
6.6).
There
was
a
significant
difference
in
the
mean
proportions
of
atretic
follicles
among
treatments
(
Kruskal­
Wallis,
H
=
14.26,
p
=
0.001,
df
=
2).
The
proportion
of
atretic
follicles
was
greater
for
females
from
the
High
concentration
than
for
those
from
the
Control
treatment
or
the
Low
concentration.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
223
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Treatment
atretic_
follicles
Figure
6.6.
Boxplot
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Corpora
Lutea
 
The
mean
proportion
of
corpora
lutea
per
300
follicles
(
counted
per
fish)
ranged
from
0.001
for
females
in
the
High
concentration
to
0.012
for
females
in
the
Control
treatment
(
Figure
6.7).
There
were
significant
differences
in
the
mean
proportion
of
corpora
lutea
among
treatments
(
Kruskal­
Wallis,
H
=
11.24,
p
=
0.004,
df
=
2).
The
value
for
the
Control
treatment
was
significantly
greater
than
those
of
the
other
two
treatments.

Low
High
Control
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
Treatment
corpora_
lutea
Figure
6.7.
Boxplot
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
224
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Observations
 
One
female
from
the
Control
treatment
was
observed
to
have
abnormal­
appearing
Stage
2
ova
that
had
crenulated
surfaces.
Six
females
from
the
High
concentration
had
vacuoles
in
the
nuclei
of
some
Stage
3
and/
or
Stage
4
cells.
One
additional
female
from
the
High
concentration
had
inflammatory
cells
in
the
ovary.

6.1.6
Male
Gonad
Histology
General
Testes
Staging
 
Testes
from
24
males
exposed
to
fadrozole
during
the
EPA
14­
Day
Fadrozole
assay
were
examined
to
determine
the
general
developmental
condition.
Males
in
all
treatments
had
welldeveloped
testes,
showing
Stage
4
and
Stage
5
development
(
see
Methods
for
description
of
developmental
stages).
All
of
the
96
microscopic
fields
examined
in
the
8
Control
treatment
males
showed
Stage
4
(
49
fields)
or
Stage
5
(
47
fields)
development.
All
of
the
96
microscopic
fields
examined
in
the
8
Low­
concentration
treatment
males
showed
Stage
4
(
67
fields)
or
Stage
5
(
29
fields)
development.
All
of
the
96
microscopic
fields
examined
in
the
8
High­
concentration
treatment
males
showed
Stage
4
(
40
fields)
or
Stage
5
(
56
fields)
development.
Statistical
analysis
of
the
mean
staging
from
12
microscopic
fields
per
fish
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
3.15,
p
=
0.207,
df
=
2).

Quantitative
Testicular
Staging
 
One
hundred
cells
in
each
of
three
section
per
male
were
examined
to
quantitatively
determine
the
developmental
condition
of
the
testes.
The
developmental
stage
all
treatment
testes
ranged
from
Stage
2a
to
Stage
5
(
Figure
6.8).
Variability
within
treatments
for
each
stage
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
197%
(
Table
6.6).
Statistical
analyses
showed
that
there
were
significant
differences
among
treatments
in
the
proportion
of
cells
in
developmental
Stage
4.
The
proportion
of
cells
showing
Stage
4
development
was
greater
in
males
from
the
Low
concentration
than
in
males
from
the
Control
treatment
or
the
High
concentration.
The
probability
value
calculated
for
Stage
5
was
slightly
above
the
critical
limit
of
0.050
(
Table
E14FAD
MHist­
1).
The
greatest
difference
was
between
the
mean
proportion
of
cells
showing
Stage
5
development
in
males
from
the
High
concentration
(
0.68)
and
the
Low
concentration
(
0.40).
Therefore,
there
was
no
effect
on
testicular
developmental
stage
associated
with
fadrozole
dose.

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.

Control
(
n
=
8)
Low
(
n
=
8)
High
(
n
=
8)
Kruskal­
Wallis
Stage
Mean
Stdev
CV
Mean
Stdev
CV
Value
Stdev
CV
H
p
1
0
0
 
0
0
 
0
0
 
 
 
2a
0.007
0.010
148%
0.003
0.006
197%
0.003
0.003
95%
0.93
0.629
2b
0.022
0.020
91%
0.026
0.013
48%
0.014
0.012
88%
4.27
0.118
3a
0.120
0.114
95%
0.208
0.131
63%
0.115
0.108
94%
3.40
0.183
3b
0.160
0.108
68%
0.154
0.092
60%
0.085
0.067
78%
2.89
0.235
4
0.098
0.058
59%
0.207
0.093
45%
0.102
0.011
11%
8.22
0.016*
5
0.592
0.252
43%
0.403
0.198
49%
0.681
0.158
23%
5.81
0.055
*
p
<
0.05
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
225
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1
2a
2b
3a
3b
4
5
Testicular
Stage
Proportion
Control
Low
High
Figure
6.8.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
14­
Day
Fadrozole
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.

Tubule
Diameter
 
The
diameter
of
the
seminiferous
tubules
of
males
from
the
Control
treatment
ranged
from
145.0
µ
m
to
248.3
µ
m
(
Figure
6.9).
Tubule
diameters
of
males
from
the
two
test
concentrations
ranged
from
114.7
µ
m
to
240.3
µ
m.
No
significant
differences
in
the
mean
tubule
diameter
per
treatment
(
Table
6.7)
were
detected
(
Kruskal­
Wallis,
H
=
4.07,
p
=
0.131,
df
=
2).
The
achieved
power
for
this
endpoint
was
13%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
67
(
Table
6.7).

Table
6.7.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
167.9
35.8
21%
13%
67
low
8
151.2
23.0
15%
high
8
177.2
34.5
19%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
226
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
250
200
150
100
Treatment
diameter
Figure
6.9.
Boxplot
of
seminiferous
tubule
diameter
(
µ
m)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

Observations
 
One
male
from
the
Control
treatment
showed
Sertoli
cell
proliferation
with
sperm
phagocytosis.
The
same
male
also
showed
focal
tubule
disorganization,
multifocal
proliferation
of
secondary
spermatogonia,
and
sperm
clumping
in
the
tubule
lamina.
One
male
from
the
Low
concentration
and
three
males
from
the
High
concentration
were
observed
to
have
multifocal
Leydig
cell
proliferation.
One
male
from
the
Low
concentration
had
an
abnormal­
appearing
arrangement
of
components
of
spermatogenesis.
Another
male
from
the
Low
concentration
showed
tubules
in
developmental
Stage
5
that
appeared
to
have
a
smaller
diameter
than
normal
and
had
a
lower
than
normal
density
of
sperm
in
tubule
lumina.
Ovatestes,
containing
three
Stage
3
ova,
were
detected
in
one
male
from
the
High
concentration.
One
male
of
the
males
from
the
High
concentration
that
had
multifocal
Leydig
cell
proliferation
also
showed
focal
disruption
of
tubule
developmental
sequence,
with
the
proliferation
of
developmental
Stage
2b
cells
in
mature
tubules
containing
developmental
Stage
5
cells
and
had
occasional
foci
of
two
or
three
necrotic
Stage
3a
or
Stage
3b
cells.
Another
male
from
the
High
concentration
had
multifocal
areas
of
three
or
four
abnormal
developmental
Stage
3b
and
Stage
4
cells
with
the
Stage
4
cell
clumps
appearing
to
be
inside
cell
wall
of
the
stage
3b
cells.
No
testicular
atrophy
was
recorded
and
no
ovatestes
were
observed
for
any
treatment.

6.1.7
Plasma
Steroid
Concentrations
Estradiol
 
Estradiol
concentrations
in
Control­
treatment
females
used
during
the
EPA
14­
Day
fadrozole
assay
ranged
from
1,164
pg/
mL
to
5,523
pg/
mL
(
Figure
6.10).
Among
females
exposed
to
the
two
fadrozole
concentrations,
estradiol
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
2,158
pg/
mL.
A
significant
difference
in
the
mean
estradiol
concentration
per
treatment
(
Table
6.8)
was
detected
(
Kruskal­
Wallis,
H
=
36.86,
p
<
0.001,
df
=
2).
The
mean
estradiol
concentration
in
females
from
the
High
concentration
was
less
than
that
in
females
from
the
Control
treatment
and
the
Low
concentration.
The
achieved
power
for
this
endpoint
was
100%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
3
(
Table
6.8).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
227
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.8.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
15
2,064
1,176
57%
100%
3
low
16
1,088
468
43%
high
16
39
86
218%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
15.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
6000
5000
4000
3000
2000
1000
0
treatment
Estradiol
Figure
6.10.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisks
represent
probable
outliers.

Estradiol
was
not
detected
in
males
from
the
Control
treatment
(
8
individuals)
or
in
those
exposed
to
the
Low­
and
High­
fadrozole
concentrations
(
8
individuals
per
treatment).

Testosterone
 
Testosterone
concentrations
in
Control­
treatment
females
used
during
the
EPA
14­
Day
fadrozole
assay
ranged
from
221
pg/
mL
to
2,644
pg/
mL
(
Figure
6.11).
Among
females
exposed
to
the
two
fadrozole
concentrations,
testosterone
concentrations
ranged
from
732
pg/
mL
to
4,992
pg/
mL.
Significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
6.9)
were
detected
(
Kruskal­
Wallis,
H
=
14.91,
p
=
0.001,
df
=
2).
The
mean
testosterone
concentration
in
females
from
the
Control
treatment
was
less
than
those
in
females
from
the
Low
and
High
concentrations.
The
achieved
power
for
this
endpoint
was
96%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
9
(
Table
6.9).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
228
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.9.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
13
934
629
67%
96%
9
low
14
2,356
1,268
54%
high
16
2,355
1,175
50%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
13.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
5000
4000
3000
2000
1000
0
treatment
Testosterone
Figure
6.11.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

Testosterone
concentrations
in
Control
treatment
males
used
during
the
EPA
14­
Day
fadrozole
assay
ranged
from
198
pg/
mL
to
3,569
pg/
mL
(
Figure
6.12).
Among
males
exposed
to
the
two
fadrozole
concentrations,
testosterone
concentrations
ranged
from
1,182
pg/
mL
to
7,124
pg/
mL.
A
significant
difference
in
the
mean
testosterone
concentration
per
treatment
(
Table
6.10)
was
detected
(
Kruskal­
Wallis,
H
=
7.12,
p
=
0.029,
df
=
2).
The
mean
testosterone
concentration
in
males
from
the
Control
treatment
was
less
than
those
in
males
from
the
High
concentration.
The
achieved
power
for
this
endpoint
was
68%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
11
(
Table
6.10).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
229
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.10.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
1,790
997
56%
68%
11
low
8
3,169
833
26%
high
8
4,431
2,294
52%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
7000
6000
5000
4000
3000
2000
1000
0
treatment
Testosterone
Figure
6.12.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

11­
ketotestosterone
 
11­
ketotesosterone
was
detected
in
only
1
of
11
Control­
treatment
females
used
during
the
EPA
14­
Day
fadrozole
assay
(
Figure
6.13).
Among
females
exposed
to
the
two
fadrozole
concentrations,
testosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
1,528
pg/
mL.
No
significant
differences
in
the
mean
11­
ketotestosterone
concentration
per
treatment
(
Table
6.11)
were
detected
(
Kruskal­
Wallis,
H
=
5.69,
p
=
0.058,
df
=
2).
However,
the
calculated
probability
was
slightly
greater
than
the
selected
critical
limit
(
0.050)
for
the
test.
The
greatest
difference
in
mean
11­
ketotestosterone
concentration
was
between
females
from
the
Control
treatment
(
60
pg/
mL)
and
those
from
the
High
concentration
(
426
pg/
mL).
The
mean
value
for
Control
females
was
also
much
less
than
that
for
females
from
the
Low
concentration
(
400
pg/
mL).
The
achieved
power
for
this
endpoint
was
54%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
19
(
Table
6.11).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
230
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.11.
Summary
statistics
and
power
estimates
for
female
11­
ketotestosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
11
60
197
332%
54%
19
low
11
400
516
129%
high
14
426
430
101%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
11.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
1500
1000
500
0
treatment
11­
keto
Figure
6.13.
Boxplot
of
female
11­
ketotestosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

11­
ketotesosterone
concentrations
in
Control
treatment
males
used
during
the
EPA
14­
Day
fadrozole
assay
ranged
from
959pg/
mL
to
41,357
pg/
mL
(
Figure
6.14g).
Among
males
exposed
to
the
two
fadrozole
concentrations,
11­
ketotesosterone
concentrations
ranged
from
1,665
pg/
mL
to
103,500
pg/
mL.
A
significant
difference
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
6.12)
was
detected
(
Kruskal­
Wallis,
H
=
6.54,
p
=
0.038,
df
=
2).
The
mean
11­
ketotestosterone
concentration
in
males
from
the
Control
treatment
was
less
than
that
in
males
from
the
Low
concentration.
The
achieved
power
for
this
endpoint
was
64%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
11
(
Table
6.12).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
231
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.12.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
14,376
13,061
91%
64%
11
low
8
56,077
31,055
55%
high
8
41,690
36,973
89%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
100000
50000
0
treatment
11­
keto
Figure
6.14.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

6.1.8
Fecundity
Total
Fecundity
 
Variability
among
treatments
in
the
total
number
of
eggs
produced
during
the
EPA
14­
Day
Fadrozole
assay
was
moderate
to
high
(
Figure
6.15).
Total
counts
in
the
Control
treatment
ranged
from
3,063
eggs
to
5,252
eggs
and
from
2,164
eggs
to
3,408
eggs
in
the
Low
concentration.
Total
counts
for
three
replicates
of
the
High
fadrozole
concentration
treatment
were
similar,
ranging
from
644
eggs
to
1,041
eggs,
whereas
the
total
number
of
eggs
produced
in
the
fourth
replicate
was
32.
Egg
production
by
females
in
the
High
concentration
stopped
after
Day
4
of
the
assay.
Significant
differences
in
the
mean
numbers
of
eggs
(
square­
root
transformed
)
produced
per
treatment
(
Table
6.13)
were
detected
(
Kruskal­
Wallis,
H
=
9.27,
p
=
0.010,
df
=
2).
The
mean
numbers
of
eggs
produced
by
females
in
the
Control
treatment
and
the
Low
concentration
were
greater
than
the
mean
numbers
of
eggs
laid
by
females
in
the
High
concentration.
The
achieved
power
for
this
assay
was
100%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
3
(
Table
6.13).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
232
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.13.
Summary
statistics
and
power
estimates
for
fecundity
data
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
4
4462
961
22%
100%
3
low
4
2517
602
24%
high
4
609
422
69%
1
Calculated
from
square­
root
transformed
data;
with
sample
size
=
4.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
square­
root
transformed
data.

0
1,000
2,000
3,000
4,000
5,000
6,000
0
2
4
6
8
10
12
14
Day
Cumulative
Number
of
Eggs
Control
Low
High
Figure
6.15.
Total
egg
production
by
replicate
per
treatment
for
the
EPA
14­
Day
fadrozole
assay.

Fecundity
per
Female
Reproductive
Day
 
During
the
EPA
14­
Day
Fadrozole
assay,
the
maximum
number
of
female
reproductive
days
was
achieved
for
all
treatments
(
Table
6.14).
The
number
of
eggs
produced
per
female
reproductive
day
in
the
Control
treatment
varied
from
54.7
eggs
to
93.8
eggs
and
from
38.3
eggs
to
60.9
eggs
in
the
Low
concentration
(
Figure6.16).
For
the
High
concentration,
the
number
of
eggs
produced
per
female
reproductive
day
ranged
from
0.6
eggs
to
18.6
eggs.
Because
no
fish
died
during
the
assay,
the
statistical
results
reported
here
are
the
same
as
those
reported
for
total
fecundity.
Females
in
the
High
concentration
produced
significantly
fewer
eggs
per
reproductive
day
than
those
in
the
Control
treatment
and
the
Low
concentration
(
Kruskal­
Wallis,
H
=
9.27,
p
=
0.010,
df
=
2).
The
achieved
power
for
this
assay
was
82%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
4
(
Table
6.14).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
233
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.14.
Summary
statistics
and
power
estimates
for
fecundity
per
female
reproductive
day
for
the
EPA
14­
Day
Fadrozole
assay.

Level
Mean
Number
of
Reproductive
Days
1
N
Mean
Stdev
CV
Achieved
Power
2
Sample
Size
Required
3
control
56
4
79.7
17.2
22%
82%
4
low
56
4
45.0
10.8
24%
high
56
4
10.9
7.5
69%
1
Maximum
number
=
56.
2
Calculated
from
natural
log
transformed
data;
with
sample
size
=
4.
3
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
100
90
80
70
60
50
40
30
20
10
0
treatment
eggs/
ReproDay
Figure
6.16.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Eggs
on
Tiles/
Dishes
 
The
mean
number
of
eggs
laid
on
the
tiles
among
the
treatments
during
the
EPA
14­
Day
Fadrozole
assay
varied
from
551
eggs
for
the
High
concentration
to
4,015
eggs
for
the
Control
treatment
(
Appendix
E,
Table
4.3).
The
number
of
eggs
on
dishes
ranged
from
59
eggs
for
the
High
concentration
to
448
eggs
for
the
Control
treatment.
Because
of
the
variability
in
the
total
number
of
eggs
laid
per
treatment,
the
proportional
difference
in
the
number
of
eggs
on
dishes
versus
those
on
tiles
[
1 (#
eggs
on
dishes
÷
#
eggs
on
tiles)]
was
calculated
(
Appendix
E,
Figure
4.2).
There
were
no
significant
differences
in
this
mean
proportional
difference
among
treatments
(
Kruskal­
Wallis,
H
=
0.81,
p
=
0.668,
df
=
2).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
234
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
6.1.9
Fertilization
Success
Total
Fertilization
 
The
total
(
tiles
+
dishes)
fertilization
success
rates
for
most
treatment
replicates
during
the
EPA
14­
Day
Fadrozole
assay
were
high,
ranging
from
0.997
(
Control­
treatment
and
Lowconcentration
replicates)
to
1.00
(
replicates
from
all
treatments)
(
Figure
6.17).
The
proportion
of
eggs
fertilized
for
the
High
concentration
was
1.00
for
all
replicates.
No
significant
differences
in
mean
fertilization
success
rates
(
Table
6.15)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
5.93,
p
=
0.052,
df
=
2).
The
achieved
power
for
this
assay
was
48%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
7
(
Table
6.15).

Table
6.15.
Summary
statistics
and
power
estimates
for
the
proportion
of
eggs
fertilized
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
4
0.998
0.001
0.1%
48%
7
low
4
0.999
0.002
0.2%
high
4
1.000
0
0.0%
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
4.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

Low
High
Control
1.000
0.999
0.998
0.997
treatment
Total
Prop­
fert
Figure
6.17.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Fertilization
of
Eggs
on
Tiles
and
Dishes
 
The
fertilization
success
rates
for
all
treatment
replicates
for
eggs
laid
on
tiles
during
the
EPA
14­
Day
Fadrozole
assay
were
high,
ranging
from
0.996
(
Low
concentration
replicate)
to
1.00
(
High­
and
Low­
concentration
replicates)
(
Appendix
E,
Figure
4.3).
No
significant
differences
in
mean
fertilization
success
rates
(
Appendix
E,
Table
4.4)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
5.93,
p
=
0.052,
df
=
2).
The
fertilization
success
rates
for
all
treatment
replicates
for
eggs
laid
on
dishes
during
the
assay
were
high,
ranging
from
0.995
(
Control­
treatment
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
235
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
replicate)
to
1.00
(
several
replicates,
including
all
treatments)
(
Appendix
E,
Figure
4.4).
No
significant
differences
in
mean
fertilization
success
rates
(
Appendix
E,
Table
4.4)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
2.00,
p
=
0.368,
df
=
2).

6.1.10
Hatchability
and
Larval
Development
Eggs
were
collected
during
the
14­
Day
pre­
exposure
period
for
the
evaluation
of
hatchability.
The
proportion
of
fertilized
eggs
that
hatched
ranged
from
0.96
to
1.00
for
tanks
that
would
be
used
for
the
Control
treatment,
from
0.78
to
0.96
for
tanks
that
would
constitute
the
Low
concentration,
and
from
0.98
to
1.00
for
tanks
that
would
be
assigned
to
the
High
concentration
during
the
14­
Day
exposure
assay.
Among
the
tanks
evaluated
during
the
pre­
exposure
period,
but
that
were
not
used
in
the
14­
Day
assay,
the
mean
proportion
of
fertilized
eggs
that
hatched
was
0.98.
There
was
a
significant
difference
detected
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
8.42,
p
=
0.038,
df
=
3).
The
proportion
of
eggs
that
hatched
in
tanks
destined
for
the
Low
concentration
treatment
was
significantly
less
than
that
for
eggs
laid
in
tanks
that
would
be
used
for
the
High
concentration.

Eggs
were
collected
during
the
EPA
14­
Day
Fadrozole
assay
for
the
evaluation
of
hatchability.
The
proportion
of
fertilized
eggs
that
hatched
ranged
from
0.92
to
1.00
in
the
Control
treatment
and
from
0.84
to
1.00
for
the
Low
concentration
(
Figure
6.18).
No
eggs
were
available
to
evaluate
hatchability
for
the
High
concentration
because
the
evaluation
was
started
on
Day
12
of
the
assay
and
no
eggs
were
laid
in
those
tanks
after
Day
4.
There
were
no
significant
differences
between
the
Control
treatment
and
the
Low
concentration
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
0.05,
p
=
0.821,
df
=
2).
The
achieved
power
for
this
endpoint
was
5%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
>
1,000
(
Table
6.16).

Table
6.16.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
6
0.973
0.031
3%
5%
>
1000
low
7
0.959
0.062
6%
high
0
 
 
 
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
6.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
236
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
low
control
1.00
0.95
0.90
0.85
treatment
Prop­
Hatch
Figure
6.18.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Eggs
were
collected
during
the
pre­
exposure
period
for
the
evaluation
of
larval
development.
The
mean
proportion
of
larvae
that
developed
normally
(
i.
e.,
that
showed
no
morphological
abnormalities)
was
0.95
(
sd
=
0.05)
for
all
tanks
that
would
be
used
in
the
Control
treatment.
The
mean
proportion
of
normal
larvae
in
the
remaining
treatments
was
0.97
(
sd
=
0.03)
in
the
Low
concentration
and
0.98
(
sd
=
0.02)
in
the
High
concentration.
Among
the
tanks
evaluated
during
the
pre­
exposure
period,
but
that
were
not
used
in
the
21­
Day
assay,
the
mean
proportion
of
normal
larvae
was
0.97
(
sd
=
0.01).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
1.38,
p
=
0.710,
df
=
3).

Eggs
were
collected
during
the
EPA
14­
Day
Fadrozole
assay
for
the
evaluation
of
larval
development.
The
proportion
of
larvae
that
developed
normally
(
i.
e.,
that
showed
no
morphological
abnormalities)
ranged
from
0.74
to
1.00
for
the
Control
treatment
and
from
0.92
to
1.00
for
the
Low
concentrations
(
Figure
6.19).
There
were
no
larvae
available
form
the
High
concentration
for
the
evaluation
of
development.
There
were
no
significant
differences
between
the
Control
treatment
and
the
Low
concentration
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
0.08,
p
=
0.772,
df
=
1).
The
achieved
power
for
this
endpoint
was
6%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
402
(
Table
6.17).

Table
6.17.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
6
0.916
0.123
13%
6%
402
low
7
0.970
0.026
3%
high
0
 
 
 
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
6.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
237
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
low
control
1.0
0.9
0.8
treatment
Prop­
Norm
Figure
6.19.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

6.1.11
Body
Weight
The
body
weight
of
females
used
in
the
EPA
14­
Day
Fadrozole
assay
ranged
from
1.5
g
to
3.6
g.
A
significant
difference
in
mean
body
weight
among
treatments
was
detected
(
Kruskal­
Wallis,
H
=
12.33,
p
=
0.002,
df
=
2).
The
mean
body
weight
of
females
exposed
to
the
High
concentration
was
significantly
greater
than
that
of
females
in
the
Control
treatment.
The
body
weight
of
males
used
in
the
EPA
14­
Day
Fadrozole
assay
ranged
from
2.5
g
to
7.5
g.
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
2.90,
p
=
0.235,
df
=
2).

6.2
EPA
21­
Day
Assay
for
Fadrozole
The
EPA
21­
Day
Fadrozole
assay
was
conducted
from
March
10,
2003,
to
March
25,
2003
(
pre­
exposure
assay),
and
from
March
25,
2003,
to
April
15,
2003
(
exposure
assay).

6.2.1
Survival
All
males
and
females
in
all
treatments
survived
the
EPA
21­
Day
Fadrozole
assay.

6.2.2
Vitellogenin
Vitellogenin
concentrations
in
Control
treatment
females
used
during
the
EPA
21­
Day
fadrozole
assay
ranged
from
2,667,000
ng/
mL
to
10,890,000
ng/
mL
(
Figure
6.20).
Among
females
exposed
to
the
two
fadrozole
concentrations,
vitellogenin
concentrations
ranged
from
0
ng/
mL
(
not
detected)
to
2,768,500
ng/
mL.
Significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
6.18)
were
detected
(
Kruskal­
Wallis,
H
=
39.41,
p
=
<
0.001,
df
=
2).
Vitellogenin
concentrations
in
Control
treatment
females
were
significantly
greater
than
those
in
females
exposed
to
the
Low
and
High
fadrozole
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
238
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
concentrations.
Additionally,
vitellogenin
concentrations
in
females
from
the
Low
concentration
were
greater
than
those
in
females
from
the
High
concentration.
The
achieved
power
for
this
endpoint
was
100%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
3
(
Table
6.18).

Table
6.18.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
16
5,949,000
2,252,237
38%
100%
3
low
16
1,197,869
754,539
63%
high
16
4,914
10,679
217%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
16.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
10000000
5000000
0
treatment
(
y
)

Figure
6.20.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Vitellogenin
concentrations
in
Control
treatment
males
used
during
the
EPA
21­
Day
fadrozole
assay
ranged
from
168
ng/
mL
to
8,288
ng/
mL
(
Figure
6.21).
Among
males
exposed
to
the
two
fadrozole
concentrations,
vitellogenin
concentrations
ranged
from
14
ng/
mL
to
15,097
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
6.19)
were
detected
(
Kruskal­
Wallis,
H
=
0.85,
p
=
0.655,
df
=
2).
The
achieved
power
for
this
endpoint
was
13%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
67
(
Table
6.19).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
239
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.19.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
2,334
2,639
113%
13%
67
low
8
3,576
5,554
155%
high
8
2,074
2,792
135%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
15000
10000
5000
0
treatment
Fad­
VTG
Figure
6.21.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

6.2.3
Appearance
/
Secondary
Sex
Characteristics
All
of
the
females
used
during
the
EPA
21­
Day
Fadrozole
assay
exhibited
typical
female
morphology
(
no
fat
pad,
no
tubercles,
no
vertical
banding,
ovipositor
present).

All
of
the
males
used
during
the
EPA
21­
Day
Fadrozole
assay
exhibited
typical
male
morphology
(
fat
pads,
tubercles,
vertical
banding,
no
ovipositor
present).

6.2.4
Gonadosomatic
Index
The
range
of
GSI
values
calculated
for
females
in
the
all
treatments
varied
from
three­
to
seven­
fold
(
Figure
6.22),
and
the
overall
variability
within
the
treatment
was
moderate
to
high
(
CVs
=
28%
 
31%;
Table
6.20).
The
highest
value
(
GSI
=
22.2)
was
obtained
for
a
female
exposed
to
the
High­
fadrozole
concentration.
One
female
exposed
to
the
Low­
fadrozole
concentration
had
a
GSI
value
of
21.3.
A
significant
difference
in
the
mean
GSI
value
per
treatment
(
Table
6.20)
was
detected
(
Kruskal­
Wallis,
H
=
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
240
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
10.50,
p
=
0.005,
df
=
2).
The
mean
GSI
value
for
females
from
the
High
concentration
was
greater
than
that
for
females
from
the
Control
treatment.
The
achieved
power
for
this
endpoint
was
80%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
16
(
Table
6.20).

Table
6.20.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
16
10.3
2.9
28%
80%
16
low
16
13.3
4.1
31%
high
16
15.2
4.8
31%
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
16.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

Low
High
Control
22
12
2
treatment
GSI
Figure
6.22.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

The
range
of
most
GSI
values
calculated
for
males
during
the
EPA
21­
Day
Fadrozole
assay,
was
small,
ranging
from
0.7
to
1.8
(
Figure
6.23),
which
approximates
the
typical
range
for
reproductively­
active
male
fathead
minnows.
One
male
in
the
Low
concentration
had
a
GSI
value
of
2.3,
attributable
to
its
relatively
high
gonad
weight
of
0.10
g
and
moderate
body
size
(
4.5
g).
The
three
large
males
did
not
have
abnormally
high
GSI
values
(
0.8
 
1.6),
which
reflects
their
proportionally
high
gonad
weights
(
0.07
g
 
0.13
g).
There
were
significant
differences
in
mean
GSI
values
(
Table
6.21)
among
treatments
(
Kruskal­
Wallis,
H
=
10.19,
p
=
0.006,
df
=
2).
The
mean
GSI
values
for
fish
from
the
High
and
Low
concentrations
were
greater
than
the
mean
value
for
fish
from
the
Control
treatment.
The
achieved
power
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
241
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
for
this
endpoint
was
87%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
7
(
Table
6.21).

Table
6.21.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
1.00
0.20
20%
87%
7
low
8
1.37
0.45
33%
high
8
1.52
0.18
12%
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

Low
High
Control
2.5
2.0
1.5
1.0
treatment
GSI
(
y
)

Figure
6.23.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

6.2.5
Female
Gonad
Histology
Histological
analyses
were
conducted
on
the
ovaries
of
48
females
exposed
to
fadrozole
during
the
EPA
21­
Day
Assay.

General
Ovary
Staging
 
Statistical
analysis
of
the
mean
ovarian
staging
from
12
microscopic
fields
per
female
in
the
EPA
21­
Day
Fadrozole
assay
revealed
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
34.09,
p
=
<
0.001,
df
=
2).
The
mean
ovarian
stage
of
females
exposed
to
the
High
concentration
was
significantly
less
than
that
of
females
from
the
Control
treatment
or
the
Low
concentration.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
242
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Quantitative
Ovarian
Staging
 
One
hundred
cells
in
each
of
three
sections
per
female
were
examined
to
quantitatively
determine
the
developmental
stage
of
the
ovaries.
Ova
from
fish
from
the
Control
treatment
and
Low
concentration
ranged
from
Stage
1a
to
Stage
5
(
see
Methods
for
a
description
of
the
stages
(
Figure
6.24).
Ova
from
fish
from
the
High
concentration
ranged
from
Stage
1a
to
Stage
4.
Variability
within
treatments
for
each
stage,
particularly
for
Stage
5,
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
400%
(
Table
6.22).
Statistical
analyses
showed
that
within
each
of
the
five
developmental
stages,
except
Stage
5,
there
were
significant
differences
among
treatments
in
the
proportion
of
cells
occurring
in
the
stage
(
Table
6.22).
The
proportion
of
ova
in
Stage
1a
was
significantly
greater
in
the
Control­
treatment
females
than
in
High­
concentration
females.
The
proportion
of
ova
in
Stage
1b
was
significantly
greater
in
the
Control­
treatment
females
than
in
High­
or
Lowconcentration
females.
The
proportion
of
ova
in
Stage
2
was
significantly
greater
in
the
Controltreatment
and
High­
concentration
females
than
in
Low­
concentration
females.
The
proportion
of
ova
in
Stage
3
was
significantly
greater
in
the
High­
concentration
females
than
in
Control­
treatment
or
Lowconcentration
females.
The
proportion
of
ova
in
Stage
4
was
significantly
less
in
the
High­
concentration
females
than
in
Control­
treatment
or
Low­
concentration
females.

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.

Control
(
n
=
16)
Low
(
n
=
16)
High
(
n
=
16)
Kruskal­
Wallis
Stage
Mean
Stdev
CV
Mean
Stdev
CV
Value
Stdev
CV
H
p
1a
0.064
0.020
31%
0.049
0.026
53%
0.037
0.020
54%
10.43
0.005*
1b
0.246
0.059
24%
0.180
0.067
37%
0.170
0.066
39%
9.75
0.008*
2
0.230
0.030
13%
0.180
0.034
19%
0.211
0.053
25%
13.55
0.001*
3
0.243
0.055
23%
0.305
0.105
34%
0.425
0.148
35%
14.94
0.001*
4
0.180
0.118
66%
0.240
0.109
46%
0.015
0.018
115%
28.14
<
0.001**
5
0.006
0.017
288%
0.000
0.001
400%
0
0
­­
2.18
0.337
*
p
<
0.01
**
p
<
0.001
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
243
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
0.00
0.10
0.20
0.30
0.40
0.50
0.60
1a
1b
2
3
4
5
Ovarian
Stage
Proportion
Control
Low
High
Figure
6.24.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
21­
Day
Fadrozole
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.

Atretic
Follicles
 
The
mean
proportion
of
atretic
follicles
per
300
follicles
(
counted
per
fish)
ranged
from
0.008
follicles
for
females
in
the
Control
treatment
to
0.126
follicles
for
females
in
the
High
concentration
(
Figure
6.25).
One
fish
from
the
High
concentration
had
a
very
high
proportion
of
atretic
follicles
(
0.27).
A
significant
difference
in
the
proportions
of
atretic
follicles
among
treatments
was
detected
(
Kruskal­
Wallis,
H
=
24.42,
p
<
0.001,
df
=
2).
The
proportion
of
atretic
follicles
per
300
follicles
in
fish
from
the
High
concentration
was
greater
than
those
of
fish
from
the
remaining
two
treatments.
Additionally,
the
proportion
of
atretic
follicles
per
300
follicles
in
fish
from
the
Low
concentration
was
greater
than
that
in
fish
from
the
Control
treatment.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
244
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
0.3
0.2
0.1
0.0
Treatment
atretic_
follicles
Figure
6.25.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Corpora
Lutea
 
The
mean
proportion
of
corpora
lutea
per
300
follicles
(
counted
per
fish)
ranged
from
0.006
for
females
in
the
Low
concentration
to
0.024
for
females
in
the
Control
treatment
(
Figure
6.26).
Variability
within
each
treatment
was
very
high,
ranging
from
132%
for
the
Control
treatment
to
388%
for
the
High
concentration.
Despite
the
high
variability,
there
was
a
significant
difference
in
the
mean
proportion
of
corpora
lutea
among
treatments
(
Kruskal­
Wallis,
H
=
7.65,
p
=
0.022,
df
=
2).
The
proportion
of
corpora
lutea
in
fish
from
the
Control
treatment
was
significantly
greater
than
that
in
fish
from
the
High
concentration.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
245
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
0.25
0.20
0.15
0.10
0.05
0.00
Treatment
corpora_
lutea
Figure
6.26.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Observations
 
One
fish
from
the
Low
concentration
were
observed
to
have
most
of
the
atretic
follicles
at
the
margins
of
the
ovaries.
One
fish
from
the
High
concentration
were
observed
to
have
inflammatory
cells
in
the
ovary.
Five
other
fish
from
the
High
concentration
had
atretic
follicles
that
were
Stage
3
(
one
fish)
or
Stage
4
(
four
fish)
cells.

6.2.6
Male
Gonad
Histology
General
Testes
Staging
 
Testes
from
24
males
exposed
to
fadrozole
during
the
EPA
21­
Day
Fadrozole
assay
were
examined
to
determine
the
general
developmental
condition.
Males
in
all
treatments
had
welldeveloped
testes,
with
most
showing
Stage
4
and
Stage
5
development
(
see
Methods
for
description
of
developmental
stages).
All
of
the
96
microscopic
fields
examined
in
the
8
Control
treatment
males
showed
Stage
4
(
88
fields)
or
Stage
5
(
8
fields)
development.
Ninety­
four
of
the
96
microscopic
fields
examined
in
the
8
Low­
concentration
treatment
males
showed
Stage
4
(
85
fields)
or
Stage
5
(
9
fields)
development.
Two
microscopic
fields
showed
Stage
3
development.
All
of
the
96
microscopic
fields
examined
in
the
8
High­
concentration
treatment
males
showed
Stage
4
(
69
fields)
or
Stage
5
(
27
fields)
development.
Statistical
analysis
of
the
mean
staging
from
12
microscopic
fields
per
fish
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
3.92,
p
=
0.141,
df
=
2).

Quantitative
Testicular
Staging
 
One
hundred
cells
in
each
of
three
sections
per
male
were
examined
to
quantitatively
determine
the
developmental
condition
of
the
testes.
The
developmental
stage
all
treatment
testes
ranged
from
Stage
2a
to
Stage
5
(
Figure
6.27).
Variability
within
treatments
for
each
stage
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
198%
(
Table
6.23).
Statistical
analyses
showed
that
there
were
significant
differences
among
treatments
in
the
proportion
of
cells
in
any
of
the
developmental
stages
(
Table
6.23).
The
mean
proportion
of
cells
in
developmental
Stage
3a
was
greater
in
fish
from
the
Low
concentration
than
in
fish
from
the
Control
treatment.
The
observed
proportions
of
cells
in
developmental
Stage
3b
ranked
higher
in
fish
from
the
Low
concentration
and
the
Control
treatment
than
in
fish
from
the
High
concentration.
The
mean
proportion
of
cells
in
developmental
Stage
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
246
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
5
was
greater
in
fish
from
the
Low
concentration
than
in
fish
from
the
High
concentration.
Therefore,
there
did
not
appear
to
be
an
effect
on
testicular
developmental
stage
associated
with
fadrozole
dose.

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.

Control
(
n
=
8)
Low
(
n
=
8)
High
(
n
=
8)
Kruskal­
Wallis
Stage
Mean
Stdev
CV
Mean
Stdev
CV
Value
Stdev
CV
H
p
1
0
0
 
0
0
 
0
0
 
 
 
2a
0.004
0.006
147%
0.002
0.003
151%
0.001
0.002
198%
1.30
0.523
2b
0.019
0.016
84%
0.014
0.010
68%
0.010
0.008
80%
1.26
0.532
3a
0.092
0.054
59%
0.204
0.079
39%
0.141
0.091
64%
6.52
0.038*
3b
0.292
0.110
38%
0.274
0.082
30%
0.176
0.079
45%
6.18
0.046*
4
0.235
0.093
40%
0.223
0.048
22%
0.162
0.076
47%
3.51
0.173
5
0.358
0.139
39%
0.283
0.135
48%
0.510
0.199
39%
6.50
0.039*
*
p
<
0.05
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1
2a
2b
3a
3b
4
5
Testicular
Stage
Proportion
Control
Low
High
Figure
6.27.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
21­
Day
Fadrozole
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.

Tubule
Diameter
 
The
average
diameter
of
the
seminiferous
tubules
of
males
from
the
Control
treatment
ranged
from
104.2
µ
m
to
136.1
µ
m
(
Figure
6.28).
Tubule
diameters
of
males
from
the
two
test
concentrations
ranged
from
101.1
µ
m
to
202.2
µ
m.
A
significant
difference
in
the
mean
tubule
diameter
per
treatment
(
Table
6.24)
was
detected
(
Kruskal­
Wallis,
H
=
12.89,
p
=
0.002,
df
=
2).
The
mean
tubule
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
247
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
diameter
in
males
form
the
High
concentration
was
greater
than
those
in
males
from
the
Low
concentration
and
the
Control
treatment.
The
achieved
power
for
this
endpoint
was
97%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
5
(
Table
6.24).

Table
6.24.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
115.0
10.8
9%
97%
5
low
8
123.5
12.7
10%
high
8
151.8
25.0
16%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
200
150
100
Treatment
diameter
(
y
)

Figure
6.28.
Boxplot
of
male
seminiferous
tubule
diameter
(
µ
m)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Observations
 
No
Sertoli
cell
proliferation
was
observed.
Four
males
from
the
High
concentration
were
found
to
have
focal
proliferation
of
Leydig
cells.
One
male
from
the
High
concentration
had
focal
proliferation
of
secondary
spermatogonia.
Four
males
from
the
High
concentration
had
multifocal
proliferation
of
secondary
spermatogonia.
In
two
of
these
fish,
the
proliferation
extended
into
the
lumen
and
included
Stages
3b,
4,
and
5
cells.
In
one
fish,
the
proliferation
extended
into
mature
tubules.
No
testicular
atrophy
was
recorded
and
no
ovatestes
were
observed
for
any
treatment.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
248
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
6.2.7
Plasma
Steroid
Concentrations
Estradiol
 
Estradiol
concentrations
in
Control­
treatment
females
used
during
the
EPA
21­
Day
fadrozole
assay
ranged
from
372
pg/
mL
to
5,847
pg/
mL
(
Figure
6.29).
Among
females
exposed
to
the
Lowfadrozole
concentrations,
estradiol
concentrations
ranged
from
401
pg/
mL
to
2,587
pg/
mL.
No
females
from
the
High
concentration
were
available
for
estradiol
determination.
A
significant
difference
in
the
mean
estradiol
concentration
per
treatment
(
Table
6.25)
was
detected
(
Kruskal­
Wallis,
H
=
5.16,
p
=
0.023,
df
=
2).
The
mean
estradiol
concentration
in
females
from
the
Low
concentration
was
less
than
that
in
females
from
the
Control
treatment.
The
achieved
power
for
this
endpoint
was
40%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
17
(
Table
6.25).

Table
6.25.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
16
2,861
1,492
52%
40%
17
low
7
1,343
756
56%
high
0
 
 
 
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
7.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
Control
6000
5000
4000
3000
2000
1000
0
treatment
Estradiol
Figure
6.29.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Estradiol
concentrations
in
Control
treatment
males
used
during
the
EPA
21­
Day
Fadrozole
assay
ranged
from
0
pg/
mL
(
not
detected)
to
193
pg/
mL
(
Figure
6.29).
Estradiol
was
not
detected
in
males
exposed
to
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
249
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
the
Low­
fadrozole
concentrations
(
4
individuals).
No
males
from
the
High
concentration
were
available
for
estradiol
determination
(
Table
6.26).

Table
6.26.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
1
control
8
70
96
138%
 
 
low
4
0
0
 
high
0
 
 
 
1
Not
calculated.

Testosterone
 
Testosterone
concentrations
in
Control­
treatment
females
used
during
the
EPA
21­
Day
Fadrozole
assay
ranged
from
199
pg/
mL
to
3,828
pg/
mL
(
Figure
6.30).
Among
females
exposed
to
the
two
fadrozole
concentrations,
testosterone
concentrations
ranged
from
561
pg/
mL
to
7,275
pg/
mL.
Significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
6.27)
were
detected
(
Kruskal­
Wallis,
H
=
13.68,
p
=
0.001,
df
=
2).
The
mean
testosterone
concentration
in
females
from
the
Control
treatment
was
less
than
those
in
females
from
the
Low
and
High
concentrations.
The
achieved
power
for
this
endpoint
was
93%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
10
(
Table
6.27).

Table
6.27.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
16
1,096
938
86%
93%
10
low
13
1,977
834
42%
high
16
2,928
1,951
67%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
13.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
250
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
8000
7000
6000
5000
4000
3000
2000
1000
0
treatment
Testosterone
Figure
6.30.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisks
represent
probable
outliers.

Testosterone
concentrations
in
Control
treatment
males
used
during
the
EPA
21­
Day
Fadrozole
assay
ranged
from
953
pg/
mL
to
3,926
pg/
mL
(
Figure
6.31).
Among
males
exposed
to
the
two
fadrozole
concentrations,
testosterone
concentrations
ranged
from
3,282
pg/
mL
to
15,262
pg/
mL.
Significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
6.28)
were
detected
(
Kruskal­
Wallis,
H
=
14.92,
p
=
0.001,
df
=
2).
The
mean
testosterone
concentration
in
males
from
the
Control
treatment
was
less
than
those
in
males
from
the
Low
and
High
concentrations.
The
achieved
power
for
this
endpoint
was
100%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
4
(
Table
6.28).

Table
6.28.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
2,208
1,116
51%
100%
4
low
8
6,593
1,600
24%
high
8
9,030
4,252
47%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
251
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
15000
10000
5000
0
treatment
Testosterone
Figure
6.31.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

11­
ketotestosterone
 
11­
ketotesosterone
concentrations
in
Control­
treatment
females
used
during
the
EPA
21­
Day
Fadrozole
assay
ranged
from
0
pg/
mL
(
not
detected)
to
594
pg/
mL
(
Figure
6.32).
However,
11­
ketotestosterone
was
only
detected
in
2
of
the
16
females
from
the
Control
treatment
that
were
analyzed.
Among
females
exposed
to
the
two
fadrozole
concentrations,
testosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
1,966
pg/
mL.
A
significant
difference
in
the
mean
11­
ketotestosterone
concentration
per
treatment
(
Table
6.29)
was
detected
(
Kruskal­
Wallis,
H
=
9.05,
p
=
0.011,
df
=
2).
The
mean
11­
ketotestosterone
concentration
in
females
from
the
Control
treatment
was
less
than
those
in
females
from
the
High
concentration.
However,
the
achieved
power
for
this
endpoint
was
only
48%,
because
of
the
high
variability
in
the
Control
response.
The
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
22
(
Table
6.29).

Table
6.29.
Summary
statistics
and
power
estimates
for
female
11­
ketotestosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
16
71
193
274%
48%
22
low
11
369
377
102%
high
16
515
593
115%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
13.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
252
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
2000
1000
0
treatment
11­
keto
Figure
6.32.
Boxplot
of
female
11­
ketotestosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisks
represent
probable
outliers.

11­
ketotesosterone
concentrations
in
Control
treatment
males
used
during
the
EPA
21­
Day
Fadrozole
assay
ranged
from
4,697
pg/
mL
to
43,298
pg/
mL
(
Figure
6.33).
Among
males
exposed
to
the
two
fadrozole
concentrations,
11­
ketotesosterone
concentrations
ranged
from
14,292
pg/
mL
to
192,900
pg/
mL.
Significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
6.30)
were
detected
(
Kruskal­
Wallis,
H
=
14.89,
p
=
0.001,
df
=
2).
The
mean
11­
ketotestosterone
concentration
in
males
from
the
Control
treatment
was
less
than
those
in
males
from
the
Low
and
High
concentrations.
The
achieved
power
for
this
endpoint
was
100%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
4
(
Table
6.30).

Table
6.30.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
19,770
15,109
76%
100%
4
low
8
130,695
47,538
36%
high
8
77,822
54,034
69%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
253
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
200000
100000
0
treatment
11­
keto
Figure
6.33.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

6.2.8
Fecundity
Total
Fecundity
 
A
15­
day
pre­
exposure
evaluation
of
total
egg
production
was
performed.
Total
15­
day
counts
among
the
three
treatments
in
the
exposure
assay
(
individual
tank
values
summed
for
each
treatment)
ranged
from
about
17,000
eggs
to
22,000
eggs
(
Figure
6.34).
A
significant
difference
in
the
mean
15­
day
egg
production
among
the
groups
of
replicates
evaluated
during
the
pre­
exposure
assay
was
detected
(
Kruskal­
Wallis,
H
=
8.74,
p
=
0.033,
df
=
3).
However,
the
only
significant
difference
was
that
the
numbers
of
eggs
laid
in
the
tanks
that
were
not
subsequently
used
during
the
21­
Day
fadrozole
assay
were
lower
than
the
number
for
the
tanks
that
would
eventually
constitute
the
High
concentration
during
the
assay.
There
were
no
differences
in
the
numbers
of
eggs
laid
among
the
tanks
that
would
eventually
be
used
for
the
three
treatments
during
the
21­
Day
assay.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
254
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
45,000
­
14
­
12
­
10
­
8
­
6
­
4
­
2
0
2
4
6
8
10
12
14
16
18
20
Day
Cumulative
Number
of
Eggs
Control
Low
High
Figure
6.34.
Total
egg
production
per
treatment
for
the
15­
Day
pre­
exposure
assay.

During
the
EPA
21­
Day
Fadrozole
assay,
total
counts
in
the
Control
treatment
were
reasonably
consistent
among
replicates,
varying
from
5,193
eggs
to
6,131
eggs
(
Figure
6.35).
Variability
in
total
egg
production
among
Low­
concentration
replicates
was
somewhat
greater,
ranging
from
3,224
eggs
to
6,356
eggs.
Total
counts
among
the
High­
concentration
replicates
varied
from
413
eggs
to
922
eggs.
Statistical
analysis
of
square­
root
transformed
egg
counts
showed
significant
among­
treatment
differences
(
Kruskal­
Wallis,
H
=
8.00,
p
=
0.018,
df
=
2)
in
mean
total
numbers
of
eggs
produced
(
Table
6.31).
The
mean
total
number
of
eggs
produced
by
females
in
the
High
concentration
was
significantly
less
than
the
mean
total
numbers
of
eggs
produced
by
females
in
the
Control
treatment
and
the
Low
concentration.
The
achieved
power
for
this
assay
was
100%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
2
(
Table
6.31).

Table
6.31.
Summary
statistics
and
power
estimates
for
total
fecundity
data
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
4
5548
421
8%
100%
2
low
4
4587
1305
28%
high
4
654
210
32%
1
Calculated
from
square­
root
transformed
data;
with
sample
size
=
4.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
square­
root
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
255
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
0
5
10
15
20
Day
Cumulative
Number
of
Eggs
Control
Low
High
Figure
6.35.
Total
egg
production
by
replicate
per
treatment
for
the
EPA
21­
Day
fadrozole
assay.

Fecundity
per
Female
Reproductive
Day
 
During
the
15­
Day
pre­
exposure
evaluation,
the
mean
number
of
eggs
produced
per
female
reproductive
day
ranged
from
61.1
eggs/
day
for
the
tanks
that
would
be
used
for
the
Control
treatment
to
104.4
eggs/
day
for
the
tanks
that
would
be
used
for
the
High
concentration
during
the
21­
day
exposure
assay.
The
mean
number
of
eggs
produced
per
female
reproductive
day
for
the
tanks
that
would
not
be
used
during
the
21­
day
exposure
assay
was
60.6
eggs/
day.
There
were
no
significant
differences
among
treatments
in
the
mean
numbers
of
eggs
produced
per
reproductive
day
during
the
pre­
exposure
period
(
Kruskal­
Wallis,
H
=
7.65,
p
=
0.054,
df
=
2).
The
largest
difference
in
eggs
per
female
reproductive
day
was
between
the
tanks
that
would
constitute
the
High
concentration
and
those
that
were
not
subsequently
used
during
the
exposure
assay.

During
the
EPA
21­
Day
Fadrozole
assay,
the
maximum
number
of
female
reproductive
days
was
achieved
for
all
treatments
(
Table
6.32).
The
number
of
eggs
produced
per
female
reproductive
day
varied
from
61.8
eggs
to
73.0
eggs
in
the
Control
treatment
and
from
38.4
to
75.7
in
the
Low
concentration
(
Figure
6.36).
For
the
High
concentration,
the
number
of
eggs
produced
per
female
reproductive
day
ranged
from
4.9
eggs
to
11.0
eggs.
Because
no
fish
died
during
the
assay,
the
statistical
results
reported
here
are
the
same
as
those
reported
for
total
fecundity.
The
number
of
eggs
produced
per
day
by
females
in
the
High
concentration
was
significantly
less
than
the
numbers
produced
by
females
in
the
Control
treatment
and
the
Low
concentration
(
Kruskal­
Wallis,
H
=
8.00,
p
=
0.018,
df
=
2).
The
achieved
power
for
this
assay
was
100%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
2
(
Table
6.32).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
256
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.32.
Summary
statistics
and
power
estimates
for
fecundity
per
female
reproductive
day
for
the
EPA
21­
Day
Fadrozole
assay.

Level
Mean
Number
of
Reproductive
Days
1
N
Mean
Stdev
CV
Achieved
Power
2
Sample
Size
Required
3
control
84
4
66.0
5.0
8%
100%
2
low
84
4
54.6
15.5
28%
high
84
4
7.8
2.5
32%
1
Maximum
number
=
84.
2
Calculated
from
natural
log
transformed
data;
with
sample
size
=
4.
3
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
80
70
60
50
40
30
20
10
0
treatment
eggs/
ReproDay
Figure
6.36.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Eggs
on
Tiles/
Dishes
 
The
mean
number
of
eggs
laid
on
the
tiles
during
the
15­
day
pre­
exposure
assay
varied
from
2,859
eggs
for
the
tanks
that
would
be
used
for
the
Control
treatment
to
3,791
eggs
for
the
tanks
that
would
be
used
for
the
High
concentration.
The
mean
number
of
eggs
on
dishes
ranged
from
1,464
eggs
for
the
Control
treatment
to
1,781
eggs
for
the
High
concentration.
Because
of
the
variability
in
the
total
number
of
eggs
laid
per
treatment,
the
proportional
difference
in
the
number
of
eggs
on
dishes
versus
those
on
tiles
[
1 (#
eggs
on
dishes
÷
#
eggs
on
tiles)]
was
calculated.
There
were
no
significant
differences
in
the
mean
proportional
difference
among
treatments
during
the
15­
day
pre­
exposure
assay
(
Kruskal­
Wallis,
H
=
7.26,
p
=
0.064,
df
=
3).

The
mean
number
of
eggs
laid
on
the
tiles
among
the
treatments
during
the
EPA
21­
Day
Fadrozole
assay
varied
from
534
eggs
for
the
High
concentration
to
4,938
eggs
for
the
Control
treatment
(
Appendix
E;
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
257
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
4.7).
The
number
of
eggs
on
dishes
ranged
from
120
eggs
for
the
High
concentration
to
610
eggs
for
the
Control
treatment.
Because
of
the
variability
in
the
total
number
of
eggs
laid
per
treatment,
the
proportional
difference
in
the
number
of
eggs
on
dishes
versus
those
on
tiles
[
1 (#
eggs
on
dishes
÷
#
eggs
on
tiles)]
was
calculated
(
Appendix
E;
Figure
4.6).
A
significant
difference
in
this
mean
proportional
difference
among
treatments
was
detected
(
Kruskal­
Wallis,
H
=
6.96,
p
=
0.031,
df
=
2).
Proportionally
more
eggs
occurred
on
tiles
in
the
Control
treatment
than
in
the
High­
concentration
treatment.

6.2.9
Fertilization
Success
Total
Fertilization
 
Eggs
were
collected
during
the
15­
day
pre­
exposure
period
for
the
evaluation
of
fertilization
success
rate.
The
mean
proportion
of
eggs
fertilized
in
the
Control
treatment
was
0.997
[
standard
deviation
(
sd)
=
0.006],
1.000
(
sd
=
0.0003)
in
the
Low
concentration,
and
1.000
(
sd
=
0.0001)
in
the
High
concentration.
The
mean
proportion
of
eggs
fertilized
in
the
replicates
that
were
not
used
in
the
21­
day
exposure
assay
was
1.000
(
sd
=
0.0005).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
were
fertilized
(
Kruskal­
Wallis,
H
=
1.17,
p
=
0.760,
df
=
3).

The
total
(
tiles
+
dishes)
fertilization
success
rates
for
all
treatment
replicates
during
the
EPA
21­
Day
Fadrozole
assay
were
high,
ranging
from
0.995
(
High­
concentration
replicate)
to
1.00
(
Highconcentration
and
Control­
treatment
replicates)
(
Figure
6.37).
No
significant
differences
in
mean
fertilization
success
rates
(
Table
6.33)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
1.56,
p
=
0.457,
df
=
2).
The
achieved
power
for
this
assay
was
6%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
201
(
Table
6.33).

Table
6.33.
Summary
statistics
and
power
estimates
for
the
proportion
of
eggs
fertilized
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
4
0.999
0.001
0.1%
6%
201
low
4
0.999
0.001
0.1%
high
4
0.999
0.003
0.3%
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
4.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
258
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
1.000
0.999
0.998
0.997
0.996
0.995
treatment
Total
Prop­
fert
Figure
6.37.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Fertilization
of
Eggs
on
Tiles
and
Dishes
 
During
the
15­
Day
pre­
validation
assay,
there
were
no
significant
differences
in
the
fertilization
success
rates
among
treatments
for
eggs
laid
on
tiles
(
Kruskal­
Wallis,
H
=
1.17,
p
=
0.760,
df
=
3)
or
on
dishes
as
all
eggs
that
occurred
on
dishes
were
fertilized.
The
fertilization
success
rates
for
all
treatment
replicates
for
eggs
laid
on
tiles
during
the
EPA
21­
Day
Fadrozole
assay
were
high,
ranging
from
0.992
(
High­
concentration
replicate)
to
1.00
(
Highconcentration
and
Control­
treatment
replicates)
(
Appendix
E,
Figure
4.7).
No
significant
differences
in
mean
fertilization
success
rates
(
Appendix
E,
Table
4.8)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
1.56,
p
=
0.457,
df
=
2).
The
fertilization
success
rates
for
all
treatment
replicates
for
eggs
laid
on
dishes
during
the
assay
were
high,
ranging
from
0.998
(
Control
treatment
replicate)
to
1.00
(
all
High
and
Low­
concentration
replicates)
(
Appendix
E,
Figure
4.8).
No
significant
differences
in
mean
fertilization
success
rates
(
Appendix
E,
Table
4.8)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
2.00,
p
=
0.368,
df
=
2).

6.2.10
Hatchability
and
Larval
Development
Eggs
were
collected
during
the
15­
day
pre­
exposure
period
for
the
evaluation
of
hatchability.
The
mean
proportion
of
fertilized
eggs
that
hatched
in
the
Control
treatment
was
0.93
[
standard
deviation
(
sd)
=
0.14],
0.99
(
sd
=
0.02)
in
the
Low
concentration,
and
0.97
(
sd
=
0.06)
in
the
High
concentration.
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
0.50,
p
=
0.779,
df
=
2).

Eggs
were
collected
during
the
EPA
21­
Day
Fadrozole
assay
for
the
evaluation
of
hatchability.
The
proportion
of
fertilized
eggs
that
hatched
ranged
from
0.88
to
1.00
in
the
Control
treatment
and
from
0.83
to
1.00
for
the
Low
concentration
(
Figure
6.38).
No
eggs
were
available
to
evaluate
hatchability
for
the
High
concentration
because
the
evaluation
was
started
on
Day
12
of
the
assay
and
no
eggs
were
laid
in
those
tanks
after
Day
9.
There
were
no
significant
differences
between
the
Low
concentration
and
the
Control
treatment
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
0.14,
p
=
0.708,
df
=
1).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
259
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
The
achieved
power
for
this
endpoint
was
6%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
767
(
Table
6.34).

Table
6.34.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
11
0.962
0.045
5%
6%
767
low
11
0.942
0.071
8%
high
0
 
 
 
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
11.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

low
control
1.00
0.95
0.90
0.85
treatment
Prop­
Hatch
Figure
6.38.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Eggs
were
collected
during
the
pre­
exposure
period
for
the
evaluation
of
larval
development.
The
mean
proportion
of
larvae
that
developed
normally
(
i.
e.,
that
showed
no
morphological
abnormalities)
in
the
Control
treatment
was
0.97
(
sd
=
0.02).
The
mean
proportion
of
normal
larvae
in
the
remaining
treatments
was
0.99
(
sd
=
0.01)
in
the
Low
concentration
and
0.96
(
sd
=
0.03)
in
the
High
concentration.
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
3.77,
p
=
0.152,
df
=
2).

Eggs
were
collected
during
the
EPA
21­
Day
Fadrozole
assay
for
the
evaluation
of
larval
development.
The
proportion
of
larvae
that
developed
normally
(
i.
e.,
that
showed
no
morphological
abnormalities)
ranged
from
0.84
to
1.00
in
the
Control
treatment
and
from
0.70
to
1.00
for
the
Low
concentration
(
Figure
6.39).
Again,
no
larvae
were
available
in
the
High
concentration
to
evaluate
development.
There
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
260
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
were
no
significant
differences
between
the
Low
concentration
and
the
Control
treatment
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
0.76,
p
=
0.382,
df
=
1).
The
achieved
power
for
this
endpoint
was
13%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
102
(
Table
6.35).

Table
6.35.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
21­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
11
0.955
0.059
6%
13%
102
low
11
0.917
0.107
12%
high
0
 
 
 
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
11.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

low
control
1.0
0.9
0.8
0.7
treatment
Prop­
Norm
(
y
)

Figure
6.39.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

6.2.11
Body
Weight
The
body
weight
of
females
used
in
the
EPA
21­
Day
Fadrozole
assay
ranged
from
1.5
g
to
4.4
g.
There
were
significant
differences
in
mean
body
weight
among
all
treatments
(
Kruskal­
Wallis,
H
=
24.62,
p
=
<
0.001,
df
=
2).
The
mean
body
weight
of
females
in
the
High
concentration
was
greater
than
that
of
females
in
the
other
two
treatments
and
mean
body
weight
of
females
in
the
Low
concentration
was
greater
than
that
of
females
in
the
Control
treatment.
The
body
weight
of
males
used
in
the
EPA
21­
Day
Fadrozole
assay
ranged
from
3.8
g
to
9.2
g.
Three
males
were
unusually
large
(
Figure
6.40).
Two
that
DRAFT
EPA
WA
3­
8
(
Report
of
WA
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18
Study)
261
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
were
exposed
to
the
High
concentration
weighed
9.2
g
and
6.6
g.
One
male
from
the
Control
treatment
weighed
8.3
g.
However,
there
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
1.94,
p
=
0.380,
df
=
2).

Low
High
Control
9
8
7
6
5
4
treatment
fish_
wgt_
whole
Figure
6.40.
Boxplot
of
the
body
weights
of
males
by
treatment
for
the
EPA
21­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

6.3
Non­
spawning
Adult
14­
day
Assay
for
Fadrozole
The
Non­
spawning
Adult
14­
Day
Fadrozole
assay
was
conducted
from
April
1,
2003
to
April
15,
2003
(
exposure
assay).

6.3.1
Survival
All
males
and
females
in
all
treatments
survived
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

6.3.2
Vitellogenin
Vitellogenin
concentrations
in
Control
treatment
females
used
during
the
Non­
spawning
Adult
14­
Day
fadrozole
assay
ranged
from
2,245,000
ng/
mL
to
11,020,000
ng/
mL
(
Figure
6.41).
Among
females
exposed
to
the
three
fadrozole
concentrations,
vitellogenin
concentrations
ranged
from
0
ng/
mL
(
not
detected)
to
1,612,000
ng/
mL.
Significant
differences
in
the
mean
vitellogenin
concentration
among
treatments
(
Table
6.36)
were
detected
(
Kruskal­
Wallis,
H
=
32.71,
p
=
<
0.001,
df
=
2).
Mean
vitellogenin
concentrations
among
Control­
treatment
females
were
significantly
greater
than
the
mean
concentrations
for
females
exposed
to
the
High,
and
Medium
fadrozole
concentrations.
Additionally,
the
mean
concentration
of
vitellogenin
from
Low
concentration
females
was
significantly
greater
than
those
from
females
in
the
Medium
and
High
fadrozole
concentrations.
The
achieved
power
for
this
endpoint
was
100%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
3
(
Table
6.36).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
262
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.36.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
4,934,800
2,619,357
53%
100%
3
low
10
793,075
447,921
56%
medium
10
18,431
30,153
164%
high
9
8,962
17,857
199%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
9.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Mid
Low
High
Control
10000000
5000000
0
treatment
Fad­
VTG
Figure
6.41.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Vitellogenin
concentrations
in
Control
treatment
males
used
during
the
Non­
spawning
Adult
14­
Day
fadrozole
assay
ranged
from
0
ng/
mL
(
not
detected)
to
3,734
ng/
mL
(
Figure
6.42).
Among
most
males
exposed
to
the
three
fadrozole
concentrations,
vitellogenin
concentrations
ranged
from
0
ng/
mL
(
not
detected)
to
829
ng/
mL.
One
male
exposed
to
the
High
fadrozole
concentration
had
a
vitellogenin
concentration
of
6,377
ng/
mL
and
a
second
had
a
concentration
of
5,498
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
6.37)
were
detected
(
Kruskal­
Wallis,
H
=
1.67,
p
=
0.644,
df
=
2).
The
achieved
power
for
this
endpoint
was
9%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
174
(
Table
6.37).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
263
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
6.37.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
675
1,261
187%
9%
174
low
10
74
82
111%
10
192
236
123%
high
10
1,395
2,422
174%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
10.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Mid
Low
High
Control
7000
6000
5000
4000
3000
2000
1000
0
treatment
Fad­
VTG
Figure
6.42.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

6.3.3
Appearance
/
Secondary
Sex
Characteristics
Most
of
the
females
used
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
showed
normal
female
morphology.
One
female
from
the
High
concentration
had
the
fat
pad
typical
of
males.

Morphological
development
among
males
used
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
varied
among
treatments
(
Figure
6.43).
Three
males,
one
exposed
to
the
Medium
concentration
and
two
exposed
to
the
High
concentration,
lacked
tubercles.
Two
males
exposed
to
the
Medium
concentration
also
lacked
fat
pads.
Sixteen
of
the
40
males
used
during
the
assay
lacked
vertical
banding.
There
was
no
consistent
dose­
related
pattern
to
these
variations
in
morphology.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
264
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
Tubercles
Abs
Tubercles
Prs
Fat
Pad
NP
Fat
Pad
P
Fat
Pad
Pro
Vert
Banding
NP
Vert
Banding
P
Vert
Banding
Pro
Proportion
Control
Low
Mid
High
Figure
6.43.
Secondary
sex
characteristics
of
males
used
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

6.3.4
Gonadosomatic
Index
The
range
of
GSI
values
calculated
for
females
in
the
all
treatments
varied
from
about
two­
to
eight­
fold
(
Figure
6.44),
and
the
overall
within­
treatment
variability
was
moderate
(
CVs
=
2%
 
51%;
Table
6.38).
The
highest
female
GSI
value
was
21.7
(
one
fish
in
the
Low
concentration),
but
several
fish
had
GSI
values
~
19
 
20.
A
significant
difference
in
mean
GSI
values
(
Table
6.38)
among
treatments
was
detected
(
Kruskal­
Wallis,
H
=
9.21,
p
=
0.027,
df
=
3).
The
mean
GSI
value
for
females
exposed
to
the
High
fadrozole
concentration
was
significantly
less
than
the
mean
GSI
for
females
from
the
Control
treatment.
The
achieved
power
for
this
endpoint
was
59%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
15
(
Table
6.38).

Table
6.38.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
15.3
3.1
20%
59%
15
low
10
13.5
3.9
29%
medium
10
10.5
3.9
38%
high
10
10.2
5.2
51%
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
10.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
265
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
20
10
0
treatment
GSI
Figure
6.44.
Boxplot
of
female
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
asterisks
represent
probable
outliers.

The
range
of
most
GSI
values
calculated
for
males
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay,
was
large,
ranging
from
0.5
to
2.1
(
Figure
6.45),
which
approximates
the
typical
range
for
reproductively­
active
male
fathead
minnows.
Overall
within­
treatment
variability
was
moderate
to
high
(
CVs
=
30%
 
63%;
Table
6.39).
The
highest
and
lowest
male
GSI
values
were
2.4
and
2.6
(
for
one
fish
from
the
High
concentration
and
one
from
the
Control
treatment)
and
0.1
and
0.4
(
two
fish
from
the
Control
treatment),
respectively.
However,
there
were
no
significant
differences
in
mean
GSI
values
(
Table
OECD14FAD
GSI­
2)
among
treatments
(
Kruskal­
Wallis,
H
=
1.29,
p
=
0.731,
df
=
3).
The
achieved
power
for
this
endpoint
was
15%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
68
(
Table
6.39).

Table
6.39.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
1.19
0.75
63%
15%
68
low
10
1.27
0.57
45%
medium
10
1.24
0.37
30%
high
10
1.45
0.52
36%
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
10.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
266
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
2.5
2.0
1.5
1.0
0.5
0.0
treatment
GSI
Figure
6.45.
Boxplot
of
male
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

6.3.5
Female
Gonad
Histology
Histological
analyses
were
conducted
on
the
ovaries
of
40
females
exposed
to
fadrozole
during
the
Non­
Spawning
Adult
14­
Day
Assay.

General
Ovary
Staging
 
Statistical
analysis
of
the
mean
ovarian
staging
from
12
microscope
fields
per
fish
in
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
3.93,
p
=
0.269,
df
=
3).

Quantitative
Ovarian
Staging
 
One
hundred
cells
in
each
of
three
sections
per
female
were
examined
to
quantitatively
determine
the
developmental
condition
of
the
ovaries.
Ova
from
fish
in
the
Medium
and
High
concentrations
ranged
from
Stage
1a
to
Stage
5
(
see
Methods
for
a
description
of
the
stages),
whereas
ova
from
females
from
the
Control
treatment
and
Low
concentration
showed
Stage
1a
to
Stage
4
development
(
Figure
6.46).
Variability
within
treatments
for
each
stage
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
316%
(
Table
6.40).
Statistical
analyses
showed
that
there
was
a
significant
difference
among
treatments
in
the
proportion
of
cells
in
developmental
Stages
1a,
Stage
3,
and
Stage
4,
but
the
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
the
developmental
Stages
1b,
2,
and
5
(
Table
6.40).
The
proportion
of
cells
in
developmental
Stage
1a
in
the
Low
and
Medium
concentrations
were
significantly
greater
than
those
in
the
High
concentration.
The
proportion
of
cells
in
developmental
Stage
3
in
the
High
concentration
was
significantly
greater
than
that
in
the
Control
treatment.
The
proportion
of
cells
in
developmental
Stage
4
in
the
Medium
concentration
was
significantly
less
than
that
in
the
Control
treatment.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
267
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
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.

Control
(
n
=
10)
Low
(
n
=
10)
Medium
(
n
=
10)
High
(
n
=
10)
Kruskal­
Wallis
Stage
Mean
Stdev
CV
Mean
Stdev
CV
Mean
Stdev
CV
Mean
Stdev
CV
H
p
1a
0.050
0.022
45%
0.064
0.030
47%
0.075
0.028
37%
0.037
0.013
34%
13.34
0.004**
1b
0.284
0.071
25%
0.277
0.117
42%
0.277
0.075
27%
0.197
0.085
43%
7.41
0.060
2
0.159
0.057
36%
0.141
0.066
47%
0.203
0.070
34%
0.175
0.054
31%
4.55
0.208
3
0.143
0.054
38%
0.195
0.065
33%
0.226
0.088
39%
0.266
0.103
39%
9.88
0.020*
4
0.281
0.123
44%
0.247
0.156
63%
0.134
0.095
71%
0.157
0.109
69%
8.23
0.041*
5
0.000
0.000
 
0.000
0.000
 
0.000
0.001
316%
0.001
0.002
316%
2.05
0.561
0.00
0.10
0.20
0.30
0.40
0.50
0.60
1a
1b
2
3
4
5
Ovarian
Stage
Proportion
Control
Low
Mid
High
Figure
6.46.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.

Atretic
Follicles
 
The
mean
proportion
of
atretic
follicles
per
300
follicles
(
counted
per
fish)
ranged
from
0.060
follicles
for
females
in
the
Medium
concentration
to
0.166
follicles
for
females
in
the
High
concentration
treatment
(
Figure
6.47).
There
were
significant
differences
in
the
mean
proportion
of
atretic
follicles
among
treatments
(
Kruskal­
Wallis,
H
=
11.34,
p
=
0.010,
df
=
3).
The
proportion
of
atretic
follicles
in
females
from
the
High
concentration
was
greater
than
those
in
females
from
the
other
three
treatments.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
268
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
0.5
0.4
0.3
0.2
0.1
0.0
Treatment
atretic_
follicles
Figure
6.47.
Boxplot
of
atretic
follicles
per
300
follicles
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Corpora
Lutea
 
The
mean
proportion
of
corpora
lutea
per
300
follicles
(
counted
per
fish)
ranged
from
0.0003
for
females
in
the
Low
concentration
and
Control
treatment
to
0.025
for
females
in
the
Medium
concentration
(
Figure
6.48).
There
were
no
significant
differences
in
the
mean
proportion
of
corpora
lutea
among
treatments
(
Kruskal­
Wallis,
H
=
5.90,
p
=
0.116,
df
=
3).

Mid
Low
High
Control
0.25
0.20
0.15
0.10
0.05
0.00
Treatment
corpora_
lutea
Figure
6.48.
Boxplot
of
corpora
lutea
per
300
follicles
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
269
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Observations
 
One
female
from
the
Control
treatment
was
observed
to
have
many
Stage
4
cells
didn't
develop
and
became
atretic.
Six
females
from
the
High
concentration
had
atretic
follicles
that
were
Stage
4
cells.
One
of
these
females
also
had
atretic
follicles
that
were
Stage
3
cells.

6.3.6
Male
Gonad
Histology
General
Testes
Staging
 
Testes
from
40
males
exposed
to
fadrozole
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
were
examined
to
determine
the
general
developmental
condition.
However,
the
cassette
containing
a
gonad
sample
for
one
male
from
the
Control
treatment
contained
too
little
tissue
to
permit
a
definitive
evaluation
and
this
male
was
excluded
from
the
analyses.
Males
in
all
treatments
had
well­
developed
testes
with
most
showing
Stage
4
and
Stage
5
development
(
see
Methods
for
description
of
developmental
stages).
All
except
8
of
the
108
microscopic
fields
examined
in
the
9
Control
treatment
males
showed
Stage
4
(
36
fields)
or
Stage
5
(
64
fields)
development.
Most
of
the
120
microscopic
fields
examined
in
the
10
Low­
concentration
treatment
males
showed
Stage
4
(
58
fields)
or
Stage
5
(
56
fields)
development.
Six
microscopic
fields
showed
Stage
3
development.
In
the
10
Medium
concentration
males
available
for
examination,
all
of
the
120
microscopic
fields
showed
Stage
4
(
64
fields)
or
Stage
5
(
56
fields)
development.
In
the
10
High
concentration
males
available
for
examination,
115
of
the
120
microscopic
fields
showed
Stage
4
(
62
fields)
or
Stage
5
(
53
fields)
development.
Statistical
analysis
of
the
mean
staging
from
12
microscopic
fields
per
fish
revealed
that
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
0.74,
p
=
0.863,
df
=
3).

Quantitative
Testicular
Staging
 
One
hundred
cells
in
each
of
three
sections
per
male
were
examined
to
quantitatively
determine
the
developmental
condition
of
the
testes.
The
developmental
stage
all
treatment
testes
ranged
from
Stage
2a
to
Stage
5
(
Figure
6.49).
Variability
within
treatments
for
each
stage
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
175%
(
Table
6.41).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
any
of
the
developmental
stages
(
Table
6.41).
Therefore,
there
was
no
effect
on
testicular
developmental
stage
associated
with
fadrozole
dose.

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.

Control
(
n
=
9)
Low
(
n
=
10)
Medium
(
n
=
10)
High
(
n
=
10)
Kruskal­
Wallis
Stag
e
Mean
Stdev
CV
Mean
Stdev
CV
Mean
Stdev
CV
Mean
Stdev
CV
H
p
1
0
0
 
0
0
 
0
0
 
0
0
 
 
 
2a
0.004
0.005
119%
0.003
0.005
152%
0.004
0.005
115%
0.001
0.002
175%
2.54
0.467
2b
0.021
0.032
149%
0.054
0.083
154%
0.027
0.029
108%
0.040
0.055
136%
1.01
0.799
3a
0.080
0.108
135%
0.092
0.085
92%
0.049
0.048
98%
0.108
0.110
102%
1.74
0.628
3b
0.134
0.101
75%
0.120
0.078
65%
0.097
0.090
93%
0.111
0.092
83%
0.86
0.835
4
0.116
0.067
58%
0.072
0.059
81%
0.115
0.077
67%
0.116
0.051
44%
3.93
0.269
5
0.644
0.269
42%
0.659
0.238
36%
0.708
0.191
27%
0.624
0.228
37%
0.40
0.940
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
270
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1
2a
2b
3a
3b
4
5
Testicular
Stage
Proportion
Control
Low
Mid
High
Figure
6.49.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.

Tubule
Diameter
 
The
diameter
of
the
seminiferous
tubules
of
males
from
the
Control
treatment
ranged
from
121.7
µ
m
to
267.5
µ
m
(
Figure
6.50).
Tubule
diameters
of
males
from
the
three
test
concentrations
ranged
from
82.2
µ
m
to
294.7
µ
m.
No
significant
differences
in
the
mean
tubule
diameter
per
treatment
(
Table
6.42)
were
detected
(
Kruskal­
Wallis,
H
=
4.03,
p
=
0.258,
df
=
3).
The
achieved
power
for
this
endpoint
was
23%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
36
(
Table
6.42).

Table
6.42.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
9
193.8
55.4
29%
23%
36
low
10
189.2
58.4
31%
medium
10
149.0
42.1
28%
high
10
168.2
63.7
38%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
9.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
271
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
300
200
100
Treatment
diameter
Figure
6.50.
Boxplot
of
seminiferous
tubule
diameter
(
µ
m)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Observations
 
One
male
from
the
High
concentration
showed
Sertoli
cell
proliferation.
One
male
exposed
to
the
Low
concentration
and
one
male
from
the
Medium
concentration
showed
multifocal
Leydig
cell
proliferation.
Three
males
exposed
to
the
High
concentration
were
found
to
have
focal
or
multifocal
Leydig
cell
proliferation.
No
testicular
atrophy
and
no
ovatestes
were
observed
in
males
from
any
treatment.
Several
males
were
observed
to
have
abnormal
testicular
development
(
Table
6.43).

Table
6.43.
Histological
observations
for
males
exposed
to
concentrations
of
fadrozole
during
the
Nonspawning
Adult
14­
D
assay.

Fish
ID
Concentration
Leydig
Cell
Proliferation
Observations
218733
Control
None
Multifocal
basophilic
cystic
structures
with
concentric
laminations
inside
tubules
and
ducts,
up
to
45
µ
m
diameter
218736
Control
None
Transition
from
stage
3b
to
stage
4
results
in
foci
of
nuclear
fragments
and
spermatids
and
mixed
stage
nuclear
fragments
in
tubule
lumina.

Large
numbers
of
Stage
2
b
cells
in
tubules
218737
Control
None
Multifocal
proliferation
of
secondary
spermatogonia
218738
Control
None
Stage
4
ova
in
section
 
probably
an
artifact
218750
Low
None
Tubules
in
stage
5
appeared
to
have
smaller
diameter
than
normal
Multifocal
partial
divisions
of
Stage
3b
and
Stage
4
cells
in
tubule
lumina
Abnormal
developmental
sequence
 
a
high
concentration
Stage
2b
cells
and
very
few
Stage
4
cells
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
272
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Fish
ID
Concentration
Leydig
Cell
Proliferation
Observations
Tubule
lumina
occluded
in
some
cases
218756
Low
Multifocal
Multifocal
proliferation
of
secondary
spermatogonia
218762
Medium
None
Tubules
in
stage
5
appear
to
have
smaller
diameter
than
normal
Proliferation
of
state
2b
and
3a
cells
along
tubules
filled
with
mature
sperm
218763
Medium
None
Large
numbers
of
stage
2b
cells
in
tubules
218764
Medium
None
Tubules
in
stage
5
appeared
to
have
smaller
diameter
than
normal
218765
Medium
None
Cystic
structures
(
concretions)
in
some
tubule
lumina
 
irregular
to
20
µ
m
diameter
218767
Medium
None
Stage
3
ova
in
section
 
probably
an
artifact
218782
High
multifocal
Most
tubules
normal,
but
some
with
multifocal
areas
of
Sertoli
cell
proliferation
and
Leydig
cell
proliferation
Also,
possible
secondary
spermatocyte
proliferation.

218783
High
None
Multifocal
proliferation
of
secondary
spermatogonia
A
few
syncytia
of
Stage
5
cells
A
few
clumps
of
Stage
3b
and
Stage
4
cells
in
tubule
lumina
218784
High
None
Stage
3
ova
in
section
 
probably
an
artifact
218787
High
None
Necrotic
cells
in
tubule
lumina
associated
with
dense
clusters
of
sperm
Multifocal
proliferation
of
secondary
spermatogonia
218788
High
None
Multifocal
proliferation
of
secondary
spermatogonia,
filled
with
Stage
5
cells
Occasional
incomplete
division
of
Stage
3
cells
,
which
appear
necrotic
Abnormal
appearing
arrangement
of
components
of
spermatogenesis
Areas
where
tubule
lumina
occluded
with
apparent
Stage
2b
cells
and
some
necrotic
cells
in
tubule
lumina
218789
High
None
Clumps
of
Stage
5
cells
in
tubule
lumina
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
273
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
6.3.7
Plasma
Steroid
Concentrations
Estradiol
 
Estradiol
concentrations
in
Control­
treatment
females
used
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
ranged
from
0
pg/
mL
(
not
detected)
to
3,502
pg/
mL
(
Figure
6.51).
Among
females
exposed
to
the
three
fadrozole
concentrations,
estradiol
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
1,956
pg/
mL.
A
significant
difference
in
the
mean
estradiol
concentration
per
treatment
(
Table
6.44)
was
detected
(
Kruskal­
Wallis,
H
=
18.33,
p
<
0.001,
df
=
2).
The
mean
estradiol
concentration
in
females
from
the
High
concentration
was
less
than
that
in
females
from
the
Control
treatment.
The
achieved
power
for
this
endpoint
was
54%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
15
(
Table
6.44).

Table
6.44.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
1,519
1,132
75%
53%
15
low
10
727
524
72%
medium
9
320
457
143%
high
10
142
129
90%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
9.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Mid
Low
High
Control
3000
2000
1000
0
treatment
Estradiol
Figure
6.51.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisks
represent
probable
outliers.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
274
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Estradiol
was
detected
only
in
one
male
exposed
to
the
Medium
concentration
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
(
1,814
pg/
mL).
Estradiol
was
not
detected
in
males
from
the
Control
treatment
(
9
individuals)
or
in
those
exposed
to
the
Low
and
High
concentrations
(
10
individuals
per
treatment)
(
Table
6.45).

Table
6.45.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
1
control
9
0
0
 
 
 
low
10
0
0
 
medium
10
181
574
316%
high
10
0
0
 
1
Not
calculated.

Testosterone
 
Testosterone
concentrations
in
Control­
treatment
females
used
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
ranged
from
146
pg/
mL
to
2,053
pg/
mL
(
Figure
6.52).
Among
females
exposed
to
the
three
fadrozole
concentrations,
testosterone
concentrations
ranged
from
394
pg/
mL
to
7,314
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
6.46)
were
detected
(
Kruskal­
Wallis,
H
=
4.61,
p
=
0.203,
df
=
2.
The
achieved
power
for
this
endpoint
was
29%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
26
(
Table
6.46).

Table
6.46.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
9
1,095
712
65%
29%
26
low
10
1,730
822
48%
medium
8
986
407
41%
high
9
2,067
2,084
101%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
8.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
275
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
8000
7000
6000
5000
4000
3000
2000
1000
0
treatment
Testosterone
Figure
6.52.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

Testosterone
concentrations
in
Control
treatment
males
used
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
ranged
from
1,005
pg/
mL
to
2,603
pg/
mL
(
Figure
6.53).
Among
males
exposed
to
the
three
fadrozole
concentrations,
testosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
13,606
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
6.47)
were
detected
(
Kruskal­
Wallis,
H
=
3.28,
p
=
0.350,
df
=
2).
The
achieved
power
for
this
endpoint
was
21%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
41
(
Table
6.47).

Table
6.47.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
9
1,889
493
26%
21%
41
low
10
1,658
805
49%
medium
10
1,413
813
58%
high
10
3,256
3,860
119%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
9.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
276
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
14000
12000
10000
8000
6000
4000
2000
0
treatment
Testosterone
Figure
6.53.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

11­
ketotestosterone
 
11­
ketotesosterone
was
not
detected
in
Control­
treatment
females,
nor
in
females
exposed
to
the
Low
fadrozole
concentration,
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
(
Figure
6.54).
11­
ketotesosterone
was
detected
in
one
of
seven
females
exposed
to
the
Medium
concentration
(
519
pg/
mL).
Among
the
10
females
from
the
High
fadrozole
concentrations,
11­
ketotestosterone
was
detected
in
6,
and
the
concentrations
for
those
ranged
from
329
pg/
mL
to
897
pg/
mL.
Significant
differences
in
the
mean
11­
ketotestosterone
concentration
per
treatment
(
Table
6.48)
were
detected
(
Kruskal­
Wallis,
H
=
11.23,
p
=
0.011,
df
=
2).
11­
ketotestosterone
was
primarily
detected
in
females
from
the
High
concentration,
with
one
female
from
the
Medium
concentration
also
having
a
detectable
level
of
the
compound.
The
achieved
power
for
this
endpoint
was
59%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
8
(
Table
6.48).

Table
6.48.
Summary
statistics
and
power
estimates
for
female
11­
ketotestosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
7
0
0
59%
8
low
5
0
0
medium
7
74
196
264%
high
10
397
378
95%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
5.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
277
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
900
800
700
600
500
400
300
200
100
0
treatment
11­
keto
Figure
6.54.
Boxplot
of
female
11­
ketotestosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

11­
ketotesosterone
concentrations
in
Control
treatment
males
used
during
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
ranged
from
0
pg/
mL
(
not
detected)
to
19,106
pg/
mL
(
Figure
6.55).
Among
males
exposed
to
the
three
fadrozole
concentrations,
11­
ketotesosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
57,374
pg/
mL.
No
significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
6.49)
were
detected
(
Kruskal­
Wallis,
H
=
1.32,
p
=
0.724,
df
=
2).
The
achieved
power
for
this
endpoint
was
9%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
170
(
Table
6.49).

Table
6.49.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
7,585
6,499
86%
9%
170
low
10
7,422
8,541
115%
medium
10
6,101
7,505
123%
high
10
12,535
17,396
139%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
10.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
278
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
60000
50000
40000
30000
20000
10000
0
treatment
11­
keto
Figure
6.55.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

6.3.8
Body
Weight
and
Length
The
body
weight
of
females
used
in
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
ranged
from
1.7
g
to
4.8
g
(
Figure
6.56).
There
were
no
significant
differences
in
mean
body
weight
(
natural
log
transformed)
among
treatments
(
Kruskal­
Wallis,
H
=
1.05,
p
=
0.790,
df
=
3).
The
achieved
power
for
this
endpoint
was
8%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
186
(
Table
6.50).

The
body
(
fork)
length
of
females
used
in
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
ranged
from
46
mm
to
62
mm
(
Figure
6.57).
There
were
no
significant
differences
in
mean
body
weight
(
natural
log
transformed)
among
treatments
(
Kruskal­
Wallis,
H
=
1.96,
p
=
0.581,
df
=
3).
The
achieved
power
for
this
endpoint
was
18%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
53
(
Table
6.51).

Table
6.50.
Summary
statistics
and
power
estimates
for
female
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
2.9
0.8
27%
8%
186
low
10
2.7
0.6
23%
medium
10
2.9
0.7
24%
high
10
2.9
0.5
18%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
10.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
279
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
5
4
3
2
treatment
fish_
wgt_
whole
Figure
6.56.
Boxplot
of
female
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

Table
6.51.
Summary
statistics
and
power
estimates
for
female
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
54.2
3.4
6%
18%
53
low
10
54.3
5.4
10%
medium
10
55.4
4.5
8%
high
10
56.9
2.8
5%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
10.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
280
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
60
55
50
45
treatment
fish_
length_
fork
Figure
6.57.
Boxplot
of
female
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

The
body
weight
of
males
used
in
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
ranged
from
3.9
g
to
8.1
g
(
Figure
6.58).
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
3.33,
p
=
0.343,
df
=
3).
The
achieved
power
for
this
endpoint
was
21%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
46
(
Table
6.52).

The
body
(
fork)
length
of
males
used
in
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay
ranged
from
57
mm
to
80
mm
(
Figure
6.59).
There
were
no
significant
differences
in
mean
body
weight
(
natural
log
transformed)
among
treatments
(
Kruskal­
Wallis,
H
=
5.89,
p
=
0.117,
df
=
3).
The
achieved
power
for
this
endpoint
was
30%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
32
(
Table
6.53).

Table
6.52.
Summary
statistics
and
power
estimates
for
male
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
5.8
1.3
23%
21%
46
low
10
5.5
1.0
19%
medium
10
5.1
0.7
14%
high
10
5.6
0.8
13%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
10.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
281
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
8
7
6
5
4
treatment
fish_
wgt_
whole
Figure
6.58.
Boxplot
of
male
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Table
6.53.
Summary
statistics
and
power
estimates
for
male
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Fadrozole
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
70.8
6.5
9%
30%
32
low
10
70.2
4.5
6%
medium
10
66.4
3.9
6%
high
10
70.3
4.7
7%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
10.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
282
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
80
70
60
treatment
fish_
length_
fork
(
y
)

Figure
6.59.
Boxplot
of
male
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Fadrozole
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.
