Document ID: EPA-HQ-OPPT-2003-0027-0013
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-08-06T04:00Z

DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
152
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
5.0
RESULTS:
FLUTAMIDE
5.1
EPA
14­
Day
Assay
for
Flutamide
The
EPA
14­
Day
Flutamide
exposure
assay
was
conducted
from
February
18,
2003,
to
February
25,
2003
(
pre
exposure
assay),
and
from
February
25,
2003,
to
March
11,
2003
(
exposure
assay).

5.1.1
Survival
All
males
and
females
in
all
treatments
survived
the
EPA
14­
Day
Flutamide
assay.

5.1.2
Vitellogenin
Vitellogenin
concentrations
in
Control
treatment
females
used
during
the
EPA
14­
Day
Flutamide
assay
ranged
from
533,500
ng/
mL
to
6,960,500
ng/
mL
(
Figure
5.1).
Among
females
exposed
to
the
two
flutamide
concentrations,
vitellogenin
concentrations
ranged
from
1,781,000
ng/
mL
to
8,388,000
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
5.1)
were
detected
(
Kruskal­
Wallis,
H
=
3.49,
p
=
0.175,
df
=
2).
The
achieved
power
for
this
endpoint
was
41%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
35
(
Table
5.1).

Table
5.1.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
15
3,077,733
1,532,872
50%
41%
35
low
16
3,489,000
1,686,463
48%
high
15
4,188,333
1,706,450
41%
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.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
153
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
8000000
6000000
4000000
2000000
0
treatment
Flut­
VTG
Figure
5.1.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
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.

Vitellogenin
concentrations
in
Control
treatment
males
used
during
the
EPA
14­
Day
Flutamide
assay
ranged
from
0
ng/
mL
(
not
detected)
to
12,700
ng/
mL
(
Figure
5.2).
Among
most
males
exposed
to
the
two
flutamide
concentrations,
vitellogenin
concentrations
ranged
from
0
ng/
mL
(
not
detected)
to
15,212
ng/
mL.
One
male
exposed
to
the
High
flutamide
concentration
had
a
vitellogenin
concentration
of
353,600
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
5.2)
were
detected
(
Kruskal­
Wallis,
H
=
0.32,
p
=
0.850,
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
5.2).

Table
5.2.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
3,365
4,499
134%
5%
>
1,000
low
8
4,497
5,469
122%
high
8
44,563
124,870
280%
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)
154
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
300000
200000
100000
0
treatment
Flut­
VTG
Figure
5.2.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
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.

5.1.3
Appearance
/
Secondary
Sex
Characteristics
All
of
the
females
used
during
the
EPA
14­
Day
Flutamide
assay
exhibited
typical
female
morphology
(
no
fat
pad,
no
tubercles,
ovipositor
present)
with
two
exceptions.
One
female
from
the
Control
treatment
lacked
an
ovipositor.
One
female
from
the
High
concentration
that
showed
the
dark
vertical
banding
typically
found
in
males.

Most
males
used
during
the
EPA
14­
Day
Flutamide
assay
had
typical
male
morphological
features.
One
male
from
the
High
concentration
lacked
tubercles
and
a
dorsal
fat
pad.
Tubercles
were
present
in
all
other
males.
Fat
pads
were
present
or
pronounced
in
all
other
males
from
all
treatments.
Vertical
banding
was
present
or
pronounced
in
all
males.

5.1.4
Gonadosomatic
Index
The
range
of
GSI
values
calculated
for
females
in
the
Control
treatments
varied
from
1.0
to
19.7
(
Figure
5.3),
whereas
the
GSI
values
for
the
other
treatments
varied
from
3.6
to
21.5
for
females
from
the
High
concentration
and
from
8.1
to
16.3
for
females
from
the
Low
concentration.
The
overall
variability
within
each
treatment
was
low
to
moderate
(
CVs
=
17%
 
40%;
Table
5.3).
There
were
no
significant
differences
in
mean
GSI
values
(
Table
5.3)
among
treatments
(
Kruskal­
Wallis,
H
=
0.14,
p
=
0.933,
df
=
2).
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
442
(
Table
5.3).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
155
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.3.
Table
E14FL
GSI­
1.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
16
11.4
4.4
38%
8%
442
low
16
11.3
1.9
17%
high
16
12.2
4.9
40%
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
20
10
0
treatment
GSI
Figure
5.3.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
14­
Day
Flutamide
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
Flutamide
assay,
was
small,
ranging
from
0.4
to
2.1
(
Figure
5.4),
which
approximates
the
typical
range
for
reproductively­
active
male
fathead
minnows.
The
highest
and
lowest
male
GSI
values
were
2.1
(
one
fish
in
the
High
concentration)
and
0.4
(
two
fish
in
the
Control
treatment),
respectively.
Variability
was
moderate
(
CVs
=
25%
 
48%).
There
were
no
significant
differences
in
mean
GSI
values
(
Table
5.4)
among
treatments
(
Kruskal­
Wallis,
H
=
3.44,
p
=
0.180,
df
=
2).
The
achieved
power
for
this
endpoint
was
12%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
84
(
Table
5.4).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
156
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.4.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
1.1
0.5
48%
12%
84
low
8
0.8
0.2
25%
high
8
1.2
0.5
40%
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
2.0
1.5
1.0
0.5
treatment
GSI
Figure
5.4.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
14­
Day
Flutamide
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.

5.1.5
Female
Gonad
Histology
Histological
analyses
were
conducted
on
the
ovaries
of
47
females
exposed
to
flutamide
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
Flutamide
assay
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
4.73,
p
=
0.094,
df
=
2).

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
High
concentration
ranged
from
Stage
1a
to
Stage
5
(
see
Methods
for
a
description
of
the
stages),
whereas
ovaries
in
females
from
the
Low­
concentration
treatment
showed
Stage
1a
to
Stage
4
development
(
Figure
5.5).
Variability
within
treatments
for
each
stage
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
303%
(
Table
5.5).
Statistical
analyses
showed
that
there
were
no
significant
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
157
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
difference
among
treatments
in
the
proportion
of
cells
in
each
of
the
five
developmental
stages
(
Table
5.5).
Therefore,
there
was
no
effect
on
ovarian
developmental
stage
associated
with
flutamide
dose.

Table
5.5.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
EPA
14­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.

Control
(
n
=
15)
Low
(
n
=
16)
High
(
n
=
16)
Kruskal­
Wallis
Stage
Mean
Stdev
CV
Mean
Stdev
CV
Value
Stdev
CV
H
p
1a
0.079
0.030
38%
0.094
0.037
39%
0.084
0.031
37%
0.95
0.621
1b
0.332
0.082
25%
0.305
0.040
13%
0.328
0.127
39%
2.39
0.302
2
0.174
0.039
23%
0.178
0.040
23%
0.158
0.071
45%
3.12
0.210
3
0.190
0.057
30%
0.188
0.044
23%
0.158
0.074
47%
2.19
0.335
4
0.193
0.090
47%
0.223
0.085
38%
0.215
0.116
54%
1.04
0.594
5
0.022
0.064
284%
0
0
 
0.033
0.100
303%
3.14
0.208
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
1a
1b
2
3
4
5
Ovarian
Stage
Proportion
Control
Low
High
Figure
5.5.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
14­
Day
Flutamide
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
0.0004
follicles
for
females
in
the
Control
treatment
to
0.015
follicles
for
females
in
the
High
concentration
(
Figure
5.6).
There
was
a
significant
difference
in
the
mean
proportions
of
atretic
follicles
among
treatments
(
Kruskal­
Wallis,
H
=
12.88,
p
=
0.002,
df
=
2).
The
proportion
of
atretic
follicles
was
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
158
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
greater
for
females
from
the
High
concentration
than
from
the
Control
treatment
or
the
Low
concentration.

Low
High
Control
0.10
0.05
0.00
Treatment
atretic_
follicles
(
y
)

Figure
5.6.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Flutamide
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
0.009
for
females
in
the
High
concentration
to
0.010
for
females
in
the
Low
concentration
(
Figure
5.7).
There
were
no
significant
differences
in
the
mean
proportion
of
corpora
lutea
among
treatments
(
Kruskal­
Wallis,
H
=
0.56,
p
=
0.757,
df
=
2).
The
value
for
the
High
concentration
was
significantly
lower
than
those
of
the
other
two
treatments.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
159
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
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
5.7.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Flutamide
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
Control
treatment
had
only
eight
ova
present
in
the
three
ovarian
section
examined.
This
fish
was
not
included
in
the
data
analyses.
Another
fish
in
the
Control
treatment
was
observed
to
have
some
cortical
alveolus
stage
ova
(
Stage
2)
that
appeared
abnormal
because
they
had
a
very
large
yolk
body
next
to
the
nucleus.
Three
fish
exposed
to
the
High
flutamide
concentration
had
very
few
ova
or
very
few
advanced
stage
ova
in
the
ovaries.

5.1.6
Male
Gonad
Histology
General
Testes
Staging
 
Testes
from
24
males
exposed
to
flutamide
during
the
EPA
14­
Day
Flutamide
assay
were
examined
to
determine
the
general
developmental
condition.
Males
in
all
treatments
had
welldeveloped
testes
with
all
showing
Stage
4
and
Stage
5
development
(
see
Methods
for
description
of
developmental
stages).
All
of
the
98
microscopic
fields
examined
in
the
8
Control
treatment
males
showed
Stage
4
(
86
fields)
or
Stage
5
(
12
fields)
development.
All
of
the
96
microscopic
fields
examined
in
the
8
Low­
concentration
treatment
males
showed
Stage
4
(
90
fields)
or
Stage
5
(
6
fields)
development.
All
of
the
96
microscopic
fields
examined
in
the
8
High­
concentration
treatment
males
showed
Stage
4
(
81
fields)
or
Stage
5
(
15
fields)
development.
Statistical
analysis
of
the
mean
staging
from
12
microscopic
fields
per
fish
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
0.70,
p
=
0.703,
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
5.8).
Variability
within
treatments
for
each
stage
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
170%
(
Table
5.6).
Although
statistical
analyses
showed
that
there
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
any
developmental
Stage,
the
probability
value
calculated
for
Stage
2a
was
slightly
above
the
critical
limit
of
0.050
(
Table
5.6).
Therefore,
there
was
no
effect
on
testicular
developmental
stage
associated
with
flutamide
dose.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
160
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.6.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
EPA
14­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.

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
0.002
0.004
170%
0.006
0.005
83%
0.008
0.006
74%
5.82
0.054
2b
0.025
0.026
105%
0.015
0.013
85%
0.017
0.014
83%
0.43
0.805
3a
0.113
0.056
50%
0.135
0.058
43%
0.152
0.077
51%
1.86
0.394
3b
0.276
0.123
45%
0.287
0.076
26%
0.251
0.102
40%
0.59
0.746
4
0.174
0.079
45%
0.170
0.029
17%
0.124
0.068
55%
3.39
0.184
5
0.410
0.179
44%
0.386
0.085
22%
0.448
0.216
48%
0.23
0.893
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1
2a
2b
3a
3b
4
5
Testicular
Stage
Proportion
of
Cells
Control
Low
High
Figure
5.8.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
14­
Day
Flutamide
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
66.1
µ
m
to
161.9
µ
m
(
Figure
5.9).
Tubule
diameters
of
males
from
the
two
test
concentrations
ranged
from
92.2
µ
m
to
164.2
µ
m.
No
significant
differences
in
the
mean
tubule
diameter
per
treatment
(
Table
5.7)
were
detected
(
Kruskal­
Wallis,
H
=
2.42,
p
=
0.297,
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
152
(
Table
5.7).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
161
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.7.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
118.6
29.0
24%
9%
152
low
8
108.4
10.8
10%
high
8
125.1
22.2
18%
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
160
110
60
Treatment
diameter
Figure
5.9.
Boxplot
of
seminiferous
tubule
diameter
(
µ
m)
by
treatment
for
the
EPA
14­
Day
Flutamide
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
 
No
Sertoli
cell
proliferation
was
observed.
One
male
from
the
Control
treatment
was
observed
to
have
Leydig
cell
proliferation.
The
same
male
was
also
noted
to
have
disorganized
development
of
the
seminiferous
tubules
with
early
developmental
stages
shed
prematurely
into
tubule
lumina.
No
testicular
atrophy
was
recorded
and
no
ovatestes
were
observed
for
any
treatment.

5.1.7
Plasma
Steroid
Concentrations
Estradiol
 
Estradiol
concentrations
in
Control­
treatment
females
used
during
the
EPA
14­
Day
Flutamide
assay
ranged
from
0
pg/
mL
(
not
detected)
to
4,453
pg/
mL
(
Figure
5.10).
Among
females
exposed
to
the
two
flutamide
concentrations,
estradiol
concentrations
ranged
from
282
pg/
mL
to
5,837
pg/
mL.
No
significant
differences
in
the
mean
estradiol
concentration
per
treatment
(
Table
5.8)
were
detected
(
Kruskal­
Wallis,
H
=
2.02,
p
=
0.365,
df
=
2).
The
achieved
power
for
this
endpoint
was
13%,
and
the
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
162
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
115
(
Table
5.8).

Table
5.8.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
12
2,241
1,338
60%
13%
115
low
15
2,351
1,294
55%
high
15
1,693
1,221
72%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
12.
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
5.10.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
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.

Estradiol
concentrations
in
Control
treatment
males
used
during
the
EPA
14­
Day
Flutamide
assay
ranged
from
210
pg/
mL
to
540
pg/
mL
(
Figure
5.11).
Among
males
exposed
to
the
two
flutamide
concentrations,
estradiol
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
992
pg/
mL.
A
significant
difference
in
the
mean
estradiol
concentration
per
treatment
(
Table
5.9)
was
detected
(
Kruskal­
Wallis,
H
=
8.80,
p
=
0.012,
df
=
2).
The
mean
concentration
of
estradiol
in
males
from
the
High
concentration
was
less
than
that
in
males
from
the
Low
concentration.
The
achieved
power
for
this
endpoint
was
22%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
31
(
Table
5.9).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
163
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.9.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
7
302
110
36%
22%
31
low
8
400
245
61%
high
7
200
97
48%
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
High
Control
1000
500
0
treatment
Estradiol
Figure
5.11.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
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
 
Testosterone
concentrations
in
Control­
treatment
females
used
during
the
EPA
14­
Day
Flutamide
assay
ranged
from
0
pg/
mL
(
not
detected)
to
2,989
pg/
mL
(
Figure
5.12).
Among
females
exposed
to
the
two
flutamide
concentrations,
testosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
4,874
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
5.10)
were
detected
(
Kruskal­
Wallis,
H
=
2.59,
p
=
0.274,
df
=
2).
The
achieved
power
for
this
endpoint
was
7%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
271
(
Table
5.10).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
164
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.10.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
11
1,227
851
69%
7%
271
low
9
1,298
791
61%
high
10
2,613
1,884
72%
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.

Low
High
Control
5000
4000
3000
2000
1000
0
treatment
Testosterone
Figure
5.12.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
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.

Testosterone
concentrations
in
Control
treatment
males
used
during
the
EPA
14­
Day
Flutamide
assay
ranged
from
949
pg/
mL
to
6,258
pg/
mL
(
Figure
5.13).
Among
males
exposed
to
the
two
flutamide
concentrations,
testosterone
concentrations
ranged
from
1,161
pg/
mL
to
5,529
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
5.11)
were
detected
(
Kruskal­
Wallis,
H
=
0.25,
p
=
0.883,
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
760
(
Table
5.11).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
165
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.11.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
7
2,853
2,159
76%
6%
760
low
8
2,552
1,173
46%
high
7
2,715
1,357
50%
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
High
Control
6000
5000
4000
3000
2000
1000
treatment
Testosterone
Figure
5.13.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
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
females
from
the
Control
treatment
(
five
individuals),
the
Low
concentration
(
six
individuals),
and
the
High
concentration
(
six
individuals)
exposed
during
the
EPA
14­
Day
Flutamide
assay.

11­
ketotesosterone
concentrations
in
Control
treatment
males
used
during
the
EPA
14­
Day
Flutamide
assay
ranged
from
5,287
pg/
mL
to
63,011
pg/
mL
(
Figure
5.14).
Among
males
exposed
to
the
two
flutamide
concentrations,
11­
ketotesosterone
concentrations
ranged
from
3,942
pg/
mL
to
48,001
pg/
mL.
No
significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
5.12)
were
detected
(
Kruskal­
Wallis,
H
=
0.37,
p
=
0.830,
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
356
(
Table
5.12).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
166
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.12.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
7
24,269
24,145
99%
6%
356
low
8
23,583
12,787
54%
high
7
21,815
13,712
63%
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
High
Control
60000
50000
40000
30000
20000
10000
0
treatment
11­
keto
Figure
5.14.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Flutamide
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.

5.1.8
Fecundity
Total
Fecundity
 
Variability
among
treatments
in
the
total
number
of
eggs
produced
per
replicate
during
the
EPA
14­
Day
Flutamide
assay
was
very
high
(
Figure
5.15).
Total
counts
in
the
Control
treatment
ranged
from
2,758
eggs
to
4,346
eggs.
Total
counts
for
the
High
flutamide
concentration
treatment
ranged
from
1,004
eggs
to
2,687
eggs.
No
eggs
were
laid
after
Day
10
in
one
High
concentration
replicate
or
after
Day
12
in
another
replicate.
The
fecundity
among
replicates
in
the
Low
flutamide
concentration
ranged
from
3,448
eggs
to
4,876
eggs.
A
significant
difference
in
the
mean
number
of
eggs
(
square­
root
transformed
)
produced
per
treatment
(
Table
5.13)
was
detected
(
Kruskal­
Wallis,
H
=
8.77,
p
=
0.012,
df
=
2).
The
total
fecundity
of
females
in
the
High­
concentration
treatment
was
significantly
less
than
it
was
for
females
in
the
Low
concentration
or
the
Control
treatment.
The
achieved
power
for
this
assay
was
79%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
5
(
Table
5.13).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
167
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.13.
Summary
statistics
and
power
estimates
for
fecundity
data
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
4
3539
684
19%
79%
5
low
4
4501
702
16%
high
4
1690
820
48%
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
500
1,000
1,500
2,000
2,500
3,000
3,500
4,000
4,500
5,000
0
2
4
6
8
10
12
14
Day
Cumulative
Number
of
Eggs
Control
Low
High
Figure
5.15.
Total
egg
production
by
replicate
per
treatment
for
the
EPA
14­
Day
Flutamide
assay.

Fecundity
per
Female
Reproductive
Day
 
During
the
EPA
14­
Day
Flutamide
assay,
the
maximum
number
of
female
reproductive
days
was
achieved
for
all
treatments
as
no
fish
died
during
the
assay
(
Table
5.14).
The
number
of
eggs
produced
per
female
reproductive
day
in
the
Control
treatment
varied
from
49.3
eggs
to
77.6
eggs
and
from
61.6
eggs
to
87.1
eggs
in
the
Low
concentration
(
Figure
5.16).
For
the
High
concentration,
the
number
of
eggs
produced
per
female
reproductive
day
ranged
from
17.9
eggs
to
48.0
eggs,
with
fish
in
two
of
the
replicates
producing
fewer
than
20
eggs
per
day
(
17.9,
18.5).
Because
no
fish
died
during
the
assay,
the
statistical
results
reported
here
are
the
same
as
those
reported
for
total
fecundity.
A
significant
difference
in
the
mean
number
of
eggs
laid
per
day
per
treatment
(
Table
5.14)
occurred.
The
number
of
eggs
laid
per
day
in
the
High­
concentration
treatment
was
significantly
less
than
it
was
for
females
in
the
Low
concentration
or
the
Control
treatment.
The
achieved
power
for
this
assay
was
77%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
5
(
Table
5.14).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
168
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.14.
Summary
statistics
and
power
estimates
for
fecundity
per
female
reproductive
day
for
the
EPA
14­
Day
Flutamide
assay.

Level
Mean
Number
of
Reproductive
Days
1
N
Mean
Stdev
CV
Achieved
Power
2
Sample
Size
Required
3
control
56
4
63.2
12.2
19%
77%
5
low
56
4
80.4
12.5
16%
high
56
4
30.2
14.6
48%
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
90
80
70
60
50
40
30
20
treatment
eggs/
ReproDay
Figure
5.16.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
14­
Day
Flutamide
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
Flutamide
assay
varied
from
1,518
eggs
for
the
High
concentration
to
4,148
eggs
for
the
Low
concentration
(
Appendix
E,
Table
3.3).
The
number
of
eggs
on
dishes
ranged
from
172
eggs
for
the
High
concentration
to
353
eggs
for
the
Low
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.
The
proportional
difference
ranged
from
0.78
(
one
High
concentration
replicate)
to
0.96
(
one
Control­
treatment
replicate)
(
Appendix
E,
Figure
3.2).
There
were
no
significant
differences
in
this
mean
proportional
difference
among
treatments
(
Kruskal­
Wallis,
H
=
1.04,
p
=
0.595,
df
=
2).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
169
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
5.1.9
Fertilization
Success
Total
Fertilization
 
The
total
(
tiles
+
dishes)
fertilization­
success
rates
for
all
treatment
replicates
during
the
EPA
14­
Day
Flutamide
assay
were
high,
ranging
from
0.993
(
one
High­
concentration
replicate)
to
1.00
(
two
High­
concentration
replicates,
three
Control­
treatment
replicates)
(
Figure
5.17).
No
significant
differences
in
mean
fertilization­
success
rates
(
Table
5.15)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
1.53,
p
=
0.465,
df
=
2).
The
achieved
power
for
this
assay
was
17%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
22
(
Table
5.15).

Table
5.15.
Summary
statistics
and
power
estimates
for
the
proportion
of
eggs
fertilized
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
4
1.000
0.001
0.1%
17%
22
low
4
1.000
0.000
0.0%
high
4
0.997
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.

Low
High
Control
1.000
0.999
0.998
0.997
0.996
0.995
0.994
0.993
treatment
Total
Prop­
fert
Figure
5.17.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
14­
Day
Flutamide
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
most
treatment
replicates
for
eggs
laid
on
tiles
during
the
EPA
14­
Day
Flutamide
assay
were
high,
ranging
from
0.992
(
one
High
concentration
replicate)
to
1.00
(
two
High
concentration
replicates,
three
Control­
treatment
replicates)
(
Appendix
E,
Figure
3.3).
No
significant
differences
in
mean
fertilization­
success
rates
(
Appendix
E,
Table
3.4)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
1.53,
p
=
0.465,
df
=
2).
The
fertilization­
success
rates
for
all
treatment
replicates
for
eggs
laid
on
dishes
during
the
assay
were
very
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
170
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
high,
ranging
from
0.997
(
one
Control­
treatment
replicate)
to
1.00
(
all
remaining
replicates
from
all
treatments)
(
Appendix
E,
Figure
3.4).
No
significant
differences
in
mean
fertilization­
success
rates
(
Appendix
E,
Table
3.4)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
2.00,
p
=
0.368,
df
=
2).

5.1.10
Hatchability
and
Larval
Development
Eggs
were
collected
during
the
14­
day
pre­
exposure
period
for
the
evaluation
of
hatchability.
The
mean
proportion
of
fertilized
eggs
that
hatched
in
the
Control
treatment
was
0.96
(
sd
=
0.05),
0.94
(
sd
=
0.03)
in
the
Low
concentration,
and
0.98
(
sd
=
0.02)
in
the
High
concentration.
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
2.20,
p
=
0.333,
df
=
2).

Eggs
were
collected
during
the
EPA
14­
Day
Flutamide
assay
for
the
evaluation
of
hatchability.
The
proportion
of
fertilized
eggs
that
hatched
ranged
from
0.94
to
1.00
in
the
Control
treatment
and
from
0.80
to
1.00
for
the
two
test
concentrations
(
Figure
5.18).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
2.99,
p
=
0.224,
df
=
2).
The
achieved
power
for
this
endpoint
was
20%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
18
(
Table
5.16).

Table
5.16.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
4
0.98
0.03
3%
20%
18
low
5
0.97
0.04
4%
high
5
0.91
0.08
9%
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
1.0
0.9
0.8
treatment
Prop­
Hatch
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
171
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
5.18.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
14­
Day
Flutamide
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
1.00
(
sd
=
0.01).
The
mean
proportion
of
normal
larvae
in
the
remaining
treatments
was
0.97
(
sd
=
0.02)
in
the
Low
concentration
and
0.98
(
sd
=
0.02)
in
the
High
concentration.
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
3.46,
p
=
0.178,
df
=
2).

Eggs
were
collected
during
the
EPA
14­
Day
Flutamide
assay
for
the
evaluation
of
larval
development.
The
proportion
of
larvae
that
developed
normally
(
i.
e.,
that
showed
no
morphological
abnormalities)
ranged
from
0.96
to
1.00
for
the
Control
treatment
and
ranged
from
0.95
to
1.00
for
the
two
test
concentrations
(
Figure
5.19).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
3.38,
p
=
0.185,
df
=
2).
The
achieved
power
for
this
endpoint
was
12%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
34
(
Table
5.17).

Table
5.17.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
4
0.98
0.02
2%
12%
34
low
5
0.99
0.01
1%
high
5
0.97
0.02
2%
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
1.00
0.99
0.98
0.97
0.96
0.95
treatment
Prop­
Norm
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
172
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Figure
5.19.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
14­
Day
Flutamide
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.

5.1.11
Body
Weight
The
body
weight
of
females
used
in
the
EPA
14­
Day
Flutamide
assay
ranged
from
0.9
g
to
3.1
g.
There
were
no
significant
differences
in
mean
body
weight
among
treatments,
although
the
probability
was
only
slighter
greater
than
the
critical
limit
of
0.05
(
Kruskal­
Wallis,
H
=
5.72,
p
=
0.057,
df
=
2).
Females
from
the
Control
treatment
tended
to
be
larger
than
those
form
the
other
two
treatments.
The
body
weight
of
males
used
in
the
EPA
14­
Day
Flutamide
assay
ranged
from
2.8
g
to
5.6
g.
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
2.72,
p
=
0.257,
df
=
2).

5.2
EPA
21­
Day
Assay
for
Flutamide
The
EPA
21­
Day
Flutamide
assay
was
conducted
from
February
11,
2003,
to
February
25,
2003
(
preexposure
assay),
and
from
February
25,
2003,
to
March
18,
2003
(
exposure
assay).

5.2.1
Survival
All
males
and
females
in
all
treatments
survived
the
EPA
21­
Day
Flutamide
assay.

5.2.2
Vitellogenin
Vitellogenin
concentrations
in
Control
treatment
females
used
during
the
EPA
21­
Day
Flutamide
assay
ranged
from
252,800
ng/
mL
to
9,071,500
ng/
mL
(
Figure
5.20).
Among
females
exposed
to
the
two
flutamide
concentrations,
vitellogenin
concentrations
ranged
from
3,006,000
ng/
mL
to
9,830,000
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
5.18)
were
detected
(
Kruskal­
Wallis,
H
=
5.08,
p
=
0.079,
df
=
2).
The
achieved
power
for
this
endpoint
was
44%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
31
(
Table
5.18).

Table
5.18.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
16
4,917,456
2,135,773
43%
44%
31
low
15
6,182,160
1,860,122
30%
high
14
6,483,000
1,538,230
24%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
14.
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)
173
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
10000000
5000000
0
treatment
Flut­
VTG
(
y
)

Figure
5.20.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
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
Flutamide
assay
ranged
from
51
ng/
mL
to
13,485
ng/
mL
(
Figure
5.21).
Among
males
exposed
to
the
two
flutamide
concentrations,
vitellogenin
concentrations
ranged
from
0
ng/
mL
(
not
detected)
to
35,355
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
5.19)
were
detected
(
Kruskal­
Wallis,
H
=
2.95,
p
=
0.229,
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
70
(
Table
5.19).

Table
5.19.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
2,418
4,547
188%
13%
70
low
8
9,681
13,513
140%
high
8
12,497
14,887
119%
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)
174
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
30000
20000
10000
0
treatment
Flut­
VTG
Figure
5.21.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
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,
ant
the
asterisk
represents
a
probable
outlier.

5.2.3
Appearance
/
Secondary
Sex
Characteristics
All
of
the
females
used
during
the
EPA
21­
Day
Flutamide
assay
exhibited
typical
female
morphology
(
no
fat
pad,
no
tubercles,
ovipositor
present)
except
that
one
female
from
the
High
concentration
that
showed
the
dark
vertical
banding
typically
found
in
males.

Most
males
during
the
EPA
21­
Day
Flutamide
assay
had
typical
male
morphological
features.
One
male
from
the
Low
concentration
lacked
tubercles
and
a
dorsal
fat
pad.
Tubercles
were
present
in
all
other
males.
Fat
pads
were
present
or
pronounced
in
all
other
males
from
all
treatments.
Vertical
banding
was
present
or
pronounced
in
all
males.

5.2.4
Gonadosomatic
Index
The
range
of
GSI
values
calculated
for
females
in
the
Control
treatment
varied
from
2.1
to
14.3
(
Figure
5.22),
whereas
the
GSI
values
for
the
other
treatments
varied
from
8.2
to
15.7.
The
overall
variability
within
the
treatment
was
moderate
(
CVs
=
18%
 
35%;
Table
5.20).
No
significant
differences
in
the
mean
GSI
value
per
treatment
(
Table
5.20)
were
detected
(
Kruskal­
Wallis,
H
=
3.00,
p
=
0.223,
df
=
2).
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
39
(
Table
5.20).

Table
5.20.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
21­
Day
Flutamide
assay.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
175
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
16
10.0
3.5
35%
40%
39
low
16
11.8
2.2
19%
high
16
10.7
2.0
18%
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
15
10
5
treatment
GSI
Figure
5.22.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
21­
Day
Flutamide
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
range
of
most
GSI
values
calculated
for
males
during
the
EPA
21­
Day
Flutamide
assay,
was
small,
ranging
from
0.6
to
1.7
(
Figure
5.23),
which
approximates
the
typical
range
for
reproductively­
active
male
fathead
minnows.
There
were
no
significant
differences
in
mean
GSI
values
(
Table
5.21)
among
treatments
(
Kruskal­
Wallis,
H
=
1.06,
p
=
0.590,
df
=
2).
The
achieved
power
for
this
endpoint
was
17%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
48
(
Table
5.21).

Table
5.21.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
21­
Day
Flutamide
assay.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
176
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
1.0
0.3
29%
17%
48
low
8
1.1
0.3
31%
high
8
1.2
0.3
26%
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
1.5
1.0
0.5
treatment
GSI
Figure
5.23.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
21­
Day
Flutamide
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.

5.2.5
Female
Gonad
Histology
Histological
analyses
were
conducted
on
the
ovaries
of
48
females
exposed
to
flutamide
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
Flutamide
assay
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
2.10,
p
=
0.350,
df
=
2).

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
in
all
treatments
ranged
from
Stage
1a
to
Stage
5
(
see
Methods
for
a
description
of
the
stages
(
Figure
5.24).
Variability
within
treatments
for
each
stage,
particularly
for
Stage
5,
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
400%
(
Table
5.22).
Statistical
analyses
showed
that
within
each
of
the
five
developmental
stages
there
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
occurring
in
the
stage
(
Table
5.22).
Therefore,
flutamide
dose
had
no
effect
on
ovarian
developmental
stage.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
177
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.22.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
EPA
21­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.

Control
(
n
=
16)
Low
(
n
=
16)
High
(
n
=
16)
Kruskal­
Wallis
Stage
Mean
Stdev
CV
Mean
Stdev
CV
Value
Stdev
CV
H
p
1a
0.079
0.028
35%
0.082
0.040
49%
0.080
0.035
44%
0.21
0.900
1b
0.303
0.083
27%
0.270
0.078
29%
0.270
0.057
21%
1.70
0.427
2
0.182
0.027
15%
0.204
0.051
25%
0.193
0.046
24%
0.68
0.714
3
0.193
0.037
19%
0.204
0.053
26%
0.184
0.075
40%
1.88
0.391
4
0.197
0.105
53%
0.218
0.119
55%
0.206
0.110
53%
0.65
0.722
5
0.015
0.043
294%
0.010
0.038
400%
0.014
0.034
246%
1.89
0.389
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
1a
1b
2
3
4
5
Ovarian
Stage
Proportion
Control
Low
High
Figure
5.24.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
21­
Day
Flutamide
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
0.002
follicles
for
females
in
the
Low
concentration
to
0.019
follicles
for
females
in
the
Control
treatment
(
Figure
5.25).
One
fish
from
the
Control
treatment
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
=
9.92,
p
=
0.007,
df
=
2).
The
majority
of
the
observations
from
the
High
concentration
ranked
higher
than
those
of
the
remaining
treatments.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
178
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
5.25.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Flutamide
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
0.011
for
females
in
the
Low
concentration
to
0.040
for
females
in
the
High
concentration
(
Figure
5.26).
Variability
within
each
treatment
was
very
high,
ranging
from
150%
for
the
High
concentration
to
236%
for
the
Low
concentration.
There
were
no
significant
differences
in
the
mean
proportion
of
corpora
lutea
among
treatments
(
Kruskal­
Wallis,
H
=
3.90,
p
=
0.142,
df
=
2).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
179
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
0.2
0.1
0.0
Treatment
corpora_
lutea
Figure
5.26.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Flutamide
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
 
Two
fish
from
the
Control
treatment
were
observed
to
have
very
few
eggs
in
the
ovaries.
Two
fish
from
the
Low
concentration
were
observed
to
have
very
few
eggs
in
the
ovaries.
One
of
these
two
fish
was
also
noted
to
have
very
small
yolk
particles
in
some
cortical
alveoli
cells.
One
fish
from
the
High
concentration
were
observed
to
have
very
few
eggs
in
the
ovaries.
One
other
fish
from
the
High
concentration
had
extensive
epithelial
and
ovarian
debris
in
the
ovary
and
one
had
debris
from
disintegrated
vitellogenic
ova
in
the
ovary.

5.2.6
Male
Gonad
Histology
General
Testes
Staging
 
Testes
from
24
males
exposed
to
flutamide
during
the
EPA
21­
Day
Flutamide
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
(
76
fields)
or
Stage
5
(
20
fields)
development.
Ninety­
four
of
the
96
microscopic
fields
examined
in
the
8
Low­
concentration
treatment
males
showed
Stage
4
(
87
fields)
or
Stage
5
(
7
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
(
68
fields)
or
Stage
5
(
28
fields)
development.
Statistical
analysis
of
the
mean
staging
from
12
microscopic
fields
per
fish
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
4.21,
p
=
0.122,
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
5.27).
Variability
within
treatments
for
each
stage
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
129%
(
Table
5.23).
Although
statistical
analyses
showed
that
there
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
any
of
the
developmental
stages,
the
probability
value
calculated
for
Stage
3b
was
slightly
above
the
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
180
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
critical
limit
of
0.050
(
Table
5.23).
The
largest
difference
in
the
mean
proportion
of
cells
in
this
developmental
stage
was
between
the
Low­
and
High­
concentration
treatments.
Therefore,
there
was
no
effect
on
testicular
developmental
stage
associated
with
flutamide
dose.

Table
5.23.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
EPA
21­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.

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
0.003
0.004
129%
0.003
0.003
93%
0.006
0.005
79%
2.21
0.331
2b
0.009
0.009
101%
0.012
0.013
112%
0.010
0.009
96%
0.04
0.980
3a
0.123
0.096
78%
0.094
0.048
51%
0.128
0.081
64%
0.60
0.740
3b
0.205
0.118
57%
0.325
0.102
31%
0.211
0.064
30%
5.95
0.051
4
0.118
0.073
62%
0.152
0.086
56%
0.151
0.064
42%
0.60
0.740
5
0.543
0.271
50%
0.415
0.132
32%
0.495
0.168
34%
1.03
0.598
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
2a
2b
3a
3b
4
5
Testicular
Stage
Proportion
of
Cells
Control
Low
High
Figure
5.27.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
21­
Day
Flutamide
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
112.5
µ
m
to
165.6
µ
m
(
Figure
5.28).
Tubule
diameters
of
males
from
the
two
test
concentrations
ranged
from
96.7
µ
m
to
148.9
µ
m.
No
significant
differences
in
the
mean
tubule
diameter
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
181
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
per
treatment
(
Table
5.24)
were
detected
(
Kruskal­
Wallis,
H
=
5.57,
p
=
0.062,
df
=
2).
The
achieved
power
for
this
endpoint
was
63%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
12
(
Table
5.24).

Table
5.24.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
(
µ
m)
data
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
137.4
18.3
13%
63%
12
low
8
116.0
16.5
14%
high
8
132.0
11.7
9%
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
170
160
150
140
130
120
110
100
90
Treatment
diameter
Figure
5.28.
Boxplot
of
male
seminiferous
tubule
diameter
(
µ
m)
by
treatment
for
the
EPA
21­
Day
Flutamide
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.

Observations
 
One
male
in
the
High­
concentration
treatment
showed
multifocal
proliferation
of
Sertoli
cells
and
Leydig
cells.
One
male
from
the
Control
treatment
and
two
males
from
the
Low
concentration
showed
focal
proliferation
of
Leydig
cells.
No
testicular
atrophy
was
recorded
and
no
ovatestes
were
observed
for
any
treatment.

5.2.7
Plasma
Steroid
Concentrations
Estradiol
 
Estradiol
concentrations
in
Control­
treatment
females
used
during
the
EPA
21­
Day
Flutamide
assay
ranged
from
0
pg/
mL
(
not
detected)
to
3,854
pg/
mL
(
Figure
5.29).
Among
females
exposed
to
the
two
flutamide
concentrations,
estradiol
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
182
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
12,367
pg/
mL.
No
significant
differences
in
the
mean
estradiol
concentration
per
treatment
(
Table
5.25)
were
detected
(
Kruskal­
Wallis,
H
=
3.12,
p
=
0.210,
df
=
2).
The
achieved
power
for
this
endpoint
was
22%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
65
(
Table
5.25).

Table
5.25.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
15
1,225
1,260
103%
22%
65
low
14
1,570
1,172
75%
high
16
2,477
3,074
124%
1
Calculated
from
natural
log
transformed
data;
with
sample
size
=
14.
2
To
detect
a
significant
difference
from
control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
10000
5000
0
treatment
Estradiol
Figure
5.29.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
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
concentrations
in
Control
treatment
males
used
during
the
EPA
21­
Day
Flutamide
assay
ranged
from
0
pg/
mL
(
not
detected)
to
298
pg/
mL
(
Figure
5.30).
Among
males
exposed
to
the
two
flutamide
concentrations,
estradiol
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
467
pg/
mL.
Significant
differences
in
the
mean
estradiol
concentration
per
treatment
(
Table
5.26)
were
detected
(
Kruskal­
Wallis,
H
=
9.63,
p
=
0.008,
df
=
2).
The
mean
concentration
of
estradiol
in
males
from
the
High
concentration
was
less
than
those
of
males
from
the
Low
concentration
and
the
Control
treatment.
The
achieved
power
for
this
endpoint
was
79%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
8
(
Table
5.26).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
183
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.26.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
197
87
44%
79%
8
low
7
301
86
28%
high
8
68
125
185%
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
High
Control
500
400
300
200
100
0
treatment
Estradiol
Figure
5.30.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
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
 
Testosterone
concentrations
in
Control­
treatment
females
used
during
the
EPA
21­
Day
Flutamide
assay
ranged
from
0
pg/
mL
(
not
detected)
to
3,475
pg/
mL
(
Figure
5.31).
Among
females
exposed
to
the
two
flutamide
concentrations,
testosterone
concentrations
ranged
from
340
pg/
mL
to
4,612
pg/
mL.
Significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
5.27)
were
detected
(
Kruskal­
Wallis,
H
=
6.68,
p
=
0.035,
df
=
2).
The
mean
testosterone
concentration
in
females
from
the
High
concentration
was
greater
than
that
of
females
from
the
Low
concentration
and
the
Control
treatment.
The
achieved
power
for
this
endpoint
was
45%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
21
(
Table
5.27).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
184
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.27.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
1,329
1,062
80%
45%
21
low
11
1,430
886
62%
high
12
2,692
1,420
53%
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.

Low
High
Control
5000
4000
3000
2000
1000
0
treatment
Testosterone
Figure
5.31.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
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.

Testosterone
concentrations
in
Control
treatment
males
used
during
the
EPA
21­
Day
Flutamide
assay
ranged
from
1,044
pg/
mL
to
4,437
pg/
mL
(
Figure
5.32).
Among
males
exposed
to
the
two
flutamide
concentrations,
testosterone
concentrations
ranged
from
390
pg/
mL
to
6,131
pg/
mL.
A
significant
difference
in
the
mean
testosterone
concentration
per
treatment
(
Table
5.28)
was
detected
(
Kruskal­
Wallis,
H
=
7.26,
p
=
0.027,
df
=
2).
The
mean
testosterone
concentrations
in
males
from
the
Low
concentration
and
the
Control
treatment
was
less
than
that
of
males
from
the
High
concentration.
The
achieved
power
for
this
endpoint
was
48%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
16
(
Table
5.28).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
185
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.28.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
2,218
1,168
53%
48%
16
low
8
2,278
1,751
77%
high
8
4,344
1,220
28%
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
6000
5000
4000
3000
2000
1000
0
treatment
Testosterone
Figure
5.32.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
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
not
detected
in
females
from
the
Control
treatment
(
six
individuals)
or
from
the
Low
concentration
(
one
individual).
No
females
from
the
High
concentration
were
available
for
11­
ketotestosteron
analyses
exposed
during
the
EPA
21­
Day
Flutamide
assay.

11­
ketotesosterone
concentrations
in
Control
treatment
males
used
during
the
EPA
21­
Day
Flutamide
assay
ranged
from
8,555
pg/
mL
to
31,716
pg/
mL
(
Figure
5.33).
Among
males
exposed
to
the
two
flutamide
concentrations,
11­
ketotesosterone
concentrations
ranged
from
1,814
pg/
mL
to
95,944
pg/
mL.
Significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
5.29)
were
detected
(
Kruskal­
Wallis,
H
=
9.38,
p
=
0.009,
df
=
2).
The
mean
11­
ketotesosterone
concentration
in
males
from
the
High
concentration
was
greater
than
those
of
males
from
the
Low
concentration
and
the
Control
treatment.
The
achieved
power
for
this
endpoint
was
58%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
13
(
Table
5.29).

Table
5.29.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Flutamide
assay.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
186
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
16,888
8,477
50%
58%
13
low
8
23,603
28,698
122%
high
8
53,209
24,556
46%
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
5.33.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Flutamide
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.

5.2.8
Fecundity
Total
Fecundity
 
A
14­
day
pre­
exposure
evaluation
of
total
egg
production
was
performed.
Total
14­
day
counts
among
the
tanks
that
were
eventually
used
for
the
three
treatments
in
the
exposure
assay
(
individual
tank
values
summed
for
each
treatment)
ranged
from
about
12,000
eggs
to
13,000
eggs
(
Figure
5.34).
No
significant
differences
in
the
mean
14­
day
egg
production
among
the
groups
of
replicates
eventually
used
in
the
flutamide
exposure
assay
were
detected
(
Kruskal­
Wallis,
H
=
0.15,
p
=
0.926,
df
=
2).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
187
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
­
14
­
12
­
10
­
8
­
6
­
4
­
2
1
3
5
7
9
11
13
15
17
19
21
Day
Cumulative
Number
of
Eggs
Control
Low
High
Figure
5.34.
Total
egg
production
per
treatment
for
the
EPA
21­
Day
Flutamide
assay.
The
vertical
line
denotes
the
initiation
of
the
exposure
assay.

During
the
EPA
21­
Day
Flutamide
assay,
total
counts
in
the
Control
treatment
were
varied
almost
1.5
fold
among
replicates,
varying
from
3,804
eggs
to
5,391
eggs
(
Figure
5.35).
Variability
in
total
egg
production
among
Low
concentration
replicates
was
somewhat
less,
ranging
from
4,003
eggs
to
5,275
eggs.
Total
counts
among
the
High
concentration
replicates
varied
about
four­
fold,
ranging
from
874
eggs
to
3,647
eggs.
Statistical
analysis
of
square­
root
transformed
egg
counts
showed
significant
amongtreatment
differences
(
Kruskal­
Wallis,
H
=
7.38,
p
=
0.025,
df
=
2)
in
mean
total
numbers
of
eggs
produced
(
Table
5.30).
The
total
fecundity
of
females
in
the
High­
concentration
treatment
was
significantly
less
than
it
was
for
females
in
the
Low
concentration
or
the
Control
treatment.
The
achieved
power
for
this
assay
was
83%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
4
(
Table
5.30).

Table
5.30.
Summary
statistics
and
power
estimates
for
total
fecundity
data
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
4
4677
772
17%
83%
4
low
4
4777
552
12%
high
4
2147
1192
56%
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)
188
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
0
5
10
15
20
Day
Cumulative
Number
of
Eggs
Control
Low
High
Figure
5.35.
Total
egg
production
by
replicate
per
treatment
for
the
EPA
21­
Day
Flutamide
assay.

Fecundity
per
Female
Reproductive
Day
 
During
the
14­
Day
pre­
exposure
evaluation,
the
mean
number
of
eggs
produced
per
female
reproductive
day
ranged
from
54.4
eggs/
day
for
the
tanks
that
would
be
used
for
the
Low­
concentration
treatment
to
58.8
eggs/
day
for
the
tanks
that
would
be
used
for
the
High
concentration
during
the
21­
day
exposure
assay.
There
were
no
significant
differences
among
treatments
in
the
numbers
of
eggs
produced
per
reproductive
day
during
the
pre­
exposure
period
(
Kruskal­
Wallis,
H
=
0.11,
p
=
0.990,
df
=
2).

During
the
EPA
21­
Day
Flutamide
assay,
the
maximum
number
of
female
reproductive
days
was
achieved
for
all
treatments
(
Table
5.31).
The
number
of
eggs
produced
per
female
reproductive
day
varied
from
45.3
eggs
to
64.2
eggs
in
the
Control
treatment
and
from
47.7
to
62.8
in
the
Low
concentration
(
Figure
5.36).
For
the
High
concentration,
the
number
of
eggs
produced
per
female
reproductive
day
ranged
from
10.4
eggs
to
43.4
eggs,
although
fish
in
two
of
the
replicates
yielded
fewer
than
20
eggs
per
day
(
10.4,
19.1).
Because
no
fish
died
during
the
assay,
the
statistical
results
reported
here
are
the
same
as
those
reported
for
total
fecundity.
A
significant
difference
in
the
mean
number
of
eggs
laid
per
day
per
treatment
(
Table
5.31)
occurred.
The
number
of
eggs
laid
per
day
in
the
Highconcentration
treatment
was
significantly
less
than
it
was
for
females
in
the
Low
concentration
or
the
Control
treatment.
The
achieved
power
for
this
assay
was
73%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
5
(
Table
5.31).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
189
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.31.
Summary
statistics
and
power
estimates
for
fecundity
per
female
reproductive
day
for
the
EPA
21­
Day
Flutamide
assay.

Level
Mean
Number
of
Reproductive
Days
1
N
Mean
Stdev
CV
Achieved
Power
2
Sample
Size
Required
3
control
84
4
55.7
9.2
17%
73%
5
low
84
4
56.9
6.6
12%
high
84
4
25.6
14.2
56%
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
70
60
50
40
30
20
10
treatment
eggs/
ReproDay
Figure
5.36.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
21­
Day
Flutamide
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
14­
day
pre­
exposure
assay
varied
from
1,944
eggs
for
the
tanks
that
would
be
used
for
the
Low
concentration
to
2,355
eggs
for
the
tanks
that
would
be
used
for
the
Control
treatment.
The
mean
number
of
eggs
on
dishes
ranged
from
704
eggs
for
the
Control
treatment
to
1,223
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
14­
day
pre­
exposure
assay
(
Kruskal­
Wallis,
H
=
5.73,
p
=
0.126,
df
=
3).

The
mean
number
of
eggs
laid
on
the
tiles
among
the
treatments
during
the
EPA
21­
Day
Flutamide
assay
varied
from
1,343
eggs
for
the
High
concentration
to
3,463
eggs
for
the
Low­
concentration
treatment
(
Appendix
E,
Table
3.7).
The
number
of
eggs
on
dishes
ranged
from
207
eggs
for
the
Control
treatment
to
350
eggs
for
the
High
concentration.
The
proportional
difference
ranged
from
0.16
(
one
High
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
190
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
concentration
replicate)
to
0.87
(
one
Control­
treatment
replicate)
(
Appendix
E,
Figure
3.6).
There
was
a
significant
difference
in
the
mean
proportional
difference
among
treatments
(
Kruskal­
Wallis,
H
=
8.35,
p
=
0.015,
df
=
2).
The
mean
proportional
difference
in
the
number
of
eggs
on
dishes
versus
those
on
tiles
for
the
High
concentration
was
significantly
lower
than
those
for
the
Low
concentration
and
the
Control
treatment.

5.2.9
Fertilization
Success
Total
Fertilization
 
Eggs
were
collected
during
the
14­
day
pre­
exposure
period
for
the
evaluation
of
fertilization­
success
rate.
The
mean
proportion
of
eggs
fertilized
in
the
Control
treatment
was
0.972
[
standard
deviation
(
sd)
=
0.053],
0.999
(
sd
=
0.004)
in
the
Low
concentration,
and
0.999
(
sd
=
0.005)
in
the
High
concentration.
The
mean
proportion
of
eggs
fertilized
in
the
replicates
that
were
not
used
in
the
21­
day
validation
assay
was
0.997
(
sd
=
0.004).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
5.47,
p
=
0.140,
df
=
3).

The
total
(
tiles
+
dishes)
fertilization­
success
rates
for
most
treatment
replicates
during
the
EPA
21­
Day
Flutamide
assay
were
high,
ranging
from
0.994
(
one
Low­
concentration
replicate)
to
1.00
(
two
Lowconcentration
replicates)
(
Figure
5.37).
One
High­
concentration
replicate
had
a
relatively
low
proportion
of
eggs
fertilized
(
0.857%).
No
significant
differences
in
mean
fertilization­
success
rates
(
Table
5.32)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
4.32,
p
=
0.115,
df
=
2).
The
achieved
power
for
this
assay
was
20%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
17
(
Table
5.32).

Table
5.32.
Summary
statistics
and
power
estimates
for
the
proportion
of
eggs
fertilized
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
4
0.999
0.001
0.1%
20%
17
low
4
0.998
0.003
0.3%
high
4
0.961
0.070
7%
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)
191
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
1.00
0.95
0.90
0.85
treatment
Total
Prop­
fert
Figure
5.37.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
21­
Day
Flutamide
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
14­
Day
pre­
validation
assay,
there
were
no
significant
differences
in
the
fertilization­
success
rates
among
treatments
for
eggs
laid
in
tiles
(
Kruskal­
Wallis,
H
=
7.44,
p
=
0.059,
df
=
3)
or
on
dishes
(
Kruskal­
Wallis,
H
=
4.85,
p
=
0.183,
df
=
3).
The
fertilization­
success
rates
for
all
treatment
replicates
for
eggs
laid
on
tiles
during
the
EPA
21­
Day
Flutamide
assay
were
high,
ranging
from
0.993
(
one
Low­
concentration
replicate)
to
1.00
(
three
Low­
and
one
High­
concentration
replicates)
(
Appendix
E,
Figure
3.7).
No
significant
differences
in
mean
fertilization­
success
rates
for
eggs
laid
on
tiles
(
Appendix
E,
Table
3.8)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
1.33,
p
=
0.516,
df
=
2).
The
fertilization­
success
rates
for
most
treatment
replicates
for
eggs
laid
on
dishes
during
the
assay
were
high,
ranging
from
0.972
(
one
High
concentration
replicate)
to
1.00
(
several
replicates;
including
all
treatments)
(
Appendix
E,
Figure
3.8).
One
High­
concentration
replicate
had
a
relatively
low
proportion
of
eggs
(
laid
on
tiles)
that
were
fertilized
(
0.693%).
No
significant
differences
in
mean
fertilization­
success
rates
(
Appendix
E,
Table
3.8)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
4.91,
p
=
0.086,
df
=
2).

5.2.10
Hatchability
and
Larval
Development
Eggs
were
collected
during
the
14­
day
pre­
exposure
period
for
the
evaluation
of
hatchability.
The
mean
proportion
of
fertilized
eggs
that
hatched
in
the
Control
treatment
was
0.98
[
standard
deviation
(
sd)
=
0.03],
1.00
(
sd
=
0.01)
in
the
Low
concentration,
and
1.00
(
sd
=
0.01)
in
the
High
concentration.
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
1.04,
p
=
0.594,
df
=
2).

Eggs
were
collected
during
the
EPA
21­
Day
Flutamide
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
0.33
to
1.00
for
the
two
test
concentrations
(
Figure
5.38).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
1.15,
p
=
0.563,
df
=
2).
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
44
(
Table
5.33).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
192
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.33.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
9
0.98
0.03
3%
18%
44
low
9
0.97
0.04
4%
high
8
0.89
0.23
26%
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
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
treatment
Prop­
Hatch
Figure
5.38.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
21­
Day
Flutamide
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.

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
0.99
(
sd
=
0.01).
The
mean
proportion
of
normal
larvae
in
the
remaining
treatments
was
0.98
(
sd
=
0.03)
in
the
Low
concentration
and
0.98
(
sd
=
0.02)
in
the
High
concentration.
There
were
no
significant
differences
among
treatments
in
the
proportion
of
normal
larvae
(
Kruskal­
Wallis,
H
=
0.71,
p
=
0.703,
df
=
2).

Eggs
were
collected
during
the
EPA
21­
Day
Flutamide
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
two
test
concentrations
(
Figure
5.39).
There
was
a
significant
difference
among
treatments
in
the
proportion
of
normal
larvae
(
Kruskal­
Wallis,
H
=
8.17,
p
=
0.017,
df
=
2).
The
Mean
proportion
of
larvae
that
developed
normally
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
193
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
was
significantly
less
in
the
High
concentration
than
it
was
in
the
Control
treatment.
The
achieved
power
for
this
endpoint
was
75%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
9
(
Table
5.34).

Table
5.34.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
21­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
9
0.97
0.05
5%
75%
9
low
9
0.96
0.04
4%
high
8
0.89
0.11
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
1.0
0.9
0.8
0.7
treatment
Prop­
Norm
Figure
5.39.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
21­
Day
Flutamide
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.

5.2.11
Body
Weight
The
body
weight
of
females
used
in
the
EPA
21­
Day
Flutamide
assay
ranged
from
1.1
g
to
2.8
g.
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
5.19,
p
=
0.075,
df
=
2).
The
body
weight
of
males
used
in
the
EPA
21­
Day
Flutamide
assay
ranged
from
3.1
g
to
5.6
g.
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
3.80,
p
=
0.150,
df
=
2).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
194
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
5.3
Non­
spawning
Adult
14­
Day
Assay
for
Flutamide
The
Non­
spawning
Adult
14­
Day
Flutamide
assay
was
conducted
from
March
10,
2003
to
March
24,
2003
(
exposure
assay).

5.3.1
Survival
All
males
and
females
in
all
treatments
survived
the
Non­
spawning
Adult
14­
Day
Flutamide
assay.

5.3.2
Vitellogenin
Vitellogenin
concentrations
in
Control
treatment
females
used
during
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
ranged
from
1,252,000
ng/
mL
to
5,983,000
ng/
mL
(
Figure
5.40).
Among
females
exposed
to
the
two
flutamide
concentrations,
vitellogenin
concentrations
ranged
from
3,325,000
ng/
mL
to
10,905,000
ng/
mL.
Significant
differences
in
the
mean
vitellogenin
concentration
among
treatments
(
Table
5.35)
were
detected
(
Kruskal­
Wallis,
H
=
10.88,
p
=
0.012,
df
=
2).
Mean
vitellogenin
concentrations
among
females
exposed
to
the
High
and
Medium
flutamide
concentrations
were
greater
than
the
mean
concentration
for
Control
treatment
females.
The
achieved
power
for
this
endpoint
was
82%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
10
(
Table
5.35).

Table
5.35.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
4,189,350
1,516,107
36%
82%
10
low
10
6,147,250
2,219,643
36%
Medium
10
7,342,100
2,699,666
37%
High
10
7,546,150
2,137,789
28%
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)
195
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
10000000
5000000
0
treatment
Flut­
VTG
Figure
5.40.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
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
Non­
spawning
Adult
14­
Day
Flutamide
assay
ranged
from
0
ng/
mL
(
not
detected)
to
1,523
ng/
mL
(
Figure
5.41).
Among
most
males
exposed
to
the
two
flutamide
concentrations,
vitellogenin
concentrations
ranged
from
0
ng/
mL
(
not
detected)
to
3,285
ng/
mL.
One
male
exposed
to
the
Low­
flutamide
concentration
had
a
vitellogenin
concentration
of
10,575
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
5.36)
were
detected
(
Kruskal­
Wallis,
H
=
3.66,
p
=
0.300,
df
=
2).
The
achieved
power
for
this
endpoint
was
12%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
94
(
Table
5.36).

Table
5.36.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
243
461
190%
12%
94
low
10
1,337
3,342
250%
Medium
10
631
1,325
210%
High
10
76
89
117%
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)
196
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
10000
5000
0
treatment
Flut­
VTG
Figure
5.41.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
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.

5.3.3
Appearance
/
Secondary
Sex
Characteristics
All
females
used
during
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
showed
normal
female
morphology.

Morphological
development
among
males
used
during
the
flutamide
Non­
spawning
Adult
14­
day
assay
varied
among
treatments
(
Figure
5.42).
One
male
exposed
to
the
Medium
concentration
exhibited
a
female
body
shape.
Fourteen
of
the
40
males
used
during
the
assay
lacked
tubercles.
Most
of
these
males
also
lacked
a
fat
pad
and
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)
197
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
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
5.42.
Secondary
sex
characteristics
of
males
used
during
the
EPA
Non­
spawning
Adult
14­
Day
Flutamide
assay.

5.3.4
Gonadosomatic
Index
The
range
of
GSI
values
calculated
for
females
in
the
Control
treatments
varied
from
5.5
to
24.8
(
Figure
5.43),
whereas
the
GSI
values
for
the
other
treatments
varied
from
3.8
to
19.7.
The
overall
withintreatment
variability
was
moderate
(
CVs
=
29%
 
43%;
Table
5.37).
There
were
no
significant
differences
in
mean
GSI
values
(
Table
5.37)
among
treatments
(
Kruskal­
Wallis,
H
=
2.78,
p
=
0.426,
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
55
(
Table
5.37).

Table
5.37.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
13.7
5.6
41%
18%
55
low
10
11.7
4.5
38%
medium
10
13.0
5.6
43%
high
10
10.4
3.0
29%
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)
198
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
25
15
5
treatment
GSI
Figure
5.43.
Boxplot
of
female
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
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
most
males
during
the
Non­
spawning
Adult
14­
Day
Flutamide
assay,
was
large,
varying
from
0.3
to
2.7
(
Figure
5.44),
which
approximates
the
typical
range
for
reproductively­
active
male
fathead
minnows.
One
male
from
the
Low
concentration
had
an
exceptionally
high
GSI
value
of
21.3.
This
male
was
small
(
3.2
g)
and
had
a
hypertrophied
gonad
(
0.68
g)
that
was
much
larger
than
the
average
gonad
weight
(
0.068
g,
sd
=
0.031)
for
the
other
males
used
during
this
test.
This
male
was
excluded
from
all
analyses.
Overall
within­
treatment
variability
was
moderate
to
high
(
CVs
=
31%
 
50%;
Table
5.38).
The
highest,
excluding
the
male
mentioned
above,
and
lowest
male
GSI
values
were
2.6
 
2.7
(
for
two
fish
in
the
High
concentration)
and
0.3
 
0.4
(
three
fish,
one
from
each
treatment
except
the
Medium
concentration),
respectively.
However,
there
were
no
significant
differences
in
mean
GSI
values
(
Table
5.38)
among
treatments
(
Kruskal­
Wallis,
H
=
1.23,
p
=
0.746,
df
=
3).
The
achieved
power
for
this
endpoint
was
10%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
108
(
Table
5.38).

Table
5.38.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
1.3
0.5
39%
10%
108
low
9
1.4
0.7
50%
medium
10
1.3
0.4
31%
high
10
1.6
0.8
47%
1
Calculated
from
arcsine
square­
root
transformed
data;
with
sample
size
=
9.
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)
199
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
3
2
1
0
treatment
GSI
Figure
5.44.
Boxplot
of
male
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
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.

5.3.5
Female
Gonad
Histology
Histological
analyses
were
conducted
on
the
ovaries
of
40
females
exposed
to
flutamide
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
Flutamide
assay
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
1.08,
p
=
0.782,
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
three
exposure
treatments
ranged
from
Stage
1a
to
Stage
5
(
see
Methods
for
a
description
of
the
stages),
whereas
ova
from
females
from
the
Control
treatment
showed
Stage
1a
to
Stage
4
development
(
Figure
5.45).
Variability
within
treatments
for
each
stage
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
316%
(
Table
5.39).
Although
statistical
analyses
showed
that
there
was
a
significant
difference
among
treatments
in
the
proportion
of
cells
in
developmental
Stages
1b
and
3,
there
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
the
developmental
Stages
1a,
2,
4,
and
5
(
Table
5.39).
The
proportion
of
cells
in
developmental
Stages
1b
and
3
in
the
High
concentration
were
significantly
greater
than
those
in
the
Low­
concentration
treatments.
There
was
no
consistent
pattern
of
significant
difference
associated
with
flutamide
dose.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
200
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.39.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.

Control
(
n
=
10)
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
1a
0.087
0.028
32%
0.072
0.030
42%
0.062
0.022
36%
0.091
0.021
23%
6.79
0.079
1b
0.256
0.072
28%
0.229
0.075
33%
0.265
0.073
27%
0.326
0.058
18%
8.45
0.038
*
2
0.183
0.049
27%
0.195
0.055
28%
0.209
0.071
34%
0.213
0.049
23%
2.23
0.526
3
0.136
0.045
33%
0.171
0.054
31%
0.141
0.039
28%
0.103
0.046
45%
8.25
0.041
*
4
0.287
0.115
40%
0.226
0.082
36%
0.238
0.103
43%
0.168
0.071
42%
6.56
0.087
5
0
0
 
0.010
0.033
316%
0.001
0.003
316%
0.016
0.052
316%
1.06
0.787
*
p
<
0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
1a
1b
2
3
4
5
Ovarian
Stage
Proportion
Control
Low
Mid
High
Figure
5.45.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Flutamide
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
0.012
follicles
for
females
in
the
High
concentration
to
0.030
follicles
for
females
in
the
Medium
concentration
treatment
(
Figure
5.46).
There
were
no
significant
differences
in
the
mean
proportion
of
atretic
follicles
among
treatments
(
Kruskal­
Wallis,
H
=
2.83,
p
=
0.419,
df
=
3).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
201
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
0.3
0.2
0.1
0.0
Treatment
atretic_
follicles
Figure
5.46.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
Nonspawning
Adult
14­
Day
Flutamide
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.

Corpora
Lutea
 
The
mean
proportion
of
corpora
lutea
per
300
follicles
(
counted
per
fish)
ranged
from
0.001
for
females
in
the
Medium
concentration
to
0.016
for
females
in
the
Low
concentration
(
Figure
5.47).
There
was
a
significant
differences
in
the
mean
proportion
of
corpora
lutea
among
treatments
(
Kruskal­
Wallis,
H
=
10.81,
p
=
0.013,
df
=
3).
The
mean
proportion
of
corpora
lutea
in
ovaries
from
females
in
the
High
concentration
was
greater
than
those
for
females
from
the
Medium
and
Low
concentrations,
but
did
not
differ
from
that
for
females
from
the
Control
treatment.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
202
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
0.15
0.10
0.05
0.00
Treatment
corpora_
lutea
Figure
5.47.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
Nonspawning
Adult
14­
Day
Flutamide
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
Control
treatment
was
observed
to
have
interstitial
inflammation.
The
ovary
of
one
fish
exposed
to
the
Low­
flutamide
concentration
had
a
zone
of
only
Stage
5
eggs
and
another
zone
with
developing
ova,
containing
mix
of
all
stages,
including
Stage
5.
The
Stage
5
eggs
were
abnormally
sequestered
in
the
ovary
of
this
fish.
One
fish
exposed
to
the
High­
flutamide
concentration
had
Stage
5
eggs
segregated
in
the
ovary.

5.3.6
Male
Gonad
Histology
General
Testes
Staging
 
Testes
from
39
males
exposed
to
flutamide
during
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
were
examined
to
determine
the
general
developmental
condition.
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
1
of
the
120
microscopic
fields
examined
in
the
10
Control
treatment
males
showed
Stage
4
(
45
fields)
or
Stage
5
(
74
fields)
development.
Most
of
the
120
microscopic
fields
examined
in
the
9
Low­
concentration
treatment
males
showed
Stage
4
(
49
fields)
or
Stage
5
(
47
fields)
development.
Twelve
microscopic
fields
showed
Stage
1
development.
In
the
10
Medium
concentration
males
available
for
examination,
112
of
the
120
microscopic
fields
showed
Stage
4
(
64
fields)
or
Stage
5
(
48
fields)
development.
In
the
10
High
concentration
males
available
for
examination,
108
of
the
120
microscopic
fields
showed
Stage
4
(
53
fields)
or
Stage
5
(
55
fields)
development.
Statistical
analysis
of
the
mean
staging
from
12
microscopic
fields
per
fish
revealed
that
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
3.35,
p
=
0.341,
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
5.48).
Variability
within
treatments
for
each
stage
was
very
high
as
indicated
by
CVs
that
ranged
as
high
as
216%
(
Table
5.40).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
any
of
the
developmental
stages
(
Table
5.40).
Therefore,
there
was
no
effect
on
testicular
developmental
stage
associated
with
flutamide
dose.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
203
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.40.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments.

Control
(
n
=
10)
Low
(
n
=
8)
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
0.002
0.004
211%
0.003
0.004
142%
0.001
0.003
211%
0.001
0.002
216%
1.41
0.703
2b
0.008
0.010
131%
0.013
0.015
112%
0.034
0.048
140%
0.015
0.030
200%
5.83
0.120
3a
0.074
0.112
152%
0.043
0.030
72%
0.069
0.098
141%
0.107
0.203
190%
0.01
1.000
3b
0.104
0.134
129%
0.124
0.066
54%
0.110
0.117
106%
0.115
0.064
56%
3.01
0.390
4
0.057
0.068
120%
0.077
0.072
94%
0.099
0.099
100%
0.059
0.057
96%
1.54
0.673
5
0.755
0.250
33%
0.741
0.148
20%
0.686
0.280
41%
0.703
0.272
39%
1.34
0.720
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1
2a
2b
3a
3b
4
5
Testicular
Stage
Proportion
of
Cells
Control
Low
Mid
High
Figure
5.48.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Flutamide
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
92.2
µ
m
to
194.4
µ
m
(
Figure
5.49).
Tubule
diameters
of
males
from
the
three
test
concentrations
ranged
from
52.5
µ
m
to
254.4
µ
m.
No
significant
differences
in
the
mean
tubule
diameter
per
treatment
(
Table
5.41)
were
detected
(
Kruskal­
Wallis,
H
=
0.22,
p
=
0.974,
df
=
3).
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
1,361
(
Table
5.41).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
204
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.41.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
Non­
spawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
147.1
37.0
25%
5%
1,361
low
9
144.8
45.4
31%
medium
10
146.6
37.6
26%
high
10
153.1
51.3
34%
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
250
150
50
Treatment
diameter
Figure
5.49.
Boxplot
of
seminiferous
tubule
diameter
(
µ
m)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
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
for
any
treatment.
One
male
exposed
to
the
Medium
flutamide
concentration
showed
multifocal
Leydig
cell
proliferation.
Three
males
exposed
to
the
High
concentration
were
found
to
have
focal
or
multifocal
Leydig
cell
proliferation.
One
male
from
the
Medium
flutamide
concentration
had
an
egg
embedded
in
the
testes.
Testicular
atrophy
was
observed
for
one
of
the
High
concentration
males
(
Fish
ID
218632
in
Table
5.42)
that
showed
multifocal
Leydig
cell
proliferation.
Several
males
exposed
to
flutamide
had
abnormal
testicular
conditions
(
Table
5.42).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
205
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.42.
Histological
observations
for
males
exposed
to
concentrations
of
flutamide
during
the
Nonspawning
Adult
14­
D
assay.

Fish
ID
Concentration
Leydig
Cell
Proliferation
Observations
218604
Low
None
Premature
release
of
secondary
spermatocytes
(
stage
3b)
in
lumina.
High
concentration
of
spermatogonia
in
mature
tubules.

218620
Medium
None
Premature
release
of
secondary
spermatocytes
(
stage
3b)
in
lumina.
Some
released
stage
3b
cells
necrotic.
Lumina
generally
occluded.

218623
Medium
None
Multifocal
necrotic
primary
and
secondary
spermatocytes
(
3a
and
3b)
in
crypts
of
tubules
218624
Medium
None
Premature
release
of
secondary
spermatocytes
(
stage
3b)
in
lumina.
Multifocal
necrosis
of
secondary
spermatocytes
(
stage
3b).
218625
Medium
Multifocal
Basophilic
nodules
in
testicular
duct.

218626
Medium
None
Focal
area
proliferation
and
sequestration
of
sperm
cells.
218627
Medium
None
Nodules
in
spermatic
ducts
and
some
tubules.

218628
High
Focal
Multifocal
but
not
extensive
necrosis
of
secondary
spermatocytes
(
stage
3b).

218632
High
Multifocal
Multifocal
tubules
with
encysted
structures
that
were
possibly
abnormal
oocytes.
Multifocal
but
sparse
necrotic
spermatogonia.
One
ova
in
section,
probably
an
artifact.

218635
High
Multifocal
Tubule
lumina
occluded.
Multifocal
necrosis
of
secondary
spermatocytes
(
stage
3b).

218636
High
None
Multifocal
necrosis
of
secondary
spermatocytes
(
stage
3b).

5.3.7
Plasma
Steroid
Concentrations
Estradiol
 
Estradiol
concentrations
in
Control­
treatment
females
used
during
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay
ranged
from
105
pg/
mL
to
2,990
pg/
mL
(
Figure
5.50).
Among
females
exposed
to
the
three
flutamide
concentrations,
estradiol
concentrations
ranged
from
478
pg/
mL
to
5,591
pg/
mL.
No
significant
differences
in
the
mean
estradiol
concentration
per
treatment
(
Table
5.43)
were
detected
(
Kruskal­
Wallis,
H
=
3.06,
p
=
0.383,
df
=
2).
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
196
(
Table
5.43).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
206
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.43.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
1,638
850
52%
8%
196
low
10
1,371
842
61%
medium
10
1,909
1,423
75%
high
10
1,180
400
34%
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
6000
5000
4000
3000
2000
1000
0
treatment
Estradiol
Figure
5.50.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
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.

Estradiol
was
not
detected
in
the
10
Control­
treatment
males
used
during
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay
(
Figure
5.51).
Among
males
exposed
to
the
three
flutamide
concentrations,
estradiol
was
not
detected
in
the
9
Low­
concentration
males
or
the
10
Medium
concentration
males
(
Table
5.44).
Estradiol
was
detected
in
two
of
the
eight
males
from
the
High
concentration
that
were
analyzed.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
207
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.44.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
1
control
10
0
0
 
 
 
low
9
0
0
 
medium
10
0
0
 
high
8
65
124
191%
1
Not
calculated.

Mid
Low
High
Control
300
200
100
0
treatment
Estradiol
Figure
5.51.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
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.

Testosterone
 
Testosterone
concentrations
in
Control­
treatment
females
used
during
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay
ranged
from
771
pg/
mL
to
3,053
pg/
mL
(
Figure
5.52).
Among
females
exposed
to
the
three
flutamide
concentrations,
testosterone
concentrations
ranged
from
145
pg/
mL
to
4,057
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
5.45)
were
detected
(
Kruskal­
Wallis,
H
=
5.51,
p
=
0.138,
df
=
2).
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
32
(
Table
5.45).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
208
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.45.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
1,565
857
55%
23%
32
low
8
880
475
54%
medium
9
2,340
1,478
63%
high
10
1,235
816
66%
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.

Mid
Low
High
Control
4000
3000
2000
1000
0
treatment
Testosterone
Figure
5.52.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
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
Flutamide
assay
ranged
from
607
pg/
mL
to
4,044
pg/
mL
(
Figure
5.53).
Among
males
exposed
to
the
three
flutamide
concentrations,
testosterone
concentrations
ranged
from
513
pg/
mL
to
6,506
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
5.46)
were
detected
(
Kruskal­
Wallis,
H
=
5.00,
p
=
0.172,
df
=
2).
The
achieved
power
for
this
endpoint
was
28%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
26
(
Table
5.46).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
209
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.46.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
2,096
1,180
56%
28%
26
low
10
1,737
1,787
103%
medium
10
1,045
405
39%
high
8
2,042
1,596
78%
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.

Mid
Low
High
Control
7000
6000
5000
4000
3000
2000
1000
0
treatment
Testosterone
Figure
5.53.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
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.

11­
ketotestosterone
 
11­
ketotesosterone
was
detected
in
one
of
seven
Control­
treatment
females
and
in
one
of
five
females
from
the
Low
concentration
during
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay
(
Figure
5.54).
11­
ketotesosterone
was
not
detected
in
females
from
the
Medium
concentration
(
six
individuals)
and
the
High
concentration
(
five
individuals)
exposed
during
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.
No
significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
5.47)
were
detected
(
Kruskal­
Wallis,
H
=
1.98,
p
=
0.577,
df
=
2).
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
91
(
Table
5.47).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
210
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.47.
Summary
statistics
and
power
estimates
for
female
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
7
70
184
265%
8%
91
low
5
86
192
223%
medium
6
0
0
 
high
5
0
0
 
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.

Mid
Low
High
Control
500
400
300
200
100
0
treatment
11­
keto
Figure
5.54.
Boxplot
of
female
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
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
Flutamide
assay
ranged
from
2,467
pg/
mL
to
29,425
pg/
mL
(
Figure
5.55).
Among
males
exposed
to
the
three
flutamide
concentrations,
11­
ketotesosterone
concentrations
ranged
from
1,816
pg/
mL
to
89,227
pg/
mL.
No
significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
5.48)
were
detected
(
Kruskal­
Wallis,
H
=
0.67,
p
=
0.879,
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
295
(
Table
5.48).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
211
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.48.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
8
10,190
8,876
87%
6%
295
low
10
19,906
27,524
138%
medium
10
6,502
2,949
45%
high
7
13,341
12,900
97%

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.

Mid
Low
High
Control
100000
50000
0
treatment
11­
keto
Figure
5.55.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
Spawning
Adult
14­
Day
Flutamide
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
asterisk
represent
probable
outliers.

5.3.8
Body
Weight
and
Length
The
body
weight
of
females
used
in
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
ranged
from
1.9
g
to
3.7
g
(
Figure
5.56).
There
were
no
significant
differences
in
mean
body
weight
(
natural
log
transformed)
among
treatments
(
Kruskal­
Wallis,
H
=
3.90,
p
=
0.272,
df
=
3).
The
achieved
power
for
this
endpoint
was
36%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
26
(
Table
5.49).

The
body
(
fork)
length
of
females
used
in
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
ranged
from
46
mm
to
61
mm
(
Figure
5.57).
There
were
no
significant
differences
in
mean
body
length
(
natural
log
transformed)
among
treatments
(
Kruskal­
Wallis,
H
=
1.74,
p
=
0.627,
df
=
3).
The
achieved
power
for
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
212
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
this
endpoint
was
10%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
123
(
Table
5.50).

Table
5.49.
Summary
statistics
and
power
estimates
for
female
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
2.9
0.5
19%
36%
26
low
10
2.6
0.4
15%
medium
10
2.5
0.3
13%
high
10
2.5
0.4
15%
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
3.5
3.0
2.5
2.0
treatment
fish_
wgt_
whole
Figure
5.56.
Boxplot
of
female
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
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.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
213
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.50.
Summary
statistics
and
power
estimates
for
female
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
53.6
5.3
10%
10%
123
low
10
53.4
3.3
6%
medium
10
53.1
2.7
5%
high
10
55.0
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.

Mid
Low
High
Control
60
55
50
45
treatment
fish_
length_
fork
Figure
5.57.
Boxplot
of
female
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
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
Flutamide
assay
ranged
from
2.8
g
to
6.9
g
(
Figure
5.58).
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
7.08,
p
=
0.069,
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
28
(
Table
5.51).

The
body
(
fork)
length
of
males
used
in
the
Non­
spawning
Adult
14­
Day
Flutamide
assay
ranged
from
54
mm
to
75
mm
(
Figure
5.59).
There
were
no
significant
differences
in
mean
body
length
(
natural
log
transformed)
among
treatments
(
Kruskal­
Wallis,
H
=
5.42,
p
=
0.143,
df
=
3).
The
achieved
power
for
this
endpoint
was
17%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
52
(
Table
5.52).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
214
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
5.51.
Summary
statistics
and
power
estimates
for
male
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
5.0
0.5
10%
30%
28
low
9
4.9
0.9
18%
medium
10
5.3
0.8
16%
high
10
4.3
0.7
16%
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
7
6
5
4
3
treatment
fish_
wgt_
whole
Figure
5.58.
Boxplot
of
male
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
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.

Table
5.52.
Summary
statistics
and
power
estimates
for
male
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Flutamide
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
control
10
65.4
1.8
3%
17%
52
low
9
68.0
4.0
6%
medium
10
68.2
4.6
7%
high
10
64.2
5.0
8%
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)
215
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
75
65
55
treatment
fish_
length_
fork
Figure
5.59.
Boxplot
of
male
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Flutamide
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.