DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
21
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
3.0
RESULTS:
METHOXYCHLOR
3.1
EPA
14­
Day
Assay
for
Methoxychlor
The
EPA
14­
Day
Methoxychlor
assay
was
conducted
from
September
29,
2002,
to
October
14,
2002
(
pre­
exposure
assay),
and
from
October
14,
2002,
to
October
28,
2002
(
exposure
assay).

3.1.1
Survival
All
males
in
the
Control
treatment
and
all
males
and
females
in
the
Low­
concentration
treatment
survived
the
EPA
14­
Day
Methoxychlor
assay.
However,
two
females
in
the
Control
treatment
died
(
88%
survival).
Both
males
in
two
High­
concentration
tanks
died
before
the
end
of
the
14­
day
exposure
and
those
tanks
were
terminated
(
male
survival
=
50%).
In
the
two
High­
concentration
tanks
that
completed
the
assay,
six
of
the
eight
females
survived
(
75%).

3.1.2
Vitellogenin
Vitellogenin
concentrations
in
Control­
treatment
females
used
during
the
EPA
14­
Day
Methoxychlor
assay
ranged
from
229,750
ng/
mL
to
5,470,500
ng/
mL
(
Figure
3.1).
Among
females
exposed
to
the
two
methoxychlor
concentrations,
vitellogenin
concentrations
ranged
from
2,595
ng/
mL
to
2,177,500
ng/
mL.
Significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
3.1)
were
detected
(
Kruskal­
Wallis,
H
=
10.91,
p
=
0.004,
df
=
2).
Vitellogenin
concentrations
in
Control­
treatment
females
were
significantly
greater
than
those
in
females
exposed
to
the
Low
concentration.
The
achieved
power
for
this
endpoint
was
47%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
10
(
Table
3.1).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
22
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
6000000
5000000
4000000
3000000
2000000
1000000
0
treatment
Meth­
VTG
Boxplots
of
Meth­
VTG
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.
1.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
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
3.1.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
14
2,136,139
1,685,109
79%
47%
10
Low
13
449,451
564,871
126%
High
5
1,174,907
877,963
75%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
5.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Vitellogenin
concentrations
in
Control­
treatment
males
used
during
the
EPA
14­
Day
Methoxychlor
assay
ranged
from
0
ng/
mL
(
not
detected)
to
711
ng/
mL
(
Figure
3.2).
Among
males
exposed
to
the
two
methoxychlor
concentrations,
vitellogenin
concentrations
ranged
from
0
ng/
mL
to
1,130,000
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
3.2)
were
detected
(
Kruskal­
Wallis,
H
=
0.31,
p
=
0.856,
df
=
2).
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
51
(
Table
3.2).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
23
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
1000000
500000
0
treatment
Meth­
VTG
Boxplots
of
Meth­
VTG
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.2.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
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
3.2.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
7
181
250
138%
10%
51
Low
7
362
509
141%
High
4
562,500
649,532
115%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
4.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

3.1.3
Appearance
/
Secondary
Sex
Characteristics
All
of
the
females
used
during
the
EPA
14­
Day
Methoxychlor
assay
exhibited
typical
female
morphology
(
no
fat
pad,
no
tubercles,
ovipositor
present)
except
that
three
females
from
the
Control
treatment
lacked
ovipositors.

Most
males
used
during
the
EPA
14­
Day
Methoxychlor
assay
had
typical
male
morphological
features.
One
male
from
the
Control
treatment
lacked
a
dorsal
fat
pad.
Six
males,
at
least
one
from
each
treatment,
lacked
vertical
banding.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
24
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
3.1.4
Gonadosomatic
Index
The
range
of
GSI
values
calculated
for
females
in
all
treatments
varied
from
three­
to
five­
fold
(
Figure
3.3),
and
the
overall
variability
within
the
treatment
was
moderate
(
CVs
=
32%
 
46%)
(
Table
3.3).
The
highest
female
GSI
values
were
about
24
(
one
fish
each
in
the
Control
treatment
and
the
High
concentration).
There
were
no
significant
differences
in
mean
GSI
values
among
treatments
(
Kruskal­
Wallis,
H
=
4.77,
p
=
0.092,
df
=
2)
(
Table
3.3).
The
achieved
power
for
this
endpoint
was
19%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
31
(
Table
3.3).

Low
High
Control
25
15
5
treatment
GSI
Boxplots
of
GSI
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.3.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
25
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.3.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
EPA
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
14
14.2
4.5
32%
19%
31
Low
16
10.7
4.0
37%
High
6
14.3
6.5
46%
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
6.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

The
range
of
most
GSI
values
calculated
for
males
during
the
EPA
14­
Day
Methoxychlor
assay
was
small,
ranging
from
0.7
to
1.7
(
Figure
3.4),
which
approximates
the
typical
range
for
reproductivelyactive
male
fathead
minnows.
The
highest
and
lowest
male
GSI
values
were
1.6
to
1.7
(
two
fish
in
the
Control
treatment,
one
in
the
Low
concentration)
and
0.5
(
one
fish
in
the
Low
concentration),
respectively.
There
were
no
significant
differences
in
mean
GSI
values
among
treatments
(
Kruskal­
Wallis,
H
=
2.35,
p
=
0.308,
df
=
2)
(
Table
3.4).
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
49
(
Table
3.4).

Low
High
Control
1.7
1.5
1.3
1.1
0.9
0.7
0.5
treatment
GSI
Figure
3.4.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
14­
Day
Methoxychlor
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.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
26
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.4.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
EPA
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
8
1.12
0.36
32%
10%
49
Low
7
0.92
0.36
39%
High
4
1.26
0.10
8%
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
4.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

3.1.5
Female
Gonad
Histology
Histological
analyses
were
conducted
on
the
ovaries
of
35
females
exposed
to
methoxychlor
during
the
EPA
14­
day
assay.
Of
these
35
fish,
12
were
observed
to
have
moderate­
to­
diffuse
macrophage
infiltration
into
the
ovaries.
Tissues
from
three
fish
with
this
condition
were
evaluated
with
a
Gram
stain
and
an
acid­
fast
stain
that
demonstrated
acid­
fast­
staining
structures
consistent
with
mycobacteria.
All
other
fish
with
a
similar
macrophage
infiltration
in
the
ovaries
were
also
presumed
to
have
mycobacteriosis.
At
the
time
of
the
histological
examination,
it
could
not
be
determined
whether
the
condition
was
exacerbated
by
the
chemical
exposure.
Because
Control­
treatment
fish
were
infected,
it
was
clear
that
the
infection
was
distributed
throughout
the
population.
To
determine
whether
the
infection
affected
the
results,
the
analyses
were
conducted
once
with
all
fish
included
and
then
repeated
with
the
infected
fish
excluded.
The
results
of
the
second
set
of
analyses
(
with
infected
fish
removed)
were
reported
only
if
they
resulted
in
a
change
in
the
pattern
of
statistical
significance
obtained
for
the
analyses
that
included
all
fish.

General
Ovary
Staging
 
Statistical
analysis
of
the
mean
ovarian
staging
from
12
microscopic
fields
per
fish
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
4.99,
p
=
0.082,
df
=
2).
When
infected
fish
were
excluded
from
the
analysis,
there
was
a
statistical
difference
among
treatments
in
the
mean
ovarian
staging
(
Kruskal­
Wallis,
H
=
10.36,
p
=
0.006,
df
=
2).
The
mean
ovarian
stage
of
females
from
the
High
concentration
was
greater
than
that
for
females
from
the
Low
concentration,
but
not
for
females
from
the
Control
treatment.
Therefore,
there
was
no
significant
pattern
associated
with
the
methoxychlor
dose.

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
the
Control
and
High­
concentration
treatments
ranged
from
Stage
1a
to
Stage
5
(
see
Section
2:
Methods
for
a
description
of
the
stages),
whereas
ova
from
females
from
the
Low­
concentration
treatment
showed
Stage
1a
to
Stage
4
development
(
Figure
3.5).
Variability
within
treatments
for
each
stage
was
very
high,
as
indicated
by
CVs
that
ranged
as
high
as
245%
(
Table
3.5).
Although
statistical
analyses
showed
that
there
was
a
significant
difference
among
treatments
in
the
proportion
of
cells
in
developmental
Stage
5,
there
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
the
developmental
Stages
1a,
1b,
2,
3,
and
4
(
Table
3.5).
The
proportion
of
cells
in
developmental
Stage
5
in
the
Low
concentration
was
significantly
lower
than
that
in
the
Control
treatment.
Therefore,
there
was
no
consistent
pattern
of
significant
difference
associated
with
the
methoxychlor
dose.
There
were
no
changes
in
this
pattern
of
statistical
significance
when
the
infected
fish
were
excluded
from
the
analysis.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
27
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
1a
1b
2
3
4
5
Ovarian
Stage
Proportion
of
Cells
Control
Low
High
Figure
3.5.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
14­
Day
Methoxychlor
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.

Table
3.5.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
EPA
14­
Day
Methoxychlor
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments
Control
(
n
=
16)
Low
(
n
=
16)
High
(
n
=
4)
Kruskal­
Wallis
Stage
Mean
SD
CV
Mean
SD
CV
Value
SD
CV
H
p
1a
0.079
0.040
51%
0.101
0.045
45%
0.102
0.058
57%
1.33
0.514
1b
0.319
0.089
28%
0.392
0.074
19%
0.386
0.117
30%
4.44
0.108
2
0.170
0.062
37%
0.159
0.045
28%
0.173
0.022
13%
0.88
0.644
3
0.150
0.062
41%
0.144
0.044
30%
0.118
0.070
60%
0.53
0.765
4
0.227
0.086
38%
0.185
0.063
34%
0.199
0.128
65%
1.64
0.440
5
0.010
0.016
167%
0
0
 
0.002
0.004
245%
8.27
0.016*
*
p
<
0.05
Atretic
Follicles
 
The
mean
proportion
of
atretic
follicles
per
300
follicles
(
counted
per
fish)
ranged
from
0.02
for
females
in
the
High
concentration
to
0.04
for
females
in
the
Control
treatment
(
Figure
3.6).
There
were
no
significant
differences
in
the
proportions
of
atretic
follicles
among
treatments
(
Kruskal­
Wallis,
H
=
2.17,
p
=
0.337,
df
=
2).
There
was
no
change
in
this
pattern
of
statistical
significance
when
the
infected
fish
were
excluded
from
the
analysis.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
28
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
0.4
0.3
0.2
0.1
0.0
treatment
atretic_
follicles
Boxplots
of
atretic_
follicles
by
treatmen
Figure
3.6.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
asterisks
represent
probable
outliers.

Corpora
Lutea
 
The
mean
proportion
of
corpora
lutea
per
300
follicles
(
counted
per
fish)
ranged
from
none
for
females
in
the
High
concentration
to
0.06
for
females
in
the
Low
concentration
(
Figure
3.7).
There
was
a
significant
difference
in
the
proportions
of
corpora
lutea
among
treatments
(
Kruskal­
Wallis,
H
=
6.78,
p
=
0.034,
df
=
2).
The
value
for
the
High
concentration
was
significantly
lower
than
those
of
the
other
two
treatments.
There
was
no
change
in
this
pattern
of
statistical
significance
when
the
infected
fish
were
excluded
from
the
analysis.

Low
High
Control
0.06
0.05
0.04
0.03
0.02
0.01
0.00
treatment
corpora_
lutea
Boxplots
of
corpora_
lutea
by
treatmen
Figure
3.7.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
and
the
horizontal
line
is
the
median
value.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
29
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
3.1.6
Male
Gonad
Histology
Testes
Staging
by
Microscopic
Field
 
Testes
from
males
exposed
to
methoxychlor
during
the
EPA
14­
Day
Methoxychlor
assay
were
examined
to
determine
their
general
developmental
condition.
Males
in
all
treatments
had
well­
developed
testes
with
most
showing
Stage
4
and
Stage
5
development
(
see
Section
2:
Methods
for
description
of
developmental
stages).
All
of
the
96
microscopic
fields
examined
in
the
8
Control­
treatment
males
showed
Stage
4
(
72
fields)
or
Stage
5
(
24
fields)
development.
All
of
the
84
fields
examined
in
the
7
Low­
concentration
treatment
males
showed
Stage
4
development.
In
the
4
Highconcentration
males
available
for
examination,
all
48
microscopic
fields
examined
showed
Stage
4
(
42
fields)
or
Stage
5
(
6
fields)
development.
Statistical
analysis
of
the
mean
staging
from
12
sections
per
fish
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
1.99,
p
=
0.371,
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
of
the
Control­
treatment
testes
ranged
from
Stage
1
to
Stage
5,
whereas
testes
in
males
from
the
Low­
and
Highconcentration
treatments
showed
Stage
2a
to
Stage
5
development
(
Figure
3.8).
Variability
within
treatments
for
each
stage
was
very
high,
as
indicated
by
CVs
that
ranged
as
high
as
209%
(
Table
3.6).
Statistical
analyses
showed
that
there
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
any
developmental
Stage
(
Table
3.6).

0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
1
2a
2b
3a
3b
4
5
Testicular
Stage
Proportion
of
Cells
Control
Low
High
Figure
3.8.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
EPA
14­
Day
Methoxychlor
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
30
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.6.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
EPA
14­
Day
Methoxychlor
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments
Control
(
n
=
8)
Low
(
n
=
7)
High
(
n
=
4)
Kruskal­
Wallis
Stage
Mean
SD
CV
Mean
SD
CV
Value
SD
CV
H
p
1
0.005
0.010
209%
0
 
 
0
 
 
2.90
0.234
2a
0.018
0.019
106%
0.002
0.013
199%
0.013
0.011
88%
4.24
0.120
2b
0.042
0.051
123%
0.060
0.058
154%
0.058
0.036
63%
1.15
0.562
3a
0.172
0.147
85%
0.234
0.183
36%
0.183
0.161
88%
1.03
0.597
3b
0.188
0.096
51%
0.226
0.169
32%
0.169
0.046
27%
2.30
0.317
4
0.090
0.050
56%
0.089
0.181
28%
0.181
0.162
90%
0.30
0.859
5
0.487
0.239
49%
0.390
0.380
41%
0.380
0.109
29%
0.72
0.696
Tubule
Diameter
 
The
diameter
of
the
seminiferous
tubules
of
males
from
the
Control
treatment
ranged
from
86.4
µ
m
to
181.7
µ
m
(
Figure
3.9).
Tubule
diameters
of
males
from
the
two
test
concentrations
ranged
from
78.3
µ
m
to
155.6
µ
m.
No
significant
differences
in
the
mean
tubule
diameter
per
treatment
were
detected
(
Kruskal­
Wallis,
H
=
3.24,
p
=
0.198,
df
=
2)
(
Table
3.7).
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
30
(
Table
3.7).

Low
High
Control
180
130
80
treatment
diameter
Boxplots
of
diameter
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.9.
Boxplot
of
seminiferous
tubule
diameter
(
µ
m)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
31
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.7.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
EPA
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
8
118.0
28.7
24%
13%
30
Low
7
114.2
24.3
21%
High
4
137.4
21.0
15%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
4.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Observations
 
No
Sertoli
cell
or
Leydig
cell
proliferation
was
observed.
No
testicular
atrophy
was
recorded,
and
no
ovatestes
were
observed
for
any
treatment.

3.1.7
Plasma
Steroid
Concentrations
Estradiol
 
Estradiol
concentrations
in
Control­
treatment
females
used
during
the
EPA
14­
Day
Methoxychlor
assay
ranged
from
0
pg/
mL
(
not
detected)
to
4,643
pg/
mL
(
Figure
3.10).
Among
females
exposed
to
the
two
methoxychlor
concentrations,
estradiol
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
1,349
pg/
mL.
No
significant
differences
in
the
mean
estradiol
concentration
per
treatment
(
Table
3.8)
were
detected
(
Kruskal­
Wallis,
H
=
5.94,
p
=
0.051,
df
=
2).
However,
the
calculated
probability
value
was
only
slightly
above
the
critical
limit
of
0.050.
The
greatest
difference
in
mean
estradiol
concentration
was
between
females
from
the
Low
concentration
(
353
pg/
mL)
and
those
from
the
Control
treatment
(
1,670
pg/
mL).
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
28
(
Table
3.8).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
32
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
5000
4000
3000
2000
1000
0
treatment
Estradiol
Boxplots
of
Estradio
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.10.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

Table
3.8.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
9
1,670
1,568
94%
20%
28
Low
9
353
418
118%
High
6
455
411
90%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
6.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Estradiol
concentrations
in
Control­
treatment
males
used
during
the
EPA
14­
Day
Methoxychlor
assay
ranged
from
191
pg/
mL
to
518
pg/
mL
(
Figure
3.11).
Among
males
exposed
to
the
two
methoxychlor
concentrations,
estradiol
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
177
pg/
mL.
A
significant
difference
in
the
mean
estradiol
concentration
per
treatment
(
Table
3.9)
was
detected
(
Kruskal­
Wallis,
H
=
9.18,
p
=
0.010,
df
=
2).
The
mean
estradiol
concentration
in
males
from
the
Low
concentration
was
less
than
that
in
males
from
the
Control
treatment.
An
estradiol
concentration
was
obtainable
from
only
one
male
exposed
to
the
High
concentration
of
methoxychlor.
This
value
(
155
pg/
mL)
was
much
less
than
the
mean
Control­
treatment
value
(
299
pg/
mL).
The
achieved
power
for
this
endpoint
was
11%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
12
(
Table
3.9).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
33
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
500
400
300
200
100
0
treatment
Estradiol
Boxplots
of
Estradio
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.
11.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
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
3.9.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
7
299
111
37%
11%
12
Low
5
86
81
94%
High
1
155
1
Calculated
from
natural
log
transformed
data;
sample
size
=
2
(
smallest
allowable).
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Testosterone
 
Testosterone
concentrations
in
Control­
treatment
females
used
during
the
EPA
14­
Day
Methoxychlor
assay
ranged
from
0
pg/
mL
(
not
detected)
to
8,269
pg/
mL
(
Figure
3.12).
Among
females
exposed
to
the
two
methoxychlor
concentrations,
testosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
7,931
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
3.10)
were
detected
(
Kruskal­
Wallis,
H
=
0.11,
p
=
0.947,
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
602
(
Table
3.10).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
34
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
treatment
Testosterone
Boxplots
of
Testoste
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.12.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
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
3.10.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
9
2,719
3,284
121%
5%
602
Low
6
1,972
3,016
153%
High
5
1,689
1,632
97%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
5.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Testosterone
concentrations
in
Control­
treatment
males
used
during
the
EPA
14­
Day
Methoxychlor
assay
ranged
from
940
pg/
mL
to
10,682
pg/
mL
(
Figure
3.13).
Among
males
exposed
to
the
two
methoxychlor
concentrations,
testosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
4,546
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
3.11)
were
detected
(
Kruskal­
Wallis,
H
=
2.10,
p
=
0.349,
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
12
(
Table
3.11).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
35
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
10000
5000
0
treatment
Testosterone
Boxplots
of
Testoste
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.13.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
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
3.11.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
6
4,307
3,854
89%
20%
12
Low
6
1,531
1,843
120%
High
3
2,202
1,279
58%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
3.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

11­
Ketotestosterone
 
11­
ketotesosterone
was
only
detected
in
one
of
six
Control­
treatment
females
used
during
the
EPA
14­
Day
Methoxychlor
assay
(
Figure
3.14).
Among
females
exposed
to
the
two
methoxychlor
concentrations,
11­
ketotesosterone
was
only
detected
in
one
individual
from
each
treatment
(
four
females
analyzed
for
each
treatment).
No
significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
3.12)
were
detected
(
Kruskal­
Wallis,
H
=
0.05,
p
=
0.977,
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
691
(
Table
3.12).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
36
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
500
400
300
200
100
0
treatment
11­
keto
Boxplots
of
11­
keto
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.14.
Boxplot
of
female
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
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,
ad
the
asterisk
represents
a
probable
outlier.

Table
3.12.
Summary
statistics
and
power
estimates
for
female
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
6
79
194
245%
5%
691
Low
4
76
152
200%
High
4
99
197
200%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
4.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

11­
Ketotesosterone
concentrations
in
Control­
treatment
males
used
during
the
EPA
14­
Day
Methoxychlor
assay
ranged
from
7,660
pg/
mL
to
53,265
pg/
mL
(
Figure
3.15).
Among
males
exposed
to
the
two
methoxychlor
concentrations,
11­
ketotesosterone
concentrations
ranged
from
1,660
pg/
mL
to
25,610
pg/
mL.
No
significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
3.13)
were
detected
(
Kruskal­
Wallis,
H
=
1.51,
p
=
0.471,
df
=
2).
The
achieved
power
for
this
endpoint
was
14%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
18
(
Table
3.13).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
37
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
50000
40000
30000
20000
10000
0
treatment
11­
keto
Boxplots
of
11­
keto
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.15.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
14­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisk
represents
a
probable
outlier.

Table
3.13.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
8
18,346
14,922
81%
14%
18
Low
7
10,613
10,576
100%
High
3
14,812
6,803
46%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
3.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

3.1.8
Body
Weight
The
body
weight
of
females
used
in
the
EPA
14­
Day
Methoxychlor
assay
ranged
from
0.8
g
to
2.3
g.
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
3.42,
p
=
0.181,
df
=
2).
The
body
weight
of
males
used
in
the
EPA
14­
Day
Methoxychlor
assay
ranged
from
2.3
g
to
5.3
g.
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
0.59,
p
=
0.
744,
df
=
2).

3.1.9
Fecundity
Total
Fecundity
 
Variability
among
treatments
in
the
total
number
of
eggs
produced
during
the
EPA
14­
Day
Methoxychlor
assay
was
very
high
(
Figure
3.16).
Total
counts
in
the
Control
treatment
ranged
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
38
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
more
than
five­
fold,
varying
from
686
eggs
to
3,852
eggs.
Total
counts
for
three
replicates
of
the
High
methoxychlor
concentration
treatment
were
similar,
ranging
from
124
eggs
to
311
eggs,
whereas
the
total
number
of
eggs
produced
in
the
fourth
replicate
was
1,360.
Both
males
in
two
of
the
three
High
methoxychlor
replicates
with
low
total
egg
counts
died
after
Day
6
or
Day
7
and
each
tank
was
terminated.
In
the
third
replicate
having
low
egg
numbers,
eggs
were
laid
only
on
Day
2
and
Day
6.
There
was
about
a
three­
fold
difference
between
the
minimum
and
maximum
number
of
eggs
produced
among
replicates
in
the
Low
methoxychlor
concentration.
No
significant
differences
in
the
mean
number
of
eggs
(
square­
root
transformed
)
produced
per
treatment
were
detected
(
1­
way
ANOVA,
F
=
3.32,
p
=
0.083,
df
=
2,
9)
(
Table
3.14).
The
achieved
power
for
this
assay
was
39%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
9
(
Table
3.14).

0
500
1,000
1,500
2,000
2,500
3,000
3,500
4,000
4,500
0
2
4
6
8
10
12
14
16
Day
Cumulative
Number
of
Eggs
Control
Low
High
Figure
3.16.
Total
Egg
Production
by
Replicate
per
Treatment
for
the
EPA
14­
Day
Methoxychlor
Assay
Table
3.14.
Summary
Statistics
and
Power
Estimates
for
Fecundity
Data
for
the
EPA
14­
Day
Methoxychlor
Assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
4
1820
1423
78%
39%
9
Low
4
1590
689
43%
High
4
519
567
109%
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
4.
2
Required
size
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)
39
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Fecundity
per
Female
Reproductive
Day
 
During
the
EPA
14­
Day
Methoxychlor
assay,
the
maximum
number
of
female
reproductive
days
was
achieved
for
the
Low
concentration,
whereas
55.3
female
reproductive
days
were
achieved
in
the
Control
treatment
and
40.0
female
reproductive
days
(
71%
of
the
maximum)
were
achieved
in
the
High
concentration
(
Table
3.15).
The
number
of
eggs
produced
per
female
reproductive
day
in
the
Control
treatment
varied
from
12.3
eggs
to
68.8
eggs
and
from
16.4
eggs
to
45.1
eggs
in
the
Low
concentration
(
Figure
3.17).
For
the
High
concentration,
the
number
of
eggs
produced
per
female
reproductive
day
ranged
from
4.4
eggs
to
24.7
eggs,
with
fish
in
three
of
the
replicates
producing
fewer
than
15
eggs
per
day
(
4.4,
5.3,
13.0).
No
significant
difference
in
the
number
of
eggs
produced
per
female
reproductive
day
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
3.50,
p
=
0.174,
df
=
2).
The
achieved
power
for
this
assay
was
36%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
10
(
Table
3.15).

Low
High
Control
70
60
50
40
30
20
10
0
treatment
eggs/
ReproDay
Boxplots
of
eggs/
Rep
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.17.
Boxplot
of
the
Number
of
Eggs
Produced
per
Female
Reproductive
Day
by
Treatment
for
the
EPA
14­
Day
Methoxychlor
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
3.15.
Summary
Statistics
and
Power
Estimates
for
Fecundity
per
Female
Reproductive
Day
for
the
EPA
14­
Day
Methoxychlor
Assay
Level
Mean
Number
of
Reproductive
Days
1
N
Mean
SD
CV
Achieved
Power
2
Sample
Size
Required
3
Control
55.3
4
32.9
25.4
77%
36%
10
Low
56.0
4
28.4
12.3
43%
High
40.0
4
11.9
9.4
79%
1
Maximum
number
=
56.
2
Calculated
from
natural
log
transformed
data;
sample
size
=
4.
3
Size
required
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)
40
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Eggs
on
Tiles/
Dishes
 
The
mean
number
of
eggs
laid
on
the
tiles
among
the
treatments
during
the
EPA
14­
Day
Methoxychlor
assay
varied
from
442
eggs
for
the
High
concentration
to
1,574
eggs
for
the
Control
treatment
(
Appendix
E,
Table
1.3).
The
number
of
eggs
on
dishes
ranged
from
77
eggs
for
the
High
concentration
to
245
eggs
for
the
Control
treatment.
Because
of
the
variability
in
the
total
number
of
eggs
laid
per
treatment,
the
proportional
difference
in
the
number
of
eggs
on
dishes
versus
those
on
tiles
(
1 [#
eggs
on
dishes
÷
#
eggs
on
tiles])
was
calculated.
The
proportional
difference
ranged
from
0.25
(
one
High­
concentration
replicate)
to
0.97
(
one
Control­
treatment
replicate)
(
Appendix
E,
Figure
1.2).
There
were
no
significant
differences
in
this
mean
proportional
difference
among
treatments
(
Kruskal­
Wallis,
H
=
0.73,
p
=
0.694,
df
=
2).

3.1.10
Fertilization
Success
Total
Fertilization
 
The
total
(
tiles
and
dishes)
fertilization­
success
rates
for
most
treatment
replicates
during
the
EPA
14­
Day
Methoxychlor
assay
were
high,
ranging
from
0.932
(
one
Control­
treatment
replicate)
to
1.00
(
two
High­
concentration
replicates,
one
Control­
treatment
replicate)
(
Figure
3.18).
The
fertilization­
success
rate
for
eggs
in
one
High
concentration
was
very
low,
0.557.
No
significant
differences
in
mean
fertilization­
success
rates
(
Table
3.16)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
0.47,
p
=
0.791,
df
=
2).
The
achieved
power
for
this
assay
was
8%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
78
(
Table
3.16).

Low
High
Control
1.0
0.9
0.8
0.7
0.6
treatment
Total
Prop­
fert
Boxplots
of
Total
Pr
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.18.
Boxplot
of
the
Proportion
of
Eggs
Fertilized
by
Treatment
for
the
EPA
14­
Day
Methoxychlor
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.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
41
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.16.
Summary
Statistics
and
Power
Estimates
for
the
Proportion
of
Eggs
Fertilized
for
the
EPA
14­
Day
Methoxychlor
Assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
4
0.981
0.034
3%
8%
78
Low
4
0.991
0.009
1%
High
4
0.888
0.221
25%
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
4.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

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
Methoxychlor
assay
were
high,
ranging
from
0.911
(
one
Control­
treatment
replicate)
to
1.00
(
two
High­
concentration
replicates,
one
Control­
treatment
replicate)
(
Appendix
E,
Figure
1.3).
One
High­
concentration
replicate
had
a
very
low
fertilization­
success
rate,
0.294.
No
significant
differences
in
mean
fertilization­
success
rates
(
Appendix
E,
Table
1.4)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
0.27,
p
=
0.872,
df
=
2).
The
fertilization­
success
rates
for
all
treatment
replicates
for
eggs
laid
on
dishes
during
the
assay
were
high,
ranging
from
0.908
(
one
Highconcentration
replicate)
to
1.00
(
several
replicates;
including
all
treatments)
(
Appendix
E,
Figure
1.4).
No
significant
differences
in
mean
fertilization­
success
rates
(
Appendix
E,
Table
1.4)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
1.11,
p
=
0.573,
df
=
2).

3.1.11
Hatchability
and
Larval
Development
Eggs
were
collected
during
the
pre­
exposure
period
for
the
evaluation
of
hatchability.
The
proportion
of
fertilized
eggs
that
hatched
was
1.00
for
all
tanks
in
the
Control
treatment
and
Low
concentration.
The
mean
proportion
of
fertilized
eggs
that
hatched
in
the
High
concentration
was
0.99
(
sd
=
0.01).
The
proportion
of
fertilized
eggs
that
hatched
in
the
tanks
evaluated
during
the
pre­
exposure
period
but
not
used
in
the
14­
day
assay
was
1.00
in
all
tanks.
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
2.75,
p
=
0.432,
df
=
3).

Eggs
were
collected
on
Days
7
through
9
and
Day
12
during
the
EPA
14­
Day
Methoxychlor
assay
for
the
evaluation
of
hatchability.
The
proportion
of
fertilized
eggs
that
hatched
ranged
from
0.67
to
1.00
in
the
Control
treatment
and
from
0.84
to
0.94
for
the
two
test
concentrations
(
Figure
3.19).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
1.68,
p
=
0.432,
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
37
(
Table
3.17).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
42
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
1.0
0.9
0.8
0.7
treatment
Prop­
Hatch
Boxplots
of
Prop­
Hat
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.19.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
14­
Day
Methoxychlor
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
3.17.
Summary
statistics
and
power
estimates
for
the
proportion
of
fertile
eggs
that
hatched
for
the
EPA
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
4
0.91
0.16
18%
7%
37
Low
4
0.89
0.04
4%
High
1
0.89
 
 
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
2.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

Eggs
were
collected
during
the
pre­
exposure
period
for
the
evaluation
of
larval
development.
The
proportion
of
larvae
that
developed
normally
(
i.
e.,
that
showed
no
morphological
abnormalities)
was
1.00
for
all
tanks
in
the
Control
treatment.
The
mean
proportion
of
normal
larvae
in
the
remaining
treatments
was
1.00
(
sd
=
0)
in
the
Low
concentration
and
0.99
(
sd
=
0.01)
in
the
High
concentration.
The
mean
proportion
of
normal
larvae
in
the
tanks
evaluated
during
the
pre­
exposure
period
but
not
used
in
the
14­
day
assay
was
1.00
(
sd
=
0).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
normal
larvae
(
Kruskal­
Wallis,
H
=
2.46,
p
=
0.483,
df
=
3).

Eggs
were
collected
on
Days
7
through
9
and
Day
12
during
the
EPA
14­
Day
Methoxychlor
assay
for
the
evaluation
of
larval
development.
The
proportion
of
larvae
that
developed
normally
(
i.
e.,
that
showed
no
morphological
abnormalities)
was
1.00
for
all
replicates
of
the
Control
treatment
and
ranged
from
0.93
to
1.00
for
the
two
test
concentrations
(
Figure
3.20).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
normal
larvae
(
Kruskal­
Wallis,
H
=
5.00,
p
=
0.082,
df
=
2).
The
achieved
power
for
this
endpoint
was
19%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
6
(
Table
3.18).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
43
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
1.00
0.99
0.98
0.97
0.96
0.95
0.94
0.93
0.92
treatment
Prop­
Norm
Boxplots
of
Prop­
Nor
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.20.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
14­
Day
Methoxychlor
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
3.18.
Summary
statistics
and
power
estimates
for
the
proportion
of
normal
larvae
for
the
EPA
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
4
1.00
0
0%
19%
6
Low
4
0.97
0.03
3%
High
1
0.97
 
 
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
2.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

3.2
EPA
21­
Day
Assay
for
Methoxychlor
The
EPA
21­
Day
Methoxychlor
assay
was
conducted
from
September
29,
2002,
to
October
14,
2002
(
pre­
exposure
assay),
and
from
October
14,
2002,
to
November
4,
2002
(
exposure
assay).

3.2.1
Survival
All
males
and
females
in
the
Control
treatment
and
Low
concentration
survived
the
EPA
21­
Day
Methoxychlor
assay.
High
male
mortality
occurred
in
three
replicates
of
the
High
methoxychlor
concentration.
Both
males
in
these
three
replicates
expired
by
Day
7,
Day
14,
or
Day
15
of
the
exposure
and
each
tank
was
terminated.
One
male
in
the
fourth
High­
concentration
tank
died
during
the
exposure.
Female
survival
in
the
High
concentration
could
not
be
evaluated
because
the
three
tanks
in
which
both
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
44
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
males
died
were
terminated
before
the
end
of
the
assay.
All
females
in
the
one
High­
concentration
tank
that
ran
21
days
survived.

3.2.2
Vitellogenin
Vitellogenin
concentrations
in
most
Control­
treatment
females
used
during
the
EPA
21­
Day
Methoxychlor
assay
ranged
from
107,850
ng/
mL
to
14,220,000
ng/
mL
(
Figure
3.21).
Among
females
exposed
to
the
two
methoxychlor
concentrations,
vitellogenin
concentrations
ranged
from
373,200
ng/
mL
to
7,519,000
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
3.21)
were
detected
(
Kruskal­
Wallis,
H
=
5.29,
p
=
0.071,
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
36
(
Table
3.19).

Low
High
Control
15000000
10000000
5000000
0
treatment
Meth­
VTG
Boxplots
of
Meth­
VTG
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.21.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
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
3.19.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
16
4,928,594
4,073,229
83%
12%
36
Low
16
2,299,294
2,326,186
101%
High
4
2,477,975
1,330,838
54%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
4.
2
Size
required
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)
45
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Vitellogenin
concentrations
in
Control­
treatment
males
used
during
the
EPA
21­
Day
Methoxychlor
assay
ranged
from
0
ng/
mL
(
not
detected)
to
366
ng/
mL
(
Figure
3.22).
Among
males
exposed
to
the
Lowmethoxychlor
concentration,
vitellogenin
concentrations
ranged
from
0
ng/
mL
(
not
detected)
to
3,395
ng/
mL.
Only
one
male
from
the
High
concentration
was
available
for
analysis.
It
had
a
vitellogenin
concentration
of
10,890,000
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
3.20)
were
detected
(
Kruskal­
Wallis,
H
=
5.52,
p
=
0.063,
df
=
2).
The
achieved
power
for
this
endpoint
was
71%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
3
(
Table
3.20).

Low
High
Control
10000000
5000000
0
treatment
Meth­
VTG
Boxplots
of
Meth­
VTG
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.22.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
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
3.20.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
7
112
157
140%
71%
3
Low
7
866
1,214
140%
High
1
10,890,000
0
0%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
2
(
smallest
allowable).
2
Size
required
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)
46
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
3.2.3
Appearance
/
Secondary
Sex
Characteristics
All
of
the
females
used
during
the
EPA
21­
Day
Methoxychlor
assay
exhibited
typical
female
morphology
(
no
fat
pad,
no
tubercles,
ovipositor
present)
except
that
two
females
from
the
Control
treatment
lacked
ovipositors.

All
males
used
during
the
EPA
21­
Day
Methoxychlor
assay
had
typical
male
morphological
features
(
tubercles,
fat
pads,
vertical
banding,
no
ovipositor).

3.2.4
Gonadosomatic
Index
The
range
of
GSI
values
calculated
for
females
in
all
treatments
varied
from
three­
to
five­
fold
(
Figure
3.23),
and
the
overall
variability
within
the
treatment
was
moderate
to
high
(
CVs
=
41%
to
86%)
(
Table
3.21).
Several
fish
had
unusually
high
GSI
values.
The
highest
value
obtained
for
a
female
exposed
to
the
High
methoxychlor
concentration
had
a
GSI
value
of
51.1.
The
GSI
values
for
the
other
three
fish
in
this
treatment
ranged
from
12.0
to
14.0.
One
female
exposed
to
the
Low
concentration
had
a
GSI
of
37.8
and
two
control
animals
had
GSI
values
of
about
26.
The
total
body
weight
of
all
four
females
were
well
within
the
normal
range
for
the
test
animals,
and
it
appears
the
high
GSI
value
resulted
from
each
fish
having
unusually
high
gonad
weights.
The
gonad
weights
of
these
four
fish
ranged
from
0.52
g
to
0.82
g,
well
above
the
average
values
for
the
remaining
fish
(
0.2
g).
No
significant
differences
in
the
mean
GSI
value
per
treatment
were
detected
(
Kruskal­
Wallis,
H
=
1.34,
p
=
0.512,
df
=
2)
(
Table
3.21).
The
achieved
power
for
this
endpoint
was
15%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
25
(
Table
3.21).

Low
High
Control
50
40
30
20
10
treatment
GSI
Boxplots
of
GSI
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.23.
Boxplot
of
female
GSI
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
47
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.21.
Summary
Statistics
and
Power
Estimates
for
Female
Gonadosomatic
Index
Data
for
the
EPA
21­
Day
Methoxychlor
Assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
16
14.1
5.8
41%
15%
25
Low
16
13.7
7.2
52%
High
4
22.4
19.2
86%
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
4.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

The
range
of
most
GSI
values
calculated
for
males
during
the
EPA
21­
Day
Methoxychlor
assay
was
small,
ranging
from
0.7
to
1.9
(
Figure
3.24),
which
approximates
the
typical
range
for
reproductivelyactive
male
fathead
minnows.
One
male
in
the
Low
concentration
had
a
GSI
value
of
2.6,
attributable
to
its
relatively
high
gonad
weight
of
0.06
g
and
small
body
size
(
2.4
g).
There
were
no
significant
differences
in
mean
GSI
values
among
treatments
(
Kruskal­
Wallis,
H
=
1.58,
p
=
0.
454,
df
=
2)
(
Table
3.22).
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
203
(
Table
3.22).

Low
High
Control
2.5
1.5
0.5
treatment
GSI
Boxplots
of
GSI
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.24.
Boxplot
of
male
GSI
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
48
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.22.
Summary
Statistics
and
Power
Estimates
for
Male
Gonadosomatic
Index
Data
for
the
EPA
21­
Day
Methoxychlor
Assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
8
1.33
0.33
25%
5%
203
Low
8
1.23
0.59
48%
High
1
1.25
 
 
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
2.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

3.2.5
Female
Gonad
Histology
Histological
analyses
were
conducted
on
the
ovaries
of
36
females
exposed
to
methoxychlor
during
the
EPA
21­
day
assay.
Of
these
36
fish,
10
were
observed
to
have
moderate­
to­
diffuse
macrophage
infiltration
into
the
ovaries.
Tissues
from
three
fish
with
this
condition
were
evaluated
with
a
Gram
stain
and
acid­
fast
stain
that
demonstrated
acid­
fast
staining
structures
consistent
with
mycobacteria.
All
other
fish
with
a
similar
macrophage
infiltration
in
the
ovaries
were
also
presumed
to
have
mycobacteriosis.
At
the
time
of
the
histological
examination,
it
could
not
be
determined
whether
the
condition
was
exacerbated
by
the
chemical
exposure.
Because
Control­
treatment
fish
were
infected,
it
was
clear
that
the
infection
was
distributed
throughout
the
population.
To
determine
whether
the
infection
affected
the
results,
the
analyses
were
conducted
once
with
all
fish
included
and
then
were
repeated
with
the
infected
fish
excluded.
The
results
of
the
second
set
of
analyses
(
with
infected
fish
removed)
did
not
change
the
pattern
of
statistical
significance
obtained
for
the
analyses
that
included
all
fish.

General
Ovary
Staging
 
Statistical
analysis
of
the
mean
ovarian
staging
from
12
microscopic
fields
per
fish
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
3.97,
p
=
0.137,
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
Section
2:
Methods,
for
a
description
of
the
stages)
(
Figure
3.25).
Variability
within
treatments
for
each
stage
was
very
high,
as
indicated
by
CVs
that
ranged
as
high
as
400%
(
Table
3.23).
Although
statistical
analyses
showed
that
there
was
a
significant
difference
among
treatments
in
the
proportion
of
cells
in
developmental
Stage
2,
there
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
developmental
Stages
1a,
1b,
3,
4,
and
5
(
Table
3.23).
Therefore,
there
was
no
consistent
pattern
of
significant
difference
associated
with
methoxychlor
dose.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
49
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
1a
1b
2
3
4
5
Ovarian
Stage
Proportion
of
Cells
Control
Low
High
Figure
3.25.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
EPA
21­
Day
Methoxychlor
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.

Table
3.23.
Descriptive
Statistics
of
the
Proportion
of
Ovarian
Cells
in
each
Developmental
Stage
for
Females
from
the
EPA
21­
Day
Methoxychlor
Assay
and
Results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
Comparing
Treatments
Control
(
n
=
16)
Low
(
n
=
16)
High
(
n
=
4)
Kruskal­
Wallis
Stage
Mean
SD
CV
Mean
SD
CV
Value
SD
CV
H
p
1a
0.080
0.048
60%
0.089
0.036
40%
0.058
0.037
63%
2.05
0.359
1b
0.258
0.107
42%
0.306
0.093
30%
0.228
0.123
54%
2.98
0.226
2
0.204
0.055
27%
0.181
0.051
28%
0.232
0.035
15%
6.23
0.044*
3
0.178
0.069
39%
0.148
0.037
25%
0.234
0.169
72%
1.60
0.450
4
0.203
0.083
41%
0.200
0.106
53%
0.208
0.053
26%
0.62
0.732
5
0.000
0.001
400%
0.004
0.009
245%
0
0
 
1.98
0.371
*
p
<
0.05
Atretic
Follicles
 
The
mean
proportion
of
atretic
follicles
per
300
follicles
ranged
from
0.02
for
females
in
the
High
concentration
to
0.06
for
females
in
the
Control
treatment
(
Figure
3.26).
There
were
no
significant
differences
in
the
proportions
of
atretic
follicles
among
treatments
(
Kruskal­
Wallis,
H
=
0.08,
p
=
0.963,
df
=
2).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
50
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
Boxplots
of
atretic_
follicles
by
treatmen
Figure
3.26.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Methoxychlor
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
0.016
for
females
in
the
Control
treatment
to
0.019
for
females
in
the
Low
concentration
(
Figure
3.27).
There
were
no
significant
differences
in
the
proportions
of
corpora
lutea
among
treatments
(
Kruskal­
Wallis,
H
=
0.12,
p
=
0.941,
df
=
2).

Low
High
Control
0.10
0.05
0.00
treatment
corpora_
lutea
Boxplots
of
corpora_
lutea
by
treatmen
Figure
3.27.
Boxplot
of
the
proportion
corpora
lutea
per
300
follicles
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
51
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
3.2.6
Male
Gonad
Histology
Testes
Staging
by
Microscopic
Field
 
Testes
from
males
exposed
to
methoxychlor
during
the
EPA
21­
Day
Methoxychlor
assay
were
examined
to
determine
their
general
developmental
condition.
Males
in
all
treatments
had
well­
developed
testes
with
most
showing
Stage
4
and
Stage
5
development
(
see
Section
2:
Methods,
for
description
of
developmental
stages).
Ninety­
four
of
the
96
microscopic
fields
examined
in
the
8
Control­
treatment
males
showed
Stage
4
(
80
fields)
or
Stage
5
(
14
fields)
development.
Two
microscopic
fields
showed
Stage
3
development.
All
of
the
96
fields
examined
in
the
8
Lowconcentration
treatment
males
showed
Stage
4
(
86
fields)
or
Stage
5
(
10
fields)
development.
In
the
single
High­
concentration
male
available
for
examination,
all
12
microscopic
fields
showed
Stage
4
development.
Statistical
analysis
of
the
mean
staging
from
12
sections
per
fish
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
0.46,
p
=
0.795,
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
of
the
Control­
treatment
testes
ranged
from
Stage
2b
to
Stage
5,
whereas
testes
in
males
from
the
Low­
and
High­
concentration
treatments
showed
Stage
2a
to
Stage
5
development
(
Figure
3.28).
Variability
within
treatments
for
each
stage
was
very
high,
as
indicated
by
CVs
that
ranged
as
high
as
113%
(
Table
3.24).
Although
statistical
analyses
showed
that
there
was
a
significant
difference
among
treatments
in
the
proportion
of
cells
in
developmental
Stage
3b,
there
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
developmental
Stages
2a,
2b,
3a,
4,
and
5
(
Table
3.13).
Therefore,
there
was
no
consistent
pattern
of
significant
difference
associated
with
methoxychlor
dose.

Table
3.24.
Descriptive
Statistics
of
the
Proportion
of
Testes
Cells
in
Each
Developmental
Stage
for
Males
from
the
EPA
21­
Day
Methoxychlor
Assay
and
Results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
Comparing
Treatments
Control
(
n
=
8)
Low
(
n
=
8)
High
(
n
=
1)
Kruskal­
Wallis
Stage
Mean
SD
CV
Mean
SD
CV
Value
SD
CV
H
p
1
0
0
0%
0
0
0%
0
 
 
 
 
2a
0.006
0.006
104%
0.002
0.002
107%
0
 
 
4.18
0.124
2b
0.015
0.011
75%
0.013
0.015
113%
0.017
 
 
0.62
0.734
3a
0.105
0.060
57%
0.118
0.042
36%
0.333
 
 
2.80
0.247
3b
0.243
0.105
43%
0.329
0.086
26%
0.163
 
 
7.04
0.030
*
4
0.147
0.118
81%
0.198
0.100
50%
0.277
 
 
1.97
0.374
5
0.484
0.226
47%
0.340
0.162
48%
0.210
 
 
3.10
0.212
*
p
<
0.05
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
52
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
1
2a
2b
3a
3b
4
5
Testicular
Stage
Proportion
of
Cells
Control
Low
High
Figure
3.28.
Frequency
Histogram
Showing
the
Quantitative
Developmental
Staging
of
Testes
for
Each
Treatment
of
the
EPA
21­
Day
Methoxychlor
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
97.5
µ
m
to
176.7
µ
m
(
Figure
3.29).
Tubule
diameters
of
males
from
the
two
test
concentrations
ranged
from
97.8
µ
m
to
157.8
µ
m.
No
significant
differences
in
the
mean
tubule
diameter
per
treatment
were
detected
(
Kruskal­
Wallis,
H
=
2.29,
p
=
0.318,
df
=
2)
(
Table
3.25).
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
14
(
Table
3.25).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
53
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
180
170
160
150
140
130
120
110
100
90
treatment
diameter
Boxplots
of
diameter
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.29.
Boxplot
of
male
seminiferous
tubule
diameter
(
µ
m)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
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
3.25.
Summary
Statistics
and
Power
Estimates
for
Male
Seminiferous
Tubule
Diameter
Data
for
the
EPA
21­
Day
Methoxychlor
Assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
8
125.4
25.6
20%
10%
14
Low
8
118.3
23.0
19%
High
1
97.8
 
 
1
Calculated
from
natural
log
transformed
data;
sample
size
=
2
(
smallest
allowable).
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Observations
 
One
male
in
the
Low­
concentration
treatment
showed
interstitial
Sertoli
cell
proliferation
and
one
showed
multifocal
areas
of
Leydig
cell
proliferation.
One
male
from
the
High­
concentration
treatment
showed
mild
Leydig
cell
proliferation.
No
testicular
atrophy
was
recorded,
and
no
ovatestes
were
observed
for
any
treatment.

3.2.7
Plasma
Steroid
Concentrations
Estradiol
 
Estradiol
concentrations
in
Control­
treatment
females
used
during
the
EPA
21­
Day
Methoxychlor
assay
ranged
from
292
pg/
mL
to
6,208
pg/
mL
(
Figure
3.30).
Among
females
exposed
to
the
two
methoxychlor
concentrations,
estradiol
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
6,362
pg/
mL.
No
significant
differences
in
the
mean
estradiol
concentration
per
treatment
(
Table
3.26)
were
detected
(
Kruskal­
Wallis,
H
=
5.64,
p
=
0.060,
df
=
2).
The
achieved
power
for
this
endpoint
was
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
54
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
8%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
72
(
Table
3.26).

Table
3.26
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
16
2,681
1,861
69%
8%
72
Low
16
1,371
1,639
120%
High
4
3,927
2,854
73%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
4.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
7000
6000
5000
4000
3000
2000
1000
0
treatment
Estradiol
Boxplots
of
Estradio
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.30.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
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
Methoxychlor
assay
ranged
from
125
pg/
mL
to
420
pg/
mL
(
Figure
3.31).
Among
males
exposed
to
the
two
methoxychlor
concentrations,
estradiol
concentrations
ranged
from
62
pg/
mL
to
666
pg/
mL.
No
significant
differences
in
the
mean
estradiol
concentration
per
treatment
(
Table
3.27)
were
detected
(
Kruskal­
Wallis,
H
=
4.77,
p
=
0.092,
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
171
(
Table
3.27).

Table
3.27.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
55
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
EPA
21­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
7
306
96
31%
5%
171
Low
7
428
191
45%
High
1
263
 
 
1
Calculated
from
natural
log
transformed
data;
sample
size
=
2
(
smallest
allowable).
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
700
600
500
400
300
200
100
0
treatment
Estradiol
Boxplots
of
Estradio
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.31.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
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
most
Control­
treatment
females
used
during
the
EPA
21­
Day
Methoxychlor
assay
ranged
from
113
pg/
mL
to
6,035
pg/
mL
(
Figure
3.32).
One
Controltreatment
female
had
a
testosterone
concentration
of
27,568
pg/
mL.
Because
this
value
was
substantially
greater
than
any
other
female
testosterone
value,
the
statistical
analyses
were
performed
including
and
excluding
this
female.
Among
females
exposed
to
the
two
methoxychlor
concentrations,
testosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
9,356
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
3.28)
were
detected
when
all
measured
testosterone
values
were
included
in
the
analysis
(
Kruskal­
Wallis,
H
=
5.71,
p
=
0.058,
df
=
2).
The
calculated
probability
value
was
slightly
greater
than
the
critical
limit
of
0.050.
The
achieved
power
for
this
endpoint
when
all
measured
testosterone
values
were
included
in
the
analysis
was
12%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
36
(
Table
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
56
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
3.28).
When
the
Control­
treatment
female
that
had
a
High­
testosterone
concentration
was
excluded
from
the
analysis,
no
significant
differences
in
the
mean
testosterone
concentration
per
treatment
were
detected
when
all
measured
testosterone
values
were
included
in
the
analysis
(
Kruskal­
Wallis,
H
=
5.33,
p
=
0.070,
df
=
2).
The
achieved
power
for
this
endpoint
in
this
case
was
9%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
53.

Table
3.28.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay
with
all
females
having
a
measured
testosterone
value
included.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
12
4,335
7,479
173%
12%
36
Low
14
1,688
2,361
140%
High
4
3,964
3,310
84%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
4.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
30000
20000
10000
0
treatment
Testosterone
Boxplots
of
Testoste
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.32.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisks
represent
probable
outliers.

Testosterone
concentrations
in
Control­
treatment
males
used
during
the
EPA
21­
Day
Methoxychlor
assay
ranged
from
908
pg/
mL
to
8,956
pg/
mL
(
Figure
3.33).
Among
males
exposed
to
the
two
methoxychlor
concentrations,
testosterone
concentrations
ranged
from
1,288
pg/
mL
to
9,012
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
3.29)
were
detected
(
Kruskal­
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
57
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Wallis,
H
=
1.44,
p
=
0.487,
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
17
(
Table
3.29).

Table
3.29.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
8
5,209
3,008
58%
9%
17
Low
7
6,377
3,100
49%
High
1
1,741
 
 
1
Calculated
from
natural
log
transformed
data;
sample
size
=
2
(
smallest
allowable).
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
treatment
Testosterone
Boxplots
of
Testoste
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.33.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

11­
ketotestosterone
 
11­
ketotesosterone
concentrations
in
Control­
treatment
females
used
during
the
EPA
21­
Day
Methoxychlor
assay
ranged
from
528
pg/
mL
to
1,152
pg/
mL
(
Figure
3.34).
Among
females
exposed
to
the
two
methoxychlor
concentrations,
11­
ketotesosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
1,497
pg/
mL.
Significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
3.30)
were
detected
(
Kruskal­
Wallis,
H
=
7.93,
p
=
0.019,
df
=
2).
The
mean
concentration
of
11­
ketotestosterone
was
lower
in
females
exposed
to
the
Low
concentration
than
in
females
from
the
High
concentration
and
the
Control
treatment.
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
3
(
Table
3.30).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
58
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.30.
Summary
statistics
and
power
estimates
for
female
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
2
840
441
53%
44%
3
Low
11
162
451
279%
High
3
675
172
25%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
2.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Low
High
Control
1500
1000
500
0
treatment
11­
keto
Boxplots
of
11­
keto
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.34.
Boxplot
of
female
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
the
asterisks
represent
probable
outliers.

11­
ketotesosterone
concentrations
in
Control­
treatment
males
used
during
the
EPA
21­
Day
Methoxychlor
assay
ranged
from
14,531
pg/
mL
to
58,088
pg/
mL
(
Figure
3.35).
Among
males
exposed
to
the
two
methoxychlor
concentrations,
11­
ketotesosterone
concentrations
ranged
from
4,481
pg/
mL
to
88,994
pg/
mL.
No
significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
3.31)
were
detected
(
Kruskal­
Wallis,
H
=
3.34,
p
=
0.188,
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
18
(
Table
3.31).

Table
3.31.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
EPA
21­
Day
Methoxychlor
assay.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
59
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
35,211
16,809
48%
9%
18
Low
8
50,059
31,324
63%
High
1
12,764
1
Calculated
from
natural
log
transformed
data;
sample
size
=
2
(
smallest
allowable).
2
Size
required
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
Boxplots
of
11­
keto
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.35.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
EPA
21­
Day
Methoxychlor
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.

3.2.8
Body
Weight
The
body
weight
of
females
used
in
the
EPA
21­
Day
Methoxychlor
assay
ranged
from
1.0
g
to
2.2
g.
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
2.53,
p
=
0.282,
df
=
2).
The
body
weight
of
males
used
in
the
EPA
21­
Day
Methoxychlor
assay
ranged
from
2.3
g
to
5.3
g.
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
1.83,
p
=
0.400,
df
=
2).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
60
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
3.2.9
Fecundity
Total
Fecundity
 
Two
pre­
exposure
evaluations
of
total
egg
production
were
performed.
One
evaluation
examined
the
total
egg
production
at
15
days,
which
is
within
the
assay
guidelines;
the
other
evaluated
egg
production
at
Day
7
of
the
pre­
exposure
assay
to
determine
whether
or
not
the
shorter
preexposure
period
would
be
sufficient
to
evaluate
the
potential
reproductive
success
of
the
test
fish.
Total
15­
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
7,000
eggs
to
11,000
eggs
(
Figure
3.36).
No
significant
differences
in
the
mean
15­
day
egg
production
among
the
groups
of
replicates
that
would
be
used
in
the
methoxychlor
exposure
assay
were
detected
(
1­
way
ANOVA,
F
=
2.39,
p
=
0.124,
df
=
3,
11).
Production
at
Day
7
of
the
pre­
exposure
assay
among
the
12
replicates
that
were
eventually
used
in
the
exposure
assay
ranged
from
503
eggs
to
1923
eggs.
No
significant
differences
in
the
mean
7­
day
egg
production
among
the
groups
of
replicates
that
would
be
used
in
the
methoxychlor
exposure
assay
were
detected
(
1­
way
ANOVA,
F
=
1.97,
p
=
0.178,
df
=
3,
11).
No
significant
differences
were
detected
in
the
mean
number
of
eggs
laid
per
day
per
replicate
group
at
7
days
versus
15
days
(
Two­
Sample
t­
Test,
t
=
 
0.34,
p
=
0.368,
df
=
27).
However,
daily
within­
treatment
variation
(
indicated
by
fluctuating
coefficient
of
variation
[
CV]
values)
was
high
through
Day
9.
Therefore,
variability
during
a
7­
day
pre­
exposure
assay
is
likely
to
be
much
greater
than
that
during
a
pre­
exposure
assay
that
lasts
longer
than
9
days,
which
reduces
the
likelihood
of
reliably
choosing
replicates
with
successful
reproduction
histories
for
use
in
the
exposure
assay.

0
5,000
10,000
15,000
20,000
25,000
­
15
­
10
­
5
0
5
10
15
20
Day
Cumulative
Number
of
Eggs
Control
Low
High
Figure
3.36.
Total
egg
production
per
treatment
during
the
pre­
exposure
and
exposure
periods
of
the
EPA
21­
Day
Methoxychlor
assay.
The
vertical
line
at
Day
0
denotes
the
start
of
the
exposure
period.

During
the
EPA
21­
Day
Methoxychlor
assay,
total
counts
in
the
Control
treatment
were
reasonably
consistent
among
replicates,
varying
from
2437
eggs
to
2910
eggs
(
Figure
3.37).
Variability
in
total
egg
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
61
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
production
among
Low­
concentration
replicates
was
somewhat
greater,
ranging
from
1381
eggs
to
3055
eggs.
Total
counts
among
the
High­
concentration
replicates
varied
about
nine­
fold,
from
185
eggs
to
1744
eggs.
Statistical
analysis
of
square­
root
transformed
egg
counts
showed
significant
amongtreatment
differences
(
1­
way
ANOVA,
F
=
7.29,
p
=
0.013,
df
=
2,
9)
in
mean
total
numbers
of
eggs
produced
(
Table
3.32).
Tukey's
pairwise
comparison
identified
significant
differences
in
mean
egg
production
between
the
High
concentration
and
the
Control
treatment,
but
not
between
the
High
and
Low
concentrations
or
between
the
Low
concentration
and
the
Control
treatment.
Daily
within­
treatment
variation
(
indicated
by
fluctuating
coefficient
of
variation
values)
was
much
reduced
after
Day
9
through
Day
11,
except
for
the
High
concentration.
The
achieved
power
for
this
assay
was
81%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
4
(
Table
3.32).

0
500
1000
1500
2000
2500
3000
3500
0
5
10
15
20
25
Day
Cumulative
Number
of
Eggs
Control
Low
High
Figure
3.37.
Total
egg
production
by
replicate
per
treatment
during
the
EPA
21­
Day
Methoxychlor
assay.

Table
3.32.
Summary
Statistics
and
Power
Estimates
for
Total
Fecundity
Data
for
the
EPA
21­
Day
Methoxychlor
Assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
4
2727
209
8%
81%
4
Low
4
2039
759
37%
High
4
989
661
67%
1
Calculated
from
square­
root
transformed
data;
sample
size
=
4.
2
Required
size
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)
62
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Fecundity
per
Female
Reproductive
Day
 
During
the
15­
day
pre­
exposure
evaluation,
the
mean
number
of
eggs
produced
per
female
reproductive
day
ranged
from
28.0
eggs/
day
for
the
tanks
that
would
be
used
for
the
High­
concentration
treatment
to
46.6
eggs/
day
for
the
tanks
that
would
be
used
for
the
Low
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
=
6.06,
p
=
0.109,
df
=
3).

During
the
EPA
21­
Day
Methoxychlor
assay,
the
maximum
number
of
female
reproductive
days
(
84)
was
achieved
for
the
Control
and
Low­
concentration
treatments,
whereas
only
55
female
reproductive
days
(
65%
of
the
maximum)
were
achieved
in
the
High
concentration
(
Table
3.33).
The
number
of
eggs
produced
per
female
reproductive
day
varied
from
29.0
eggs
to
34.6
eggs
in
the
Control
treatment
and
from
16.4
to
36.4
in
the
Low
concentration
(
Figure
3.38).
For
the
High
concentration,
the
number
of
eggs
produced
per
female
reproductive
day
ranged
from
4.2
eggs
to
24.8
eggs,
although
fish
in
three
of
the
replicates
yielded
more
than
20
eggs
per
day
(
20.5,
20.8,
24.8).
No
significant
difference
in
the
number
of
eggs
produced
per
female
reproductive
day
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
4.77,
p
=
0.092,
df
=
2).
The
achieved
power
for
this
assay
was
35%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
10
(
Table
3.33).

Table
3.33.
Summary
Statistics
and
Power
Estimates
for
Fecundity
per
Female
Reproductive
Day
for
the
EPA
21­
Day
Methoxychlor
Assay
Level
Mean
Number
of
Reproductive
Days
1
N
Mean
SD
CV
Achieved
Power
2
Sample
Size
Required
3
Control
84.0
4
32.5
2.5
8%
35%
10
Low
84.0
4
24.3
9.1
37%
High
55.0
4
17.6
9.1
52%
1
Maximum
number
=
84.
2
Calculated
from
natural
log
transformed
data;
sample
size
=
4.
3
Required
size
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)
63
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
40
30
20
10
0
treatment
eggs/
ReproDay
Boxplots
of
eggs/
Rep
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.38.
Boxplot
of
the
number
of
eggs
produced
per
female
reproductive
day
by
treatment
for
the
EPA
21­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
and
the
circle
is
the
mean
value.

Eggs
on
Tiles/
Dishes
 
The
mean
number
of
eggs
laid
on
the
tiles
during
the
15­
day
pre­
exposure
assay
varied
from
1424
eggs
for
the
tanks
that
would
be
used
for
the
High
concentration
to
2612
eggs
for
the
tanks
that
would
be
used
for
the
Control
treatment.
The
mean
number
of
eggs
laid
on
the
dishes
during
the
15­
day
pre­
exposure
assay
varied
from
166
eggs
for
the
tanks
that
would
be
used
for
the
Control
treatment
to
360
eggs
for
the
tanks
that
would
be
used
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.
There
were
no
significant
differences
in
the
mean
proportional
difference
among
treatments
during
the
15­
day
preexposure
assay
(
Kruskal­
Wallis,
H
=
2.33,
p
=
0.508,
df
=
3).

The
mean
number
of
eggs
laid
on
the
tiles
among
the
treatments
during
the
EPA
21­
Day
Methoxychlor
assay
varied
from
844
eggs
for
the
High
concentration
to
2,468
eggs
for
the
Control
treatment
(
Appendix
E,
Table
1.7).
The
number
of
eggs
on
dishes
ranged
from
145
eggs
for
the
High
concentration
to
259
eggs
for
the
Control
treatment.
The
proportional
difference
ranged
from
0.27
(
one
Highconcentration
replicate)
to
0.95
to
0.96
(
also
High­
concentration
replicates)
(
Appendix
E,
Figure
1.6).
There
were
no
significant
differences
in
this
mean
proportional
difference
among
treatments
(
Kruskal­
Wallis,
H
=
0.35,
p
=
0.841,
df
=
2).

3.2.10
Fertilization
Success
Total
Fertilization
 
Eggs
were
collected
during
the
15­
day
pre­
exposure
period
for
the
evaluation
of
fertilization­
success
rate.
The
mean
proportion
of
eggs
fertilized
in
the
Control
treatment
was
0.998
(
sd
=
0.001),
0.995
(
sd
=
0.006)
in
the
Low
concentration,
and
0.997
(
sd
=
0.002)
in
the
High
concentration.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
64
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
The
mean
proportion
of
eggs
fertilized
in
the
replicates
that
were
not
used
in
the
21­
day
validation
assay
was
0.990
(
sd
=
0.015).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
0.71,
p
=
0.870,
df
=
3).

The
total
(
tiles
+
dishes)
fertilization­
success
rates
for
all
treatment
replicates
during
the
EPA
21­
Day
Methoxychlor
assay
were
high,
ranging
from
0.987
(
one
Low­
concentration
replicate)
to
1.00
(
one
Highconcentration
replicate)
(
Figure
3.39).
No
significant
differences
in
mean
fertilization­
success
rates
(
Table
3.34)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
0.73,
p
=
0.694,
df
=
2).
The
achieved
power
for
this
assay
was
10%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
46
(
Table
3.34).

Low
High
Control
1.000
0.995
0.990
treatment
Total
Prop­
fert
Boxplots
of
Total
Pr
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.39.
Boxplot
of
the
proportion
of
eggs
fertilized
by
treatment
for
the
EPA
21­
Day
Methoxychlor
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
3.34.
Summary
Statistics
and
Power
Estimates
for
the
Proportion
of
Eggs
Fertilized
for
the
EPA
21­
Day
Methoxychlor
Assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
4
0.998
0.001
0%
10%
46
Low
4
0.995
0.006
1%
High
4
0.996
0.003
0%
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
4.
2
Required
size
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)
65
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Fertilization
of
Eggs
on
Tiles
and
Dishes
 
During
the
15­
day
pre­
validation
assay,
there
were
no
significant
differences
in
the
fertilization­
success
rates
among
treatments
for
eggs
laid
in
tiles
(
Kruskal­
Wallis,
H
=
0.68,
p
=
0.879,
df
=
3)
or
on
dishes
(
Kruskal­
Wallis,
H
=
0.35,
p
=
0.950,
df
=
3).
The
fertilization­
success
rates
for
all
treatment
replicates
for
eggs
laid
on
tiles
during
the
EPA
21­
Day
Methoxychlor
assay
were
high,
ranging
from
0.984
(
one
Low­
concentration
replicate)
to
1.00
(
two
Highconcentration
replicates)
(
Appendix
E,
Figure
1.8).
No
significant
differences
in
mean
fertilizationsuccess
rates
(
Appendix
E,
Table
1.9)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
0.46,
p
=
0.793,
df
=
2).

The
fertilization­
success
rates
for
all
treatment
replicates
for
eggs
laid
on
dishes
during
the
assay
were
high,
ranging
from
0.945
(
one
High­
concentration
replicate)
to
1.00
(
several
replicates;
including
all
treatments)
(
Appendix
E,
Figure
1.9).
No
significant
differences
in
mean
fertilization­
success
rates
(
Appendix
E,
Table
1.9)
among
treatments
were
detected
(
Kruskal­
Wallis,
H
=
0.67,
p
=
0.716,
df
=
2).

3.2.11
Hatchability
and
Larval
Development
Eggs
were
collected
during
the
pre­
exposure
period
for
the
evaluation
of
hatchability.
The
proportion
of
fertilized
eggs
that
hatched
was
1.00
for
all
tanks
in
the
Control
treatment,
Low
concentration,
and
from
three
tanks
in
the
High
concentration.
The
proportion
of
fertilized
eggs
that
hatched
in
the
fourth
Highconcentration
replicate
was
0.98.
The
mean
proportion
of
fertilized
eggs
that
hatched
in
the
tanks
evaluated
during
the
pre­
exposure
period
but
not
used
in
the
21­
day
assay
was
0.97
(
sd
=
0.07).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
2.15,
p
=
0.542,
df
=
3).

Eggs
were
collected
between
Days
7
through
10,
Days
12
through
16,
and
on
Day
21
during
the
EPA
21­
Day
Methoxychlor
assay
for
the
evaluation
of
hatchability.
The
proportion
of
fertilized
eggs
that
hatched
ranged
from
0.75
to
0.96
in
the
Control
treatment
and
from
0.48
to
0.98
for
the
two
test
concentrations
(
Figure
3.40).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
eggs
that
hatched
(
Kruskal­
Wallis,
H
=
1.48,
p
=
0.476,
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
25
(
Table
3.35).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
66
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Low
High
Control
1.0
0.9
0.8
0.7
0.6
0.5
treatment
Prop­
Hatch
Boxplots
of
Prop­
Hat
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.40.
Boxplot
of
the
proportion
of
fertile
eggs
that
hatched
by
treatment
for
the
EPA
21­
Day
Methoxychlor
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
3.35.
Summary
Statistics
and
Power
Estimates
for
the
Proportion
of
Fertile
Eggs
that
Hatched
for
the
EPA
21­
Day
Methoxychlor
Assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
8
0.88
0.07
8%
8%
25
Low
8
0.74
0.20
27%
High
3
0.91
0.06
7%
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
3.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

Eggs
were
collected
during
the
pre­
exposure
period
for
the
evaluation
of
larval
development.
The
proportion
of
larvae
that
developed
normally
(
i.
e.,
that
showed
no
morphological
abnormalities)
was
1.00
for
all
tanks
in
the
Control
treatment.
The
mean
proportion
of
normal
larvae
in
the
remaining
treatments
was
0.98
(
sd
=
0.04)
in
the
Low
concentration
and
0.995
(
sd
=
0.01)
in
the
High
concentration.
The
mean
proportion
of
normal
larvae
in
the
tanks
evaluated
during
the
pre­
exposure
period
but
not
used
in
the
21­
day
assay,
was
0.95
(
sd
=
0.07).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
normal
larvae
(
Kruskal­
Wallis,
H
=
3.37,
p
=
0.337,
df
=
3).

Eggs
were
collected
between
Days
7
through
10,
Days
12
through
16,
and
on
Day
21
during
the
EPA
21­
Day
Methoxychlor
assay
for
the
evaluation
of
larval
development.
The
proportion
of
larvae
that
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
67
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
developed
normally
(
i.
e.,
that
showed
no
morphological
abnormalities)
ranged
from
0.97
to
1.00
in
the
Control
treatment
and
from
0.96
to
1.00
for
the
two
test
concentrations
(
Figure
3.41).
There
were
no
significant
differences
among
treatments
in
the
proportion
of
normal
larvae
(
Kruskal­
Wallis,
H
=
0.88,
p
=
0.643,
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
49
(
Table
3.36).

Low
High
Control
1.00
0.99
0.98
0.97
0.96
treatment
Prop­
Norm
Boxplots
of
Prop­
Nor
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.41.
Boxplot
of
the
proportion
of
normal
larvae
by
treatment
for
the
EPA
21­
Day
Methoxychlor
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
3.36.
Summary
Statistics
and
Power
Estimates
for
the
Proportion
of
Normal
Larvae
for
the
EPA
21­
Day
Methoxychlor
Assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
8
0.994
0.011
1%
6%
49
Low
8
0.994
0.014
1%
High
3
1.000
0
0%
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
3.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

3.3
Non­
spawning
Adult
14­
day
Assay
for
Methoxychlor
The
non­
spawning
adult
14­
Day
Methoxychlor
assay
was
conducted
from
October
14,
2002,
to
October
28,
2002
(
exposure
assay).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
68
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
3.3.1
Survival
Total
survival
in
the
Control
treatment
during
the
non­
spawning
adult
14­
Day
Methoxychlor
assay
was
100%.
Among
the
three
concentration
treatments,
total
survival
ranged
from
90%
(
High
concentration)
to
100%
(
Low
concentration).
One
female
died
in
the
High
and
Medium
concentrations
and
one
male
died
in
the
High
concentration.

3.3.2
Vitellogenin
Vitellogenin
concentrations
in
Control­
treatment
females
used
during
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
ranged
from
78,344
ng/
mL
to
1,620,000
ng/
mL
(
Figure
3.42).
Among
females
exposed
to
the
three
methoxychlor
concentrations,
vitellogenin
concentrations
ranged
from
13,512
ng/
mL
to
30,970,000
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
among
treatments
(
Table
3.37)
were
detected
(
Kruskal­
Wallis,
H
=
1.52,
p
=
0.677,
df
=
2).
The
achieved
power
for
this
endpoint
was
98%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
5
(
Table
3.37).

Table
3.37.
Summary
statistics
and
power
estimates
for
female
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
10
6,959,184
4,999,771
72%
11%
91
Low
10
9,617,600
9,591,390
100%
Medium
9
8,056,562
7,644,691
95%
High
9
10,758,278
7,481,935
70%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
9.
2
Size
required
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)
69
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
30000000
20000000
10000000
0
treatment
Meth­
VTG
Boxplots
of
Meth­
VTG
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.42.
Boxplot
of
female
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
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,
the
asterisk
represents
a
probable
outlier.

Vitellogenin
concentrations
in
Control­
treatment
males
used
during
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
ranged
from
0
ng/
mL
(
not
detected)
to
1,818
ng/
mL
(
Figure
3.43).
Among
males
exposed
to
the
three
methoxychlor
concentrations,
vitellogenin
concentrations
ranged
from
0
ng/
mL
(
not
detected)
to
517,020
ng/
mL.
No
significant
differences
in
the
mean
vitellogenin
concentration
per
treatment
(
Table
3.38)
were
detected
(
Kruskal­
Wallis,
H
=
2.48,
p
=
0.478,
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
47
(
Table
3.38).

Table
3.38.
Summary
statistics
and
power
estimates
for
male
vitellogenin
concentrations
(
ng/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
10
229
563
246%
18%
47
Low
10
1,566
3,242
207%
Medium
10
505
1,213
240%
High
9
158,488
219,547
139%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
9.
2
Size
required
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)
70
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
500000
400000
300000
200000
100000
0
treatment
Meth­
VTG
Boxplots
of
Meth­
VTG
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.43.
Boxplot
of
male
vitellogenin
concentration
(
ng/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
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.

3.3.3
Appearance
/
Secondary
Sex
Characteristics
All
females
used
during
the
methoxychlor
Non­
spawning
Adult
14­
day
assay
showed
normal
female
morphology.

Morphological
development
among
39
males
used
during
the
methoxychlor
Non­
spawning
Adult
14­
day
assay
varied
among
treatments
(
Figure
3.44).
Four
males
used
during
the
assay
lacked
tubercles.
Six
males
lacked
a
dorsal
fat
pad.
Thirty­
six
males
lacked
vertical
banding.
There
was
no
consistent
doserelated
pattern
to
these
variations
in
morphology.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
71
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
Tubercles
Abs
Tubercles
Prs
Fat
Pad
NP
Fat
Pad
P
Fat
Pad
Pro
Vert
Banding
NP
Vert
Banding
P
Vert
Banding
Pro
Proportion
Control
Low
Mid
High
Figure
3.44.
Secondary
sex
characteristics
of
males
used
during
the
methoxychlor
Non­
spawning
Adult
14­
Day
assay.

3.3.4
Gonadosomatic
Index
The
range
of
GSI
values
calculated
for
females
in
the
all
treatments
varied
from
two­
to
four­
fold
(
Figure
3.45),
and
the
overall
within­
treatment
variability
was
moderate
(
CVs
=
21%
to
38%)
(
Table
3.39).
The
highest
female
GSI
value
was
34.6
(
one
fish
in
the
Control
treatment),
but
several
fish
had
GSI
values
>
20.
There
were
no
significant
differences
in
mean
GSI
values
among
treatments
(
Kruskal­
Wallis,
H
=
0.91,
p
=
0.822,
df
=
3)
(
Table
3.39).
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
222
(
Table
3.39).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
72
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
35
25
15
5
treatment
GSI
Boxplots
of
GSI
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.45.
Boxplot
of
female
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Table
3.39.
Summary
statistics
and
power
estimates
for
female
gonadosomatic
index
data
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
10
20.7
6.4
31%
7%
222
Low
10
18.8
7.2
38%
Medium
9
20.4
6.5
32%
High
9
20.9
4.4
21%
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
9.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

The
range
of
most
GSI
values
calculated
for
males
during
the
non­
spawning
adult
14­
Day
Methoxychlor
assay
was
large,
ranging
from
0.8
to
2.0
(
Figure
3.46),
which
approximates
the
typical
range
for
reproductively­
active
male
fathead
minnows.
Overall
within­
treatment
variability
was
moderate
to
high
(
CVs
=
18%
to
67%)
(
Table
3.40).
The
highest
and
lowest
male
GSI
values
were
2.4
to
2.7
(
for
two
fish
in
the
Medium
concentration)
and
0.3
to
0.6
(
four
fish
in
the
Medium
concentration),
respectively.
However,
there
were
no
significant
differences
(
with
 
=
0.05)
in
mean
GSI
values
among
treatments
(
Kruskal­
Wallis,
H
=
7.20,
p
=
0.066,
df
=
3)
(
Table
3.40).
The
achieved
power
for
this
endpoint
was
33%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
26
(
Table
3.40).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
73
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
6
5
4
3
2
1
0
treatment
GSI
Boxplots
of
GSI
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.46.
Boxplot
of
male
GSI
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Table
3.40.
Summary
statistics
and
power
estimates
for
male
gonadosomatic
index
data
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
10
1.34
0.33
25%
33%
26
Low
10
1.46
0.26
18%
Medium
10
1.23
0.82
67%
High
9
2.15
1.35
63%
1
Calculated
from
arcsine
square­
root
transformed
data;
sample
size
=
9.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
arcsine
square­
root
transformed
data.

3.3.5
Female
Gonad
Histology
A
histological
analysis
was
performed
on
the
ovaries
of
39
females
exposed
to
methoxychlor
during
the
non­
spawning
adult
14­
day
assay.
Of
these
35
fish,
8
were
observed
to
have
moderate­
to­
diffuse
macrophage
infiltration
into
the
ovaries.
Tissues
from
three
fish
with
this
condition
were
evaluated
with
a
Gram
stain
and
an
acid­
fast
stain
that
demonstrated
acid­
fast­
staining
structures
consistent
with
mycobacteria.
All
other
fish
with
a
similar
macrophage
infiltration
in
the
ovaries
were
also
presumed
to
have
mycobacteriosis.
At
the
time
of
the
histological
examination,
it
could
not
be
determined
whether
the
condition
was
exacerbated
by
the
chemical
exposure.
Because
one
Control­
treatment
fish
was
infected,
it
was
clear
that
the
infection
was
distributed
throughout
the
population.
To
determine
whether
the
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
74
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
infection
affected
the
results,
the
analyses
were
conducted
once
with
all
fish
included
and
then
repeated
with
the
infected
fish
excluded.
The
results
of
the
second
set
of
analyses
(
with
infected
fish
removed)
were
reported
only
if
they
resulted
in
a
change
in
the
pattern
of
statistical
significance
obtained
for
the
analyses
that
included
all
fish.

General
Ovary
Staging
 
Statistical
analysis
of
the
mean
ovarian
staging
from
12
microscopic
fields
per
fish
revealed
no
significant
differences
among
treatments
(
Kruskal­
Wallis,
H
=
5.46,
p
=
0.141,
df
=
3).
There
was
no
change
in
this
pattern
of
statistical
significance
when
the
infected
fish
were
excluded
from
the
analysis.

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
Section
2:
Methods
for
a
description
of
the
stages)
(
Figure
3.47).
Variability
within
treatments
for
each
stage
was
very
high,
as
indicated
by
CVs
that
ranged
as
high
as
316%
(
Table
3.41).
Although
statistical
analyses
showed
that
there
was
a
significant
difference
among
treatments
in
the
proportion
of
cells
in
developmental
Stages
1a
and
4,
there
were
no
significant
differences
among
treatments
in
the
proportion
of
cells
in
the
developmental
Stages
1b,
2,
3,
and
5
(
Table
3.41).
The
proportion
of
cells
in
developmental
Stage
1a
in
the
Medium
concentration
was
significantly
lower
than
those
in
the
High­
and
Low­
concentration
treatments.
The
proportion
of
cells
in
developmental
Stage
4
in
the
Control
treatment
was
significantly
lower
than
those
in
the
Medium­
and
High­
concentration
treatments.
There
was
no
consistent
trend
of
significant
difference
associated
with
the
methoxychlor
dose.
When
infected
fish
were
excluded
from
the
analyses,
there
were
no
significant
differences
in
any
of
the
developmental
stages
among
treatments
(
Kruskal­
Wallis,
Stage
1a
 
H
=
5.18,
p
=
0.159,
df
=
3;
Stage
4
 
H
=
7.17,
p
=
0.067,
df
=
3).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
75
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
1a
1b
2
3
4
5
Ovarian
Stage
Proportion
of
Cells
Control
Low
Medium
High
Figure
3.47.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
ovaries
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.

Table
3.41.
Descriptive
statistics
of
the
proportion
of
ovarian
cells
in
each
developmental
stage
for
females
from
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments
Control
(
n
=
10)
Low
(
n
=
10)
Medium
(
n
=
9)
High
(
n
=
9)
Kruskal­
Wallis
Stage
Mean
SD
CV
Mean
SD
CV
Mean
SD
CV
Mean
SD
CV
H
p
1a
0.055
0.028
51%
0.074
0.029
39%
0.036
0.023
63%
0.073
0.021
28%
10.14
0.017*
1b
0.305
0.077
25%
0.271
0.083
31%
0.230
0.064
28%
0.269
0.046
17%
3.70
0.296
2
0.182
0.047
26%
0.183
0.048
26%
0.180
0.090
50%
0.169
0.031
19%
1.27
0.736
3
0.145
0.062
43%
0.150
0.046
30%
0.169
0.070
41%
0.133
0.057
43%
1.28
0.735
4
0.202
0.098
49%
0.293
0.124
42%
0.295
0.114
39%
0.308
0.077
25%
9.52
0.023*
5
0.002
0.004
216%
0.000
0.001
316%
0.005
0.012
236%
0
0
 
2.68
0.443
*
p
<
0.05
Atretic
Follicles
 
The
mean
proportion
of
atretic
follicles
per
300
follicles
(
counted
per
fish)
ranged
from
0.003
for
females
in
the
Kiwconcentration
to
0.10
for
females
in
the
Control
treatment
(
Figure
3.48).
There
was
a
significant
difference
in
the
proportions
of
atretic
follicles
among
treatments
(
Kruskal­
Wallis,
H
=
8.89,
p
=
0.031,
df
=
3).
The
values
for
females
in
the
Low
concentration
was
lower
than
those
for
females
in
the
Control
treatment.
However,
there
was
no
consistent
pattern
of
significant
difference
associated
with
the
methoxychlor
dose.
There
was
no
change
in
this
pattern
of
statistical
significance
when
the
infected
fish
were
excluded
from
the
analysis.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
76
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
Boxplots
of
atretic_
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.48.
Boxplot
of
the
proportion
of
atretic
follicles
per
300
follicles
by
treatment
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
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.

Corpora
Lutea
 
The
mean
proportion
of
corpora
lutea
per
300
follicles
(
counted
per
fish)
ranged
from
0.002
for
females
in
the
Medium
concentration
to
0.017
for
females
in
the
Low
concentration
(
Figure
3.49).
There
were
no
significant
differences
in
the
proportions
of
corpora
lutea
among
treatments
(
Kruskal­
Wallis,
H
=
6.19,
p
=
0.103,
df
=
3).
There
was
no
change
in
this
pattern
of
statistical
significance
when
the
infected
fish
were
excluded
from
the
analysis.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
77
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
0.05
0.04
0.03
0.02
0.01
0.00
treatment
corpora_
lutea
Boxplots
of
corpora_
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.49.
Boxplot
of
the
proportion
of
corpora
lutea
per
300
follicles
by
treatment
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
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.

3.3.6
Male
Gonad
Histology
Testes
Staging
by
Microscopic
Field
 
Testes
from
males
exposed
to
methoxychlor
during
the
nonspawning
adult
14­
Day
Methoxychlor
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
Section
2:
Methods
for
description
of
developmental
stages).
All
of
the
120
microscopic
fields
examined
in
the
10
Control­
treatment
males
showed
Stage
4
(
23
fields)
or
Stage
5
(
97
fields)
development.
All
of
the
120
microscopic
fields
examined
in
the
10
Low­
concentration
treatment
males
showed
Stage
4
(
60
fields)
or
Stage
5
(
60
fields)
development.
In
the
8
Medium­
concentration
males
available
for
examination,
all
of
the
96
microscopic
fields
showed
Stage
4
(
58
fields)
or
Stage
5
(
38
fields)
development.
In
the
8
High­
concentration
males
available
for
examination,
all
of
the
96
microscopic
fields
showed
Stage
4
(
38
fields)
or
Stage
5
(
58
fields)
development.
Statistical
analysis
of
the
mean
staging
from
12
microscopic
fields
per
fish
revealed
that
there
was
a
significant
difference
among
treatments
(
Kruskal­
Wallis,
H
=
10.54,
p
=
0.014,
df
=
3).
The
mean
testes
stage
for
males
in
the
Control
treatment
(
4.81,
sd
=
0.21)
was
significantly
greater
than
that
in
the
Medium­
concentration
(
4.40,
sd
=
0.37)
and
Low­
concentration
treatment
(
4.49,
sd
=
0.18),
but
not
greater
than
that
in
the
Highconcentration
treatment.

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
of
the
testes
from
all
treatments
ranged
from
Stage
2a
to
Stage
5
(
Figure
3.50).
Variability
within
treatments
for
each
stage
was
very
high,
as
indicated
by
CVs
that
ranged
as
high
as
186%
(
Table
3.42).
Although
statistical
analyses
showed
that
there
was
a
significant
difference
among
treatments
in
the
proportion
of
cells
in
developmental
Stage
2b,
there
were
no
significant
differences
among
treatments
in
the
proportion
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
78
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
of
cells
in
developmental
Stages
2a,
3a,
3b,
4,
and
5
(
Table
3.42).
Therefore,
there
was
no
consistent
pattern
of
significant
difference
associated
with
methoxychlor
dose.

0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1
2a
2b
3a
3b
4
5
Testicular
Stage
Proportion
of
Cells
Control
Low
Mid
High
Figure
3.50.
Frequency
histogram
showing
the
quantitative
developmental
staging
of
testes
for
each
treatment
of
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.
For
each
treatment,
the
columns
represent
the
grand
mean
proportion
of
cells
in
each
stage
and
the
bars
represent
the
standard
deviation.

Table
3.42.
Descriptive
statistics
of
the
proportion
of
testes
cells
in
each
developmental
stage
for
males
from
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
and
results
of
the
Kruskal­
Wallis
Test
(
df
=
2)
comparing
treatments
Control
(
n
=
10)
Low
(
n
=
10)
Medium
(
n
=
8)
High
(
n
=
8)
Kruskal­
Wallis
Stage
Mean
SD
CV
Mean
SD
CV
Mean
SD
CV
Mean
SD
CV
H
p
1
0
 
 
0
 
 
0
 
 
0
 
 
 
 
2a
0.003
0.003
99%
0.003
0.005
175%
0.001
0.002
186%
0.003
0.003
118%
2.41
0.491
2b
0.010
0.017
162%
0.028
0.018
65%
0.013
0.018
137%
0.008
0.004
57%
8.94
0.030*
3a
0.074
0.074
100%
0.056
0.053
94%
0.057
0.088
155%
0.025
0.022
87%
2.42
0.490
3b
0.080
0.065
81%
0.104
0.067
64%
0.125
0.085
68%
0.133
0.081
61%
3.23
0.358
4
0.073
0.114
156%
0.085
0.089
104%
0.076
0.095
126%
0.114
0.102
89%
1.79
0.617
5
0.760
0.248
33%
0.723
0.120
17%
0.729
0.250
34%
0.718
0.183
25%
1.98
0.576
*
p
<
0.05
Tubule
Diameter
 
The
diameter
of
the
seminiferous
tubules
of
males
from
the
Control
treatment
ranged
from
120.0
µ
m
to
189.2
µ
m
(
Figure
3.51).
Tubule
diameters
of
males
from
the
three
test
concentrations
ranged
from
89.2
µ
m
to
208.6
µ
m.
No
significant
differences
in
the
mean
tubule
diameter
per
treatment
were
detected
(
Kruskal­
Wallis,
H
=
4.98,
p
=
0.173,
df
=
3)
(
Table
3.43).
The
achieved
power
for
this
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
79
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
endpoint
was
26%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
29
(
Table
3.43).

Mid
Low
High
Control
200
150
100
treatment
diameter
Boxplots
of
diameter
by
treatmen
Figure
3.51.
Boxplot
of
seminiferous
tubule
diameter
(
µ
m)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Table
3.43.
Summary
statistics
and
power
estimates
for
male
seminiferous
tubule
diameter
data
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
10
154.1
23.2
15%
26%
29
Low
10
158.3
18.1
11%
Medium
8
133.1
28.4
21%
High
8
147.3
29.6
20%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
8.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Observations
 
One
male
in
the
Medium­
concentration
treatment
showed
interstitial
Sertoli
cell
proliferation
and
one
showed
multiple
foci
of
inflammatory
cells
in
the
testes.
No
testicular
atrophy
was
recorded
and
no
ovatestes
were
observed
for
any
treatment.
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
80
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
3.3.7
Plasma
Steroid
Concentrations
Estradiol
 
Estradiol
concentrations
in
Control­
treatment
females
used
during
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
ranged
from
306
pg/
mL
to
3,953
pg/
mL
(
Figure
3.52).
Among
females
exposed
to
the
two
methoxychlor
concentrations,
estradiol
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
6,336
pg/
mL.
No
significant
differences
in
the
mean
estradiol
concentration
per
treatment
(
Table
3.44)
were
detected
(
Kruskal­
Wallis,
H
=
5.52,
p
=
0.137,
df
=
2).
The
achieved
power
for
this
endpoint
was
32%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
20
(
Table
3.44).

Table
3.44.
Summary
statistics
and
power
estimates
for
female
estradiol
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
7
1,732
1,375
79%
32%
20
Low
10
517
480
93%
Medium
7
2,611
2,417
93%
High
8
1,183
1,953
165%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
7.
2
Size
required
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
Estradiol
Boxplots
of
Estradio
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.52.
Boxplot
of
female
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
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)
81
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Estradiol
concentrations
in
Control­
treatment
males
used
during
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
ranged
from
135
pg/
mL
to
312
pg/
mL
(
Figure
3.53).
Estradiol
was
not
detected
in
males
exposed
to
the
three
methoxychlor
concentrations
(
Table
3.45).
Achieved
power
was
not
calculated
for
this
endpoint.

Table
3.45.
Summary
statistics
and
power
estimates
for
male
estradiol
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
1
Control
8
229
64
28%
 
 
Low
6
0
0
 
Medium
5
0
0
 
High
5
0
0
 
1
Not
calculated
for
this
endpoint.

Mid
Low
High
Control
300
200
100
0
treatment
Estradiol
Boxplots
of
Estradio
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.53.
Boxplot
of
male
estradiol
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
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
Methoxychlor
assay
ranged
from
0
pg/
mL
(
not
detected)
to
7,781
pg/
mL
(
Figure
3.54).
Among
females
exposed
to
the
three
methoxychlor
concentrations,
testosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
9,649
pg/
mL.
A
significant
difference
in
the
mean
testosterone
concentration
per
treatment
(
Table
3.46)
was
detected
(
Kruskal­
Wallis,
H
=
8.23,
p
=
0.041,
df
=
2).
The
mean
concentration
of
testosterone
in
females
from
the
Medium
concentration
was
greater
than
that
in
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
82
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
females
from
the
Low
concentration.
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
38
(
Table
3.46).

Table
3.46.
Summary
statistics
and
power
estimates
for
female
testosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
7
3,063
2,795
91%
7%
38
Low
7
789
564
71%
Medium
4
5,379
3,168
59%
High
2
1,929
2,228
116%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
2.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Mid
Low
High
Control
10000
5000
0
treatment
Testosterone
Boxplots
of
Testoste
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.54.
Boxplot
of
female
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
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
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
ranged
from
132
pg/
mL
to
5,170
pg/
mL
(
Figure
3.55).
Among
males
exposed
to
the
three
methoxychlor
concentrations,
testosterone
concentrations
ranged
from
295
pg/
mL
to
7,802
pg/
mL.
No
significant
differences
in
the
mean
testosterone
concentration
per
treatment
(
Table
3.47)
were
detected
(
Kruskal­
Wallis,
H
=
5.45,
p
=
0.142,
df
=
2).
The
achieved
power
for
this
endpoint
was
14%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
33
(
Table
3.47).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
83
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.47.
Summary
statistics
and
power
estimates
for
male
testosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
8
2,247
1,855
83%
14%
33
Low
5
2,026
1,375
68%
Medium
7
703
635
90%
High
7
3,195
2,774
87%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
5.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Mid
Low
High
Control
8000
7000
6000
5000
4000
3000
2000
1000
0
treatment
Testosterone
Boxplots
of
Testoste
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.55.
Boxplot
of
male
testosterone
concentration
(
pg/
mL)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
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
only
detected
in
one
of
five
Control­
treatment
females
used
during
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
(
Figure
3.56).
Among
females
exposed
to
the
three
methoxychlor
concentrations,
11­
ketotesosterone
was
only
detected
in
one
individual
from
the
Low
and
Medium
concentrations
(
four
and
three
females
analyzed
for
each
treatment,
respectively).
11­
ketotesosterone
was
not
detected
in
the
single
female
from
the
High
concentration
available
for
analysis.
No
significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
3.48)
were
detected
(
Kruskal­
Wallis,
H
=
0.44,
p
=
0.932,
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
128
(
Table
3.48).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
84
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Table
3.48.
Summary
statistics
and
power
estimates
for
female
11­
ketotesosterone
concentrations
(
pg/
mL)
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
5
186
415
224%
5%
128
Low
4
80
159
200%
Medium
3
260
450
173%
High
1
0
0
 
1
Calculated
from
natural
log
transformed
data;
sample
size
=
2
(
smallest
allowable).
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Mid
Low
High
Control
1000
900
800
700
600
500
400
300
200
100
0
treatment
11­
keto
Boxplots
of
11­
keto
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.56.
Boxplot
of
female
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
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
ketotesosterone
concentrations
in
Control­
treatment
males
used
during
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
ranged
from
999
pg/
mL
to
40,100
pg/
mL
(
Figure
3.57).
Among
males
exposed
to
the
three
methoxychlor
concentrations,
11­
ketotesosterone
concentrations
ranged
from
0
pg/
mL
(
not
detected)
to
159,600
pg/
mL.
No
significant
differences
in
the
mean
11­
ketotesosterone
concentration
per
treatment
(
Table
3.49)
were
detected
(
Kruskal­
Wallis,
H
=
6.15,
p
=
0.105,
df
=
2).
The
achieved
power
for
this
endpoint
was
29%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
25
(
Table
3.49).

Table
3.49.
Summary
statistics
and
power
estimates
for
male
11­
ketotesosterone
concentrations
(
pg/
mL)
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
85
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.

Level
N
Mean
Stdev
CV
Achieved
Power
1
Sample
Size
Required
2
Control
8
14,552
13,669
94%
29%
25
Low
9
13,965
14,896
107%
Medium
10
4,257
4,364
103%
High
9
36,396
50,102
138%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
8.
2
Size
required
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

Mid
Low
High
Control
150000
100000
50000
0
treatment
11­
keto
Boxplots
of
11­
keto
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.57.
Boxplot
of
male
11­
ketotesosterone
concentration
(
pg/
mL)
by
treatment
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
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.

3.3.8
Body
Weight
and
Length
The
body
weight
of
females
used
in
the
non­
spawning
adult
14­
Day
Methoxychlor
assay
ranged
from
1.3
g
to
3.1
g
(
Figure
3.58).
There
were
no
significant
differences
in
mean
body
weight
(
natural
log
transformed)
among
treatments
(
Kruskal­
Wallis,
H
=
6.30,
p
=
0.098,
df
=
3).
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
20
(
Table
3.50).

The
body
(
fork)
length
of
females
used
in
the
non­
spawning
adult
14­
Day
Methoxychlor
assay
ranged
from
40
mm
to
55
mm
(
Figure
3.59).
There
were
no
significant
differences
in
mean
length
among
treatments
(
Kruskal­
Wallis,
H
=
1.51,
p
=
0.679,
df
=
3).
The
achieved
power
for
this
endpoint
was
11%,
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
86
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
93
(
Table
3.51).

Mid
Low
High
Control
3.2
2.2
1.2
treatment
fish_
wgt_
whole
Boxplots
of
fish_
wgt
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.58.
Boxplot
of
female
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay.
The
box
represents
the
interquartile
range,
whiskers
represent
the
data
range,
the
horizontal
line
is
the
median
value,
the
circle
is
the
mean
value,
and
asterisks
represent
probable
outliers.

Table
3.50.
Summary
statistics
and
power
estimates
for
female
body
weight
(
g)
data
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
10
2.2
0.3
15%
41%
20
Low
10
1.8
0.3
15%
Medium
9
2.0
0.5
22%
High
9
1.8
0.4
24%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
9.
2
Required
size
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)
87
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
55
50
45
40
treatment
fish_
length_
fork
Boxplots
of
fish_
len
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.59.
Boxplot
of
female
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
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
3.51.
Summary
statistics
and
power
estimates
for
female
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
10
47.8
2.6
5%
11%
93
Low
10
46.2
3.2
7%
Medium
9
47.4
3.5
7%
High
9
47.0
4.2
9%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
9.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.

The
body
weight
of
males
used
in
the
non­
spawning
adult
14­
Day
Methoxychlor
assay
ranged
from
1.6
g
to
5.5
g
(
Figure
3.60).
There
were
no
significant
differences
in
mean
body
weight
among
treatments
(
Kruskal­
Wallis,
H
=
3.80,
p
=
0.
284,
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
27
(
Table
3.52).

The
body
length
of
males
used
in
the
Non­
spawning
adult
14­
Day
Methoxychlor
assay
ranged
from
52
mm
to
70
mm
(
Figure
3.61).
There
were
no
significant
differences
in
mean
body
length
among
treatments
(
Kruskal­
Wallis,
H
=
1.73,
p
=
0.630,
df
=
3).
The
achieved
power
for
this
endpoint
was
16%,
and
the
sample
size
required
to
detect
a
significant
difference
from
the
Control
treatment
at
80%
power
was
56
(
Table
3.52).
DRAFT
EPA
WA
3­
8
(
Report
of
WA
2­
18
Study)
88
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
5.5
4.5
3.5
2.5
1.5
treatment
fish_
wgt_
whole
Boxplots
of
fish_
wgt
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.60.
Boxplot
of
male
body
weight
(
g)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
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
3.52.
Summary
statistics
and
power
estimates
for
male
body
weight
(
g)
data
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
10
2.9
0.5
17%
30%
27
Low
10
3.4
0.8
24%
Medium
10
3.2
1.1
33%
High
9
3.8
0.9
25%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
9.
2
Required
size
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)
89
Preliminary
Data:
May
be
subject
to
change
following
QA
and
Management
review
July
30,
2003
Mid
Low
High
Control
70
60
50
treatment
fish_
length_
fork
Boxplots
of
fish_
len
by
treatmen
(
means
are
indicated
by
solid
circles)

Figure
3.61.
Boxplot
of
male
body
length
(
mm)
by
treatment
for
the
Non­
spawning
Adult
14­
Day
Methoxychlor
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
3.53.
Summary
statistics
and
power
estimates
for
male
body
length
(
mm)
data
for
the
Nonspawning
Adult
14­
Day
Methoxychlor
assay
Level
N
Mean
SD
CV
Achieved
Power
1
Sample
Size
Required
2
Control
10
58.8
3.9
7%
16%
56
Low
10
60.6
4.8
8%
Medium
10
59.9
5.4
9%
High
9
61.8
4.5
7%
1
Calculated
from
natural
log
transformed
data;
sample
size
=
9.
2
Required
size
to
detect
a
significant
difference
from
Control
treatment
based
on
maximum
achieved
absolute
difference;
calculated
on
natural
log
transformed
data.
