Document ID: EPA-HQ-OPP-2003-0024-0028
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-06-03T04:00Z

Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Data
Requirement:
EPA
DP
Barcode
D288775
EPA
MRID
458677­
08
EPA
Guideline
70­
1(
Special
Study)

Test
material:
Purity:
not
reported
Common
name
Atrazine
Chemical
name:
IUPAC
CAS
name
6­
chloro­
N­
ethyl­
N'­(
1­
methylethyl)­
1,3,5­
triazine­
2,4­
diamine
CAS
No.
1912­
24­
9
Synonyms
EPA
PC
Code:
80803
Primary
Reviewer:
Thomas
M.
Steeger,
Ph.
D.,
Senior
Biologist
Date:
March
31,
2003
Environmental
Fate
and
Effects
Division,
ERB
4,
U.
S.
Environmental
Protection
Agency
Secondary
Reviewer(
s):
Joseph
E.
Tietge,
M.
S.,
Research
Aquatic
Biologist
Date:
Mid­
Continent
Ecology
Division,
National
Health
and
Environmental
Effects
Research
Laboratory
(
Duluth),
U.
S.
Environmental
Protection
Agency
Stephanie
Irene,
Ph.
D.,
Senior
Advisor
Date:
Environmental
Fate
and
Effects
Division,
ERB
3,
U.
S.
Environmental
Protection
Agency
Mary
J.
Frankenberry,
Senior
Statistician
Date:
Environmental
Fate
and
Effects
Division,
ERB
3,
U.
S.
Environmental
Protection
Agency
EPA
PC
Code
080803
Date
Evaluation
Completed:
05/
31/
2003
CITATION:
Villeneuve,
D.
L.,
K.
Coady,
M.
Hecker,
M.
B.
Murphy,
P.
D.
Jones
and
J.
P.
Giesy.
2003.
Methods
development
for
the
study
of
mechanism
of
action
of
atrazine
in
adult
and
metamorphosing
Xenopus
laevis
and
Rana
clamitans:
aromatase
induction.
Aquatic
Toxicology
Laboratory,
Michigan
State
University,
National
Food
Safety
and
Toxicology
Center,
E.
Lansing,
MI.
Sponsor:
Syngenta
Crop
Protection,
Inc.,
Laboratory
Study
ID
ECORISK
Number
MSU­
01.
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
2
of
28
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
3
of
28
EXECUTIVE
SUMMARY:

In
three
separate
studies,
two
involving
adult
male
African
clawed
frogs
(
Xenopus
laevis)
and
one
with
adult
female
Xenopus,
frogs
were
exposed
to
either
atrazine
at
25

g/
L
or
to
freshwater
under
static
renewal
conditions
with
50%
exposure
solution
changes
every
72
hours.
In
the
first
study,
15
males
were
exposed
for
26
days
(
single
tank
per
treatment);
in
the
second
study
15
males
were
exposed
for
43
days
(
single
tank
per
treatment);
and
in
the
third
study
13
females
(
6
in
one
replicate
and
7
in
the
second
replicate)
were
exposed
for
47
days.
Overall
mortality
was
3%,
7%
and
19%
in
the
26,
43
and
47­
day
exposures,
respectively;
mortality
was
primarily
associated
with
disease
(
fungal/
bacterial)
and
was
positively
correlated
(
r=
0.77)
with
the
number
of
exposure
days.
Homogenates
from
a
single
testes
and
from
brain
were
used
to
measure
aromatase
activity
(
CYP19
gene
expression)
in
males.
Aromatase
activity
in
the
testes
was
at
or
near
the
level
of
detection
(
LOD
=
0.025
fmol/
h/
mg
protein).
Mean
brain
aromatase
activity
from
atrazine­
treated
males
(
8.4
±
4.2
fmol/
h/
mg
protein)
was
not
statistically
different
(
p
=
0.678)
from
controls
(
7.1
±
4.2
fmol/
h/
mg
protein)
after
26
days
of
exposure.
Following
43
days
of
exposure,
mean
brain
aromatase
activity
in
atrazine­
treated
males
(
5.8
±
3.4
fmol/
h/
mg
protein)
was
not
statistically
different
(
p
=
0.199)
from
controls
(
10.4
±
7.1
fmol/
h/
mg
protein);
however,
in
the
second
study,
atrazine
contaminated
the
controls
at
levels
as
high
0.25

g/
L.
In
the
third
exposure
using
female
frogs,
ovarian
aromatase
activity
in
atrazine­
treated
females
averaged
4.5
±
1.7
fmol/
h/
mg
protein
and
did
not
differ
statistically
(
p=
0.447)
from
controls
(
5.4
±
2.1
fmol/
h/
mg
protein).
Mean
aromatase
activity
of
brain
homogenates
from
atrazine­
treated
females
was
7.3
±
5.0
fmol/
h/
mg
protein
did
not
differ
significantly
(
p=
0.582)
from
control
females
(
8.9
±
4.2
fmol/
h/
mg
protein).

With
a
single
tank
per
treatment
in
the
first
two
studies,
it
is
not
possible
to
document
tank
effects.
Hhowever,
the
authors
note
that
there
was
considerable
variability
in
aromatase
activity
between
frogs
receiving
the
same
treatment
with
coefficients
of
variability
at
or
exceeding
100%.
The
third
exposure
where
treatments
were
replicated
confirmed
that
tank
effects
were
high
enough
to
potentially
confound
the
ability
of
the
study
design
to
detect
treatment
effects.
Additionally,
with
only
one
atrazine
concentration
tested,
the
ability
of
the
study
to
discriminate
potential
"
low­
dose"
effects
is
limited.
Atrazine
contamination
of
controls
in
the
second
study
further
confounded
the
test's
ability
to
discriminate
subtle
effects.

The
study
recommends
that
future
testing
use
replication,
higher
sample
sizes
and
a
broader
range
of
atrazine
levels
to
test
for
potentially
subtle
effects.
Although
not
discussed
in
the
study,
water
quality
may
have
also
compromised
the
study
given
the
correlation
of
mortality
with
the
number
of
days
the
frogs
were
confined
and
the
references
to
the
diseased
state
of
the
animals
that
had
succumbed.
Apparently,
Xenopus
are
susceptible
to
bacterial
septicemia
(
red­
leg
disease)
if
poor
water
quality
persists.

Although
it
is
indicated
in
the
introduction
that
plasma
sex
steroids
were
also
analyzed,
these
data
and
associated
methods
were
not
presented
in
this
report.
The
only
mention
of
sex
steroid
measurements
was
an
indication
that
blood
samples
were
collected
by
cardiac
puncture.
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
4
of
28
I.
MATERIALS
AND
METHODS
GUIDELINE
FOLLOWED:
Nonguideline
Study
COMPLIANCE:
Not
conducted
under
full
GLP;
however,
most
practices
as
defined
by
40
CFR
Part
160,
August
19,
1989
were
established
for
this
study,
including
but
not
limited
to:

°
Written,
authorized
protocol
°
Written,
authorized
Standard
Operating
Procedures
for
all
key
procedures.
°
Organization
and
Personnel
were
sufficient
in
terms
of
number,
education,
training
and
experience.
°
Facilities
were
of
suitable
size
and
construction
°
Equipment
used
was
of
appropriate
design
and
adequate
capacity.
°
Independent
A
Inspection
was
conducted
of
raw
data.
°
Final
Report
was
written
°
Raw
data,
documentation,
records,
protocols,
and
final
report
was
archieved.

A.
MATERIALS:

1.
Test
Material
Atrazine
Description:
Not
reported
Lot
No./
Batch
No.
:
Not
reported
Purity:
Not
reported
Stability
of
compound
under
test
conditions:
Not
reported
Storage
conditions
of
test
chemicals:
Not
reported
2.
Test
organism:

Species:
African
clawed
frog
(
Xenopus
laevis)
.
Age
at
test
initiation
Adults
Weight
at
study
initiation:
(
mean
and
range)
Not
reported
Length
at
study
initiation:
(
mean
and
range)
Not
reported
Source:
Adult
X.
laevis
obtained
from
Xenopus
Express
®
(
Plant
City,
FL)
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
5
of
28
B.
STUDY
DESIGN:

Objective:
1.
To
test
hypothesis
that
waterborne
exposure
to
25

g/
L
atrazine
could
up­
regulate
aromatase
activity
in
sexually
mature
X.
laevis;
male
and
female
X.
laevis
were
tested.

2.
Test
hypothesis
that
exposure
to
atrazine
causes
decreases
in
plasma
testosterone
and
increases
in
estradiol
(
consistent
with
an
increase
in
aromatase
activity).

1.
Experimental
Conditions
a)
Range­
finding
Study:

b)
Definitive
Study
Table
1
.
Experimental
Parameters
Parameter
Details
Acclimation:
period:
Conditions:
(
same
as
test
or
not)
Feeding:
Health:
(
any
mortality
observed)
several
weeks
freshwater
(
treated
well
water)
not
reported
mortality
observed
during
study;
frog
with
leision
Duration
of
the
test
Exposure
1:
26­
day
Exposure
2:
43­
day
Exposure
3:
47­
day
Test
condition
static/
flow­
through
Type
of
dilution
system
for
flowthrough
method.

Renewal
rate
for
static
renewal
static
renewal
NA
50%
test
solution
change
every
72
hours
Aeration,
if
any
not
reported
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Parameter
Details
Page
6
of
28
Test
vessel
Material:
(
glass/
stainless
steel)
Size:
Fill
volume:
fiberglass
600
L
120
L
Source
of
dilution
water
Quality:
Treated
well
water
(
MSU­
University
Research
Containment
Facility;

Water
parameters:
Hardness
pH
Dissolved
oxygen
Total
Organic
carbon
Particulate
Matter
Ammonia
Nitrite
Metals
Pesticides
Chlorine
Temperature
{
Salinity
for
marine
or
estuarine
species}

Intervals
of
water
quality
measurement
no
water
quality
data
provided
at
all
No
mention
of
whether
water
quality
measurements
were
taken
at
all
Number
of
replicates/
groups:
negative
control:
water
treated
ones:
atrazine
at
25

g/
L
Exposure
1:
single
tank
for
each
treatment
(
25

g
atrazine/
L
plus
control)
Exposure
2:
single
tank
for
each
treatment
(
25

g
atrazine/
L
plus
control)
Exposure
3:
2
replicates
per
treatment
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Parameter
Details
Page
7
of
28
Number
of
organisms
per
replicate
/
groups:
Exposure
1:
15
adult
frogs/
treatment
Exposure
2:
15
adult
frogs/
treatment
Exposure
3:
6
adults
in
Rep
1
and
7
adults
in
Rep
2
Biomass
loading
rate
Exposure
1
and
2:
15
adults/
120
L
=
0.13
frogs/
L
Exposure
3:
6
to
7
adults/
120
L
=
0.05
­
0.06
frogs/
L
Test
concentrations:
nominal:
measured:
25

g/
L
Exposure
1:
30.6
±
2.1

g/
L
Exposure
2:
34
±
8

g/
L
Exposure
3:
32
±
16

g/
L
Solvent
(
type,
percentage,
if
used)
none
Lighting
not
reported
Feeding
not
reported
Recovery
of
chemical
Level
of
Quantitation
Level
of
Detection
ELISA
(
Envirogard
Triazine
®
;
Strategic
Diagnostics
Newark,
DE)

LOD
0.025

g/
L
(
Envirogard);
0.05

g/
L
(
Beacon)

Positive
control
{
if
used,
indicate
the
chemical
and
concentrations}
none
Other
parameters,
if
any
NA
2.
Observations:

Table
2:
Observations
Criteria
Details
Parameters
measured
including
the
sublethal
effects/
toxicity
symptoms
Catalytic
activity
of
aromatase
(
CYP19)
measured
in
gonad
and
brain
homogenates.
Blood
analyzed
for
steroid
hormone
levels
Observation
intervals
daily
Were
raw
data
included?
Yes
Other
observations,
if
any
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
8
of
28
II.
RESULTS
and
DISCUSSION:
[
All
results
discussed
in
this
section
and
the
next
are
those
reported
by
the
study
authors.
Although
supplemental
data
are
typically
used
in
a
qualitative
manner
only,
EFED
verified
spreadsheet
data
and
ran
basic
statistical
analyses
on
the
major
study
parameters.
See
attached
appendix.
If
results
appeared
to
differ
in
any
substantive
way,
the
difference
was
reported
in
the
text
below.]

Although
blood
was
collected
for
steroid
hormone
analysis,
these
data
were
not
reported
in
this
study.
Catalytic
activity
of
aromatase
(
CYP19)
was
measured
using
the
tritiated
water
release
assay
based
on
method
of
Lephart
and
Simpson
(
1991);
protein
concentration
was
determined
using
fluorescamine
assay
based
on
method
of
Kennedy
and
Jones
(
1994).
Aromatase
level
was
expressed
as
fmol/
h/
mg
protein.

Atrazine
concentrations
in
exposure
tanks
are
based
on
samples
collected
following
solution
renewal
after
72­
hrs
static
exposure
Only
50%
of
the
exposure
solutions
were
changed
at
renewal
though.

Exposure
1
(
26
days):
one
atrazine­
treated
frog
died
apparently
from
disease
(
open
lesions
on
its
legs).
Single
testes
were
taken
for
analysis;
none
of
the
testes
homogenates
had
measurable
aromatase
activity;
mean
aromatase
activity
to
the
testicular
homogenates
was
not
statistically
different
among
treatments
(
p=
0.586).
Positive
control
(
Xenopus
ovarian
tissue).

Immature
Xenopus
ovarian
exhibited
roughly
similar
aromatase
levels
as
2
mg/
mL
bovine
serum
albumin;
whereas
X.
laevis
adult
ovary
contained
roughly
26
fmol/
h/
mg
protein.
Mean
aromatase
activity
from
atrazinetreated
males
(
8.4
±
4.2
fmol/
h/
mg
protein)
was
not
statistically
different
(
p
=
0.678)
from
controls
(
7.1
±
4.2
fmol/
h/
mg
protein).

Atrazine
concentrations
averaged
30.6
±
2.1

g/
L,
while
the
control
tank
contained
less
than
0.025

g/
L
(
LOD
for
ELISA).

Exposure
2
(
43
days):
one
animal
from
each
treatment
died
of
unknown
causes.
Animals
were
reported
to
appear
healthy.
Testicular
homogenates
had
low
to
non­
detectable
levels
of
aromatase
and
again,
there
was
no
statistical
difference
(
p
=
0.764)
between
treatments.
Positive
control
X.
laevis
ovarian
homogenates
yielded
measurable
aromatase
activity,
and
activity
was
inhibited
by
addition
of
7.5

L
of
559

M
4­
androstein­
4­
ol­
3,
17­
dione
(
a
specific
inhibitor
of
aromatase).

Atrzine­
treated
mean
brain
aromatase
activity
(
5.8
±
3.4
fmol/
h/
mg
protein)
was
not
statistically
different
(
p
=
0.199)
from
controls
(
10.4
±
7.1
fmol/
h/
mg
protein).
A
negative
control
(
Rana
pipiens
ovarian
homogenate)
did
not
yield
a
significant
response
in
the
aromatase
assay.
There
were
significant
differences
among
mean
aromatase
activity
in
replicate
tanks
(
tank
effects)
within
each
treatment
group.

Atrazine
concentrations
over
the
43­
day
exposure
period
averaged
34
±
8

g/
L
but
ranged
as
high
as
50

g/
L.
Atrazine
in
control
tanks
ranged
up
to
0.26

g/
L
but
average
0.07
±
0.09

g/
L.
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
9
of
28
Exposure
3
(
47
days):
two
atrazine­
treated
and
three
control
females
died;
overall
mortality
was
19%.
Fungal
or
bacterial
infections
were
considered
the
probable
cause
of
the
mortalities
although
the
surviving
animals
are
reported
as
appearing
healthy.

Ovarian
aromatase
activity
in
atrazine­
treated
females
averaged
4.5
±
1.7
fmol/
h/
mg
protein
and
did
not
differ
statistically
(
p=
0.447)
from
controls
(
5.4
±
2.1
fmol/
h/
mg
protein).
The
report
notes
that
overall
ovarian
aromatase
activity
was
approximately
33
times
greater
than
that
observed
for
testicular
homogenates
from
Exposure
II.

The
mean
aromatase
activity
of
brain
homogenates
from
atrazine­
treated
females
was
7.3
±
5.0
fmol/
h/
mg;
protein
did
not
differ
significantly
(
p=
0.582)
from
control
females
(
8.9
±
4.2
fmol/
h/
mg
protein)
.

Atrazine
concentrations
in
each
of
the
atrazine
groups
averaged
32
±
16

g/
L
and
28
±
15

g/
L;
however
concentrations
ranged
as
high
as
70

g/
L.
Atrazine
concentrations
in
control
tanks
were
generally
less
than
the
limit
of
detection
and
averaged
0.018
±
0.02

g/
L
and
0.016
±
0.019

g/
L
in
the
two
replicates.

The
report
concludes
that
there
were
no
statistically
significant
differences
in
mean
brain
homogenate
aromatase
activity
among
treatment
groups
in
all
three
exposures
(
p=
0.75).
For
ovarian
homogenates
from
exposure
3,
there
were
no
statistically
significant
differences
between
atrazine
and
control
females.
As
a
whole
"
the
results
of
the
three
exposures
do
no
support
the
hypothesis
that
waterborne
exposure
to
approximately
25

g/
L
atrazine
causes
changes
in
brain
or
gonadal
aromatase
activity
in
adult
X.
laevis
relative
to
that
in
X.
laevis
exposed
to
laboratory
water."

C.
REPORTED
STATISTICS:

D.
VERIFICATION
OF
STATISTICAL
RESULTS:
Basic
analyses
in
attached
SAS
©
[
Statistical
Analysis
System,
Release
8.01,
Cary,
North
Carolina]
output)

E.
STUDY
DEFICIENCIES:
Water
quality
data
were
not
provided;
the
purity
of
atrazine
was
not
provided;
and
atrazine
was
detected
in
the
negative
control
in
Exposure
2.
In
Exposures
1
and
3,
mortality
was
attributed
to
disease.
The
feeding
rate
and
lighting
cycle
were
not
discussed
in
the
study.

F.
REVIEWER'S
COMMENTS:

It
is
unclear
why
a
negative
control
(
Rana
pipiens
ovarian
homogenate)
did
not
yield
a
significant
response
in
the
aromatase
assay.
In
other
words,
why
didn't
aromatase
from
a
different
species
convert
testosterone
to
estrogen
since
the
substrates
are
presumably
the
same?

In
Exposure
2,
atrazine
in
control
tanks
ranged
up
to
0.26

g/
L,
but
averaged
0.07
±
0.09

g/
L.
While
the
report
states
that
this
suggests
"
atrazine
concentrations
in
control
water
was,
on
average,
at
least
485
times
lower
than
that
in
atrazine­
treated
water,"
the
levels
detected
were
well­
above
the
detection
limit
of
the
ELISA
assay
(
LOD
=
0.025).
Atrazine
in
controls
was
at
0.1

g/
L
on
March
18,
0.25

g/
L
on
March
21,
and
0.24

g/
L
on
March
30.
Thus,
exposure
to
atrazine
in
the
controls
was
not
an
isolated
event
and
may
have
confounded
the
ability
of
the
study
to
detect
treatment­
related
effects.
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
10
of
28
The
level
of
total
mortality
was
positively
correlated
(
r=
0.77)
with
the
length
of
exposure
and
in
most
cases
was
associated
with
either
bacterial
and/
or
fungal
disease.

In
the
second
exposure,
there
were
significant
differences
among
mean
aromatase
activity
in
replicate
tanks
(
tank
effects)
within
each
treatment
group.

Although
the
report
concluded
that
"
the
results
of
the
three
exposures
do
no
support
the
hypothesis
that
waterborne
exposure
to
approximately
25

g/
L
atrazine
causes
changes
in
brain
or
gonadal
aromatase
activity
in
adult
X.
laevis
relative
to
that
in
X.
laevis
exposed
to
laboratory
water,"
the
authors
acknowledged
that
the
magnitude
of
variability
in
gonadal
and
brain
aromatase
activity
among
individual
frogs
from
the
same
treatment
limited
the
power
of
the
experiments
to
detect
subtle
differences
even
if
they
occurred.
Large
sample
sizes
would
be
required
to
test
for
subtle
effects.
Additionally,
with
only
one
tank
per
treatment
in
Exposures
1
and
2,
the
ability
to
account
for
tank
effects
is
very
limited
and
as
the
authors
noted
could
potentially
yield
a
false­
negative
or
false
positive
response.

Furthermore,
the
studies
only
examined
a
single
atrazine
exposure
concentration,
i.
e.,
25

g/
L.
The
authors
note
that
one
hypothesis
that
has
been
raised
is
that
high
concentrations
of
atrazine
may
up­
regulate
aromatase
to
the
point
where
the
estrogen
produced
causes
negative
feedback
on
the
hypothalmic­
pituitary­
axis
resulting
in
down­
regulation
of
aromatase
expression.
They
conclude
that
future
testing
should
use
a
wider
range
of
concentrations.

Although
it
is
indicated
in
the
introduction
that
plasma
sex
steroids
were
also
analyzed,
these
data
and
associated
methods
were
not
presented
in
this
report.
The
only
mention
of
sex
steroid
measurements
was
an
indication
that
blood
samples
were
collected
by
cardiac
puncture.

G.
CONCLUSIONS:

Aromatase
activity
in
testes
was
too
low
to
reliably
quantify.
While
atrazine
treatment
of
adult
male
X.
laevis
did
not
significantly
affect
brain
aromatase
activity
relative
to
controls,
the
ability
of
the
current
studies
to
detect
treatment
effects
appears
limited.
Since
mortality
over
the
three
studies
was
positively
correlated
with
the
length
of
the
study
and
because
disease
appeared
to
be
an
issue
in
two
out
of
three
studies,
water
quality
may
have
been
a
problem;
however,
water
quality
data
were
not
reported
for
the
studies.
Additionally,
the
second
study
(
Exposure
2)
was
compromised
by
the
discovery
of
atrazine
in
the
control
exposure.
With
a
single
tank
per
treatment
(
no
replicates)
and
a
single
atrazine
treatment
(
25

g/
L)
per
study,
variability
within
treatments
made
it
difficult
to
clearly
differentiate
treatment
effects
and
to
test
the
hypothesis.

H.
REFERENCES:

Kennedy,
S.
W.
and
S.
P.
Jones.
1994.
Simultaneous
measurement
of
cytochrome
P4501A
catalyitc
activity
and
total
protein­
concentration
with
fluorescence
plate
reader.
Analytical
Biochemistry
222(
1):
217
­
223.

Laphart,
E.
D.
and
E.
R.
Simpson.
1991.
Assay
of
aromatase
activity.
Methods
in
Enzymology
206:
477
­
483.
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
11
of
28
NONPARAMETRIC
COMPARISON
OF
GONADAL
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
264
Obs
SAMPLE
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
A1
0
2
11.1919
5.11863
45.735
2
A10
0
2
2.1999
0.02387
1.085
3
A11
0
2
1.8184
0.09016
4.958
4
A12
0
2
0.6189
0.14642
23.658
5
A13
0
2
18.8785
0.89090
4.719
6
A14
0
2
­
0.3838
0.34889
­
90.914
7
A15
0
2
­
1.7780
0.36618
­
20.595
8
A2
0
2
19.5097
0.08537
0.438
9
A3
0
2
1.0145
0.35979
35.465
10
A4
0
2
13.9604
0.21978
1.574
11
A7
0
2
17.5941
0.01244
0.071
12
A8
0
2
6.4782
1.36797
21.116
13
A9
0
2
17.6115
0.57149
3.245
14
C1
0
2
­
0.8158
0.25213
­
30.907
15
C10
0
2
12.0858
0.74018
6.124
16
C11
0
2
14.7327
0.07916
0.537
17
C12
0
2
3.1996
0.31515
9.850
18
C13
0
2
7.2257
0.47387
6.558
19
C14
0
2
0.5245
0.04568
8.709
20
C15
0
2
12.5170
0.11766
0.940
21
C2
0
2
­
0.0897
0.25792
­
287.405
22
C3
0
2
14.6997
5.50738
37.466
23
C4
0
2
1.7527
0.36438
20.790
24
C5
0
2
1.2051
1.46082
121.218
25
C6
0
2
13.9926
3.28426
23.471
26
C8
0
2
18.9569
0.13915
0.734
27
C9
0
2
­
0.2912
0.07412
­
25.456
MEAN
BRAIN
AROMATASE
ACTIVITY
IN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
1)
265
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
ATRAZIN
0
13
8.36265
8.27945
99.0051
2
CONTROL
0
14
7.12111
7.06613
99.2279
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
12
of
28
ANOVA
FOR
BRAIN
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
1)
266
The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
GROUP
2
ATRAZIN
CONTROL
Number
of
observations
27
Dependent
Variable:
MEAN
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
1
10.390314
10.390314
0.18
0.6780
Error
25
1471.684807
58.867392
Corrected
Total
26
1482.075121
R­
Square
Coeff
Var
Root
MSE
MEAN
Mean
0.007011
99.39913
7.672509
7.718889
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
GROUP
1
10.39031393
10.39031393
0.18
0.6780
Levene's
Test
for
Homogeneity
of
MEAN
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
GROUP
1
1928.1
1928.1
1.54
0.2255
Error
25
31216.8
1248.7
Bartlett's
Test
for
Homogeneity
of
MEAN
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
GROUP
1
0.3013
0.5831
Dunnett's
t
Tests
for
MEAN
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
25
Error
Mean
Square
58.86739
Critical
Value
of
Dunnett's
t
2.05959
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Simultaneous
GROUP
Between
95%
Confidence
Comparison
Means
Limits
CONTROL
­
ATRAZIN
­
1.242
­
7.328
4.845
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
13
of
28
NONPARAMETRIC
COMPARISON
OF
BRAIN
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
FR
270
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
MEAN
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
ATRAZIN
13
190.0
182.0
20.607442
14.615385
CONTROL
14
188.0
196.0
20.607442
13.428571
Wilcoxon
Two­
Sample
Test
Statistic
190.0000
Normal
Approximation
Z
0.3639
One­
Sided
Pr
>
Z
0.3579
Two­
Sided
Pr
>
|
Z|
0.7159
t
Approximation
One­
Sided
Pr
>
Z
0.3594
Two­
Sided
Pr
>
|
Z|
0.7188
Z
includes
a
continuity
correction
of
0.5.

Kruskal­
Wallis
Test
Chi­
Square
0.1507
DF
1
Pr
>
Chi­
Square
0.6979
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
MEAN
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
ATRAZIN
13
6.0
6.259259
1.321968
0.461538
CONTROL
14
7.0
6.740741
1.321968
0.500000
Median
Two­
Sample
Test
Statistic
6.0000
Z
­
0.1961
One­
Sided
Pr
<
Z
0.4223
Two­
Sided
Pr
>
|
Z|
0.8445
Median
One­
Way
Analysis
Chi­
Square
0.0385
DF
1
Pr
>
Chi­
Square
0.8445
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
14
of
28
MEAN
GONADAL
AROMATASE
ACTIVITY
IN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
1)
273
Obs
SAMPLE
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
A1
0
2
0.40032
1.40426
350.79
2
A10
0
2
­
0.06537
0.04967
­
75.99
3
A11
0
2
0.00952
0.22021
2314.17
4
A12
0
2
­
0.33007
0.17387
­
52.68
5
A13
0
2
­
0.04765
0.14865
­
311.96
6
A14
0
2
­
0.41950
0.00438
­
1.04
7
A15
0
2
­
0.14911
0.14151
­
94.90
8
A2
0
2
­
0.52470
0.90598
­
172.67
9
A3
0
2
­
0.39147
0.30402
­
77.66
10
A4
0
2
­
0.47445
0.00000
0.00
11
A5
0
2
0.11161
0.20003
179.21
12
A7
0
2
­
0.21175
0.02073
­
9.79
13
A8
0
2
­
0.34509
0.02312
­
6.70
14
A9
0
2
­
0.08582
0.10868
­
126.65
15
C1
0
2
­
0.59029
0.29282
­
49.61
16
C10
0
2
­
0.84140
0.11866
­
14.10
17
C11
0
2
­
0.01878
0.11230
­
597.83
18
C12
0
2
­
0.19693
0.05968
­
30.30
19
C13
0
2
­
0.05506
0.02920
­
53.03
20
C14
0
2
­
0.03542
0.36070
­
1018.23
21
C15
0
2
0.40039
0.08395
20.97
22
C2
0
2
­
0.46908
0.05875
­
12.52
23
C3
0
2
­
0.27553
0.13632
­
49.48
24
C4
0
2
­
0.38774
0.48604
­
125.35
25
C5
0
2
­
0.03013
0.00000
0.00
26
C6
0
2
­
0.27841
0.00000
0.00
27
C8
0
2
­
0.33768
0.12295
­
36.41
28
C9
0
2
­
0.21742
0.07923
­
36.44
MEAN
GONADAL
AROMATASE
ACTIVITY
IN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
1)
273
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
ATRAZIN
0
14
­
0.18025
0.25669
­
142.408
2
CONTROL
0
14
­
0.23811
0.29774
­
125.044
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
15
of
28
ANOVA
FOR
GONADAL
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
1)
274
The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
GROUP
2
ATRAZIN
CONTROL
Number
of
observations
28
Dependent
Variable:
MEAN
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
1
0.02342954
0.02342954
0.30
0.5866
Error
26
2.00900245
0.07726932
Corrected
Total
27
2.03243199
R­
Square
Coeff
Var
Root
MSE
MEAN
Mean
0.011528
­
132.8879
0.277974
­
0.209179
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
GROUP
1
0.02342954
0.02342954
0.30
0.5866
Levene's
Test
for
Homogeneity
of
MEAN
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
GROUP
1
0.00313
0.00313
0.25
0.6229
Error
26
0.3280
0.0126
Bartlett's
Test
for
Homogeneity
of
MEAN
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
GROUP
1
0.2744
0.6004
Dunnett's
t
Tests
for
MEAN
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
26
Error
Mean
Square
0.077269
Critical
Value
of
Dunnett's
t
2.05558
Minimum
Significant
Difference
0.216
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
GROUP
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
CONTROL
­
ATRAZIN
­
0.05785
­
0.27382
0.15811
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
16
of
28
NONPARAMETRIC
COMPARISON
OF
GONADAL
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
278
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
MEAN
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
ATRAZIN
14
208.0
203.0
21.763884
14.857143
CONTROL
14
198.0
203.0
21.763884
14.142857
Wilcoxon
Two­
Sample
Test
Statistic
208.0000
Normal
Approximation
Z
0.2068
One­
Sided
Pr
>
Z
0.4181
Two­
Sided
Pr
>
|
Z|
0.8362
t
Approximation
One­
Sided
Pr
>
Z
0.4189
Two­
Sided
Pr
>
|
Z|
0.8377
Z
includes
a
continuity
correction
of
0.5.

Kruskal­
Wallis
Test
Chi­
Square
0.0528
DF
1
Pr
>
Chi­
Square
0.8183
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
MEAN
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
ATRAZIN
14
8.0
7.0
1.347151
0.571429
CONTROL
14
6.0
7.0
1.347151
0.428571
Median
Two­
Sample
Test
Statistic
8.0000
Z
0.7423
One­
Sided
Pr
>
Z
0.2290
Two­
Sided
Pr
>
|
Z|
0.4579
Median
One­
Way
Analysis
Chi­
Square
0.5510
DF
1
Pr
>
Chi­
Square
0.4579
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
17
of
28
AVERAGE
ATRAZINE
CONCENTRATIONS
IN
CONTROL
EXPOSURE
TANKS
BY
DATE
280
Obs
MONTH
DAY
_
TYPE_
_
FREQ_
MEAN
1
3
3
0
2
0.03820
2
3
6
0
2
0.06553
3
3
9
0
2
0.01873
4
3
12
0
2
0.00515
5
3
15
0
2
0.03088
6
3
18
0
2
0.11453
7
3
21
0
2
0.26317
8
3
24
0
2
0.01652
9
3
30
0
2
0.22902
10
4
5
0
2
0.00473
11
4
8
0
2
0.00725
AVERAGE
ATRAZINE
CONCENTRATION
IN
CONTROL
EXPOSURE
TANK
ACROSS
ENTIRE
STUDY
(
EXP
1)
281
Obs
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
0
11
0.072156
0.092187
127.761
AVERAGE
ATRAZINE
CONCENTRATIONS
IN
25
UG/
L
EXPOSURE
1
TANKS
BY
DATE
282
Obs
MONTH
DAY
_
TYPE_
_
FREQ_
MEAN
1
3
3
0
2
22.5051
2
3
6
0
2
32.4627
3
3
9
0
2
25.4194
4
3
12
0
2
25.8225
5
3
15
0
2
38.2852
6
3
18
0
2
34.8589
7
3
21
0
2
44.3471
8
3
24
0
2
49.5251
9
3
30
0
2
35.0879
10
4
5
0
2
33.9842
11
4
8
0
2
32.9775
AVERAGE
ATRAZINE
CONCENTRATION
IN
25
UG/
L
EXPOSURE
TANK
ACROSS
ENTIRE
STUDY
283
Obs
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
0
11
34.1160
8.02663
23.5275
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
18
of
28
MEAN
BRAIN
AROMATASE
ACTIVITY
FOLLOWING
EXPOSURE
2
284
Obs
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
0
28
8.07322
9.41514
116.622
ANOVA
FOR
BRAIN
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXPOSURE
2
285
The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
GROUP
2
EXP
REF
Number
of
observations
28
Dependent
Variable:
FMOL
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
1
150.032040
150.032040
1.74
0.1988
Error
26
2243.380177
86.283853
Corrected
Total
27
2393.412216
R­
Square
Coeff
Var
Root
MSE
FMOL
Mean
0.062685
115.0583
9.288910
8.073222
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
GROUP
1
150.0320396
150.0320396
1.74
0.1988
Levene's
Test
for
Homogeneity
of
FMOL
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
GROUP
1
70229.1
70229.1
3.77
0.0632
Error
26
484906
18650.2
Bartlett's
Test
for
Homogeneity
of
FMOL
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
GROUP
1
6.2027
0.0128
Dunnett's
t
Tests
for
FMOL
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
26
Error
Mean
Square
86.28385
Critical
Value
of
Dunnett's
t
2.05558
Minimum
Significant
Difference
7.2169
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Simultaneous
GROUP
Between
95%
Confidence
Comparison
Means
Limits
REF
­
EXP
4.630
­
2.587
11.846
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
19
of
28
NONPARAMETRIC
COMPARISON
OF
BRAIN
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
FR
289
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
FMOL
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
EXP
14
186.0
203.0
21.763884
13.285714
REF
14
220.0
203.0
21.763884
15.714286
Wilcoxon
Two­
Sample
Test
Statistic
186.0000
Normal
Approximation
Z
­
0.7581
One­
Sided
Pr
<
Z
0.2242
Two­
Sided
Pr
>
|
Z|
0.4484
t
Approximation
One­
Sided
Pr
<
Z
0.2275
Two­
Sided
Pr
>
|
Z|
0.4549
Z
includes
a
continuity
correction
of
0.5.

Kruskal­
Wallis
Test
Chi­
Square
0.6101
DF
1
Pr
>
Chi­
Square
0.4347
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
FMOL
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
EXP
14
6.0
7.0
1.347151
0.428571
REF
14
8.0
7.0
1.347151
0.571429
Median
Two­
Sample
Test
Statistic
6.0000
Z
­
0.7423
One­
Sided
Pr
<
Z
0.2290
Two­
Sided
Pr
>
|
Z|
0.4579
Median
One­
Way
Analysis
Chi­
Square
0.5510
DF
1
Pr
>
Chi­
Square
0.4579
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
20
of
28
MEAN
ADULT
MALE
GONADAL
AROMATASE
ACTIVITY
IN
ATRAZINE­
TREATED
AND
CONTROL
FROGS
(
EXP
2)
291
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
ATRAZIN
0
14
0.13818
0.09607
69.525
2
CONTROL
0
14
0.15601
0.19786
126.827
ANOVA
FOR
ADULT
MALE
GONADAL
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
292
The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
GROUP
2
ATRAZIN
CONTROL
Number
of
observations
28
Dependent
Variable:
FMOL
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
1
0.00222571
0.00222571
0.09
0.7640
Error
26
0.62891899
0.02418919
Corrected
Total
27
0.63114470
R­
Square
Coeff
Var
Root
MSE
FMOL
Mean
0.003526
105.7346
0.155529
0.147094
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
GROUP
1
0.00222571
0.00222571
0.09
0.7640
Levene's
Test
for
Homogeneity
of
FMOL
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
GROUP
1
0.00540
0.00540
1.08
0.3074
Error
26
0.1296
0.00499
Bartlett's
Test
for
Homogeneity
of
FMOL
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
GROUP
1
6.0347
0.0140
Dunnett's
t
Tests
for
FMOL
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.
Alpha
0.05
Error
Degrees
of
Freedom
26
Error
Mean
Square
0.024189
Critical
Value
of
Dunnett's
t
2.05558
Minimum
Significant
Difference
0.1208
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
GROUP
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
CONTROL
­
ATRAZIN
0.01783
­
0.10300
0.13867
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
21
of
28
NONPARAMETRIC
COMPARISON
OF
ADULT
MALE
GONADAL
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZI
296
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
FMOL
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
ATRAZIN
14
224.0
203.0
21.763884
16.0
CONTROL
14
182.0
203.0
21.763884
13.0
Wilcoxon
Two­
Sample
Test
Statistic
224.0000
Normal
Approximation
Z
0.9419
One­
Sided
Pr
>
Z
0.1731
Two­
Sided
Pr
>
|
Z|
0.3462
t
Approximation
One­
Sided
Pr
>
Z
0.1773
Two­
Sided
Pr
>
|
Z|
0.3546
Z
includes
a
continuity
correction
of
0.5.

Kruskal­
Wallis
Test
Chi­
Square
0.9310
DF
1
Pr
>
Chi­
Square
0.3346
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
FMOL
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
ATRAZIN
14
9.0
7.0
1.347151
0.642857
CONTROL
14
5.0
7.0
1.347151
0.357143
Median
Two­
Sample
Test
Statistic
9.0000
Z
1.4846
One­
Sided
Pr
>
Z
0.0688
Two­
Sided
Pr
>
|
Z|
0.1376
Median
One­
Way
Analysis
Chi­
Square
2.2041
DF
1
Pr
>
Chi­
Square
0.1376
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
22
of
28
MEAN
ATRAZINE
LEVEL
BY
SAMPLING
DATE
FOR
CONTROL
(
C)
AND
ATRAZINE
(
A)
TREATED
TANKS
298
Obs
MONTH
DAY
SAMPLE
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
4
17
A1
ATRAZINE
0
2
21.3668
0.25186
1.1788
2
4
17
A2
ATRAZINE
0
2
6.8501
0.20184
2.9465
3
4
17
C1
CONTROL
0
2
0.0072
0.00365
50.6899
4
4
17
C2
CONTROL
0
2
0.0041
0.00070
17.0096
5
4
20
A1
ATRAZINE
0
2
22.4385
2.50752
11.1751
6
4
20
A2
ATRAZINE
0
2
4.5276
1.69332
37.3999
7
4
20
C1
CONTROL
0
2
0.0070
0.00132
18.7495
8
4
20
C2
CONTROL
0
2
0.0254
0.00179
7.0668
9
4
23
A1
ATRAZINE
0
2
18.2464
3.42110
18.7495
10
4
23
A2
ATRAZINE
0
2
32.0647
4.89450
15.2644
11
4
23
C1
CONTROL
0
2
0.0769
0.00724
9.4163
12
4
23
C2
CONTROL
0
2
0.0771
0.00045
0.5894
13
5
2
A1
ATRAZINE
0
2
25.6397
2.86526
11.1751
14
5
2
A2
ATRAZINE
0
2
17.2724
6.55444
37.9475
15
5
2
C1
CONTROL
0
2
0.0234
0.00963
41.2067
16
5
2
C2
CONTROL
0
2
0.0186
0.00865
46.5324
17
5
5
A1
ATRAZINE
0
2
17.7872
4.56176
25.6463
18
5
5
A2
ATRAZINE
0
2
17.4941
5.47574
31.3004
19
5
5
C1
CONTROL
0
2
0.0057
0.00027
4.7134
20
5
5
C2
CONTROL
0
2
0.0044
0.00008
1.7681
21
5
8
A1
ATRAZINE
0
2
21.1917
0.49956
2.3573
22
5
8
C1
CONTROL
0
2
0.0068
0.00052
7.6546
23
5
8
C2
CONTROL
0
2
0.0048
0.00057
11.7606
24
5
11
C1
CONTROL
0
2
0.0636
0.01413
22.2114
25
5
11
C2
CONTROL
0
2
0.0121
0.00445
36.8512
26
5
14
C1
CONTROL
0
2
0.0202
0.00107
5.3020
27
5
14
C2
CONTROL
0
2
0.0140
0.00530
37.9475
28
5
17
C1
CONTROL
0
2
0.0064
0.00300
46.5324
29
5
17
C2
CONTROL
0
2
0.0072
0.00025
3.5356
30
5
20
C1
CONTROL
0
2
0.0058
0.00219
37.9475
31
5
20
C2
CONTROL
0
2
0.0040
0.00073
18.1701
32
5
23
C1
CONTROL
0
2
0.0110
0.00672
61.1231
33
5
23
C2
CONTROL
0
2
0.0203
0.00892
43.8868
34
5
26
C1
CONTROL
0
2
0.0094
0.00094
10.0029
35
5
26
C2
CONTROL
0
2
0.0177
0.00239
13.5146
36
5
29
C1
CONTROL
0
2
0.0055
0.00197
35.7501
37
5
29
C2
CONTROL
0
2
0.0055
0.00065
11.7606
38
6
1
C1
CONTROL
0
2
0.0064
0.00101
15.8467
39
6
1
C2
CONTROL
0
2
0.0047
0.00087
18.7494
MEAN
ATRAZINE
CONCENTRATION
ACROSS
SAMPLING
DATES
FOR
EACH
OF
THE
TWO
CONTROL
(
C)
AND
ATRAZI
299
Obs
SAMPLE
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
A1
ATRAZINE
0
6
21.1117
2.8844
13.662
2
A2
ATRAZINE
0
5
15.6418
10.9158
69.786
3
C1
CONTROL
0
14
0.0182
0.0229
125.436
4
C2
CONTROL
0
14
0.0157
0.0191
121.373
MEAN
ATRAZINE
CONCENTRATION
IN
CONTROL
AND
ATRAZINE­
TREATED
TANKS
OVER
ENTIRE
STUDY
(
EXP
3
300
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
MIN
MAX
STD
CV
1
ATRAZINE
0
11
18.6254
4.52761
32.0647
7.74480
41.582
2
CONTROL
0
28
0.0170
0.00403
0.0771
0.02070
121.980
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
23
of
28
MEAN
BRAIN
AROMATASE
ACTIVITY
BY
SAMPLE
AND
NUMBER
IN
CONTROL
(
C)
AND
ATRAZINE­
TREATETED
(
A)
301
Obs
SAMPLE
NUMBER
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
A1
1
ATRAZINE
0
2
20.9131
0.52971
2.5329
2
A1
2
ATRAZINE
0
2
1.9509
0.02533
1.2982
3
A1
3
ATRAZINE
0
2
1.3884
0.00843
0.6070
4
A1
4
ATRAZINE
0
2
3.8467
0.18493
4.8075
5
A1
5
ATRAZINE
0
2
5.6692
0.02706
0.4774
6
A1
6
ATRAZINE
0
2
0.9863
0.02995
3.0367
7
A2
1
ATRAZINE
0
2
7.5578
0.14937
1.9764
8
A2
2
ATRAZINE
0
2
8.7285
1.14801
13.1524
9
A2
3
ATRAZINE
0
2
7.6883
0.05418
0.7047
10
A2
4
ATRAZINE
0
2
­
0.0514
0.00540
­
10.5056
11
A2
5
ATRAZINE
0
2
21.1885
0.02985
0.1409
12
C1
1
CONTROL
0
2
11.8105
0.01860
0.1575
13
C1
2
CONTROL
0
2
2.1175
0.20755
9.8015
14
C1
3
CONTROL
0
2
6.2637
0.10634
1.6977
15
C1
4
CONTROL
0
2
21.4478
0.23323
1.0874
16
C1
5
CONTROL
0
2
4.1599
0.02262
0.5438
17
C2
1
CONTROL
0
2
15.0054
2.98537
19.8953
18
C2
2
CONTROL
0
2
1.0907
0.04346
3.9843
19
C2
3
CONTROL
0
2
7.0063
0.10293
1.4692
20
C2
4
CONTROL
0
2
8.3976
0.10458
1.2454
21
C2
5
CONTROL
0
2
12.2114
0.15241
1.2481
MEAN
BRAIN
AROMATASE
ACTIVITY
BY
SAMPLE
IN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
3)
302
Obs
SAMPLE
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
A1
0
6
5.79244
7.61168
131.407
2
A2
0
5
9.02234
7.65441
84.838
3
C1
0
5
9.15987
7.76212
84.741
4
C2
0
5
8.74230
5.31368
60.781
MEAN
BRAIN
AROMATASE
ACTIVITY
IN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
3)
303
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
ATRAZINE
0
11
7.26057
7.43303
102.375
2
CONTROL
0
10
8.95109
6.27499
70.103
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
24
of
28
ANOVA
FOR
BRAIN
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
3)
304
The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
GROUP
2
ATRAZINE
CONTROL
Number
of
observations
21
Dependent
Variable:
MEAN
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
1
14.9695821
14.9695821
0.31
0.5820
Error
19
906.8789562
47.7304714
Corrected
Total
20
921.8485383
R­
Square
Coeff
Var
Root
MSE
MEAN
Mean
0.016239
85.65689
6.908724
8.065579
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
GROUP
1
14.96958206
14.96958206
0.31
0.5820
Levene's
Test
for
Homogeneity
of
MEAN
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
GROUP
1
1145.7
1145.7
0.31
0.5865
Error
19
71107.0
3742.5
Bartlett's
Test
for
Homogeneity
of
MEAN
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
GROUP
1
0.2554
0.6133
Dunnett's
t
Tests
for
MEAN
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
19
Error
Mean
Square
47.73047
Critical
Value
of
Dunnett's
t
2.09309
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Simultaneous
GROUP
Between
95%
Confidence
Comparison
Means
Limits
CONTROL
­
ATRAZINE
1.691
­
4.628
8.009
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
25
of
28
NONPARAMETRIC
COMPARISON
OF
BRAIN
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
FR
308
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
MEAN
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
ATRAZINE
11
107.0
121.0
14.200939
9.727273
CONTROL
10
124.0
110.0
14.200939
12.400000
Wilcoxon
Two­
Sample
Test
Statistic
124.0000
Normal
Approximation
Z
0.9506
One­
Sided
Pr
>
Z
0.1709
Two­
Sided
Pr
>
|
Z|
0.3418
t
Approximation
One­
Sided
Pr
>
Z
0.1766
Two­
Sided
Pr
>
|
Z|
0.3531
Z
includes
a
continuity
correction
of
0.5.

Kruskal­
Wallis
Test
Chi­
Square
0.9719
DF
1
Pr
>
Chi­
Square
0.3242
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
MEAN
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
ATRAZINE
11
5.0
5.238095
1.171274
0.454545
CONTROL
10
5.0
4.761905
1.171274
0.500000
Median
Two­
Sample
Test
Statistic
5.0000
Z
0.2033
One­
Sided
Pr
>
Z
0.4195
Two­
Sided
Pr
>
|
Z|
0.8389
Median
One­
Way
Analysis
Chi­
Square
0.0413
DF
1
Pr
>
Chi­
Square
0.8389
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
26
of
28
MEAN
GONADAL
AROMATASE
ACTIVITY
BY
SAMPLE
AND
NUMBER
IN
CONTROL
(
C)
AND
ATRAZINE­
TREATETED
(
310
Obs
SAMPLE
NUMBER
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
A1
1
ATRAZINE
0
2
4.3309
0.13618
3.1445
2
A1
2
ATRAZINE
0
2
3.8744
0.02522
0.6510
3
A1
3
ATRAZINE
0
2
7.3398
0.02316
0.3155
4
A1
4
ATRAZINE
0
2
5.1150
0.06204
1.2129
5
A1
5
ATRAZINE
0
2
8.3364
0.01106
0.1327
6
A1
6
ATRAZINE
0
2
7.3282
0.02467
0.3366
7
A2
1
ATRAZINE
0
2
1.7495
0.02643
1.5105
8
A2
2
ATRAZINE
0
2
4.9783
0.12473
2.5055
9
A2
3
ATRAZINE
0
2
3.3030
0.07831
2.3710
10
A2
4
ATRAZINE
0
2
0.2169
0.02263
10.4327
11
A2
5
ATRAZINE
0
2
2.8070
0.34990
12.4655
12
C1
1
CONTROL
0
2
3.5497
0.02948
0.8304
13
C1
2
CONTROL
0
2
1.6790
0.04290
2.5551
14
C1
3
CONTROL
0
2
4.5738
0.50058
10.9444
15
C1
4
CONTROL
0
2
3.9795
0.02188
0.5497
16
C1
5
CONTROL
0
2
3.1596
0.03938
1.2463
17
C2
1
CONTROL
0
2
10.6332
0.14104
1.3264
18
C2
2
CONTROL
0
2
9.6644
0.09341
0.9665
19
C2
3
CONTROL
0
2
3.3400
1.00685
30.1450
20
C2
4
CONTROL
0
2
6.0585
0.00074
0.0122
21
C2
5
CONTROL
0
2
7.5248
0.31761
4.2209
MEAN
GONADAL
AROMATASE
ACTIVITY
BY
SAMPLE
IN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
3)
311
Obs
SAMPLE
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
A1
0
6
6.05412
1.84866
30.5355
2
A2
0
5
2.61093
1.77449
67.9639
3
C1
0
5
3.38835
1.09028
32.1774
4
C2
0
5
7.44418
2.90987
39.0892
MEAN
GONADAL
AROMATASE
ACTIVITY
IN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
3)
312
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
ATRAZINE
0
11
4.48904
2.49031
55.4753
2
CONTROL
0
10
5.41627
2.97674
54.9592
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
27
of
28
ANOVA
FOR
GONADAL
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
FROGS
(
EXP
3)
313
The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
GROUP
2
ATRAZINE
CONTROL
Number
of
observations
21
Dependent
Variable:
MEAN
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
1
4.5034675
4.5034675
0.60
0.4468
Error
19
141.7650045
7.4613160
Corrected
Total
20
146.2684720
R­
Square
Coeff
Var
Root
MSE
MEAN
Mean
0.030789
55.40006
2.731541
4.930574
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
GROUP
1
4.50346755
4.50346755
0.60
0.4468
Levene's
Test
for
Homogeneity
of
MEAN
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
GROUP
1
28.6086
28.6086
0.49
0.4920
Error
19
1107.1
58.2672
Bartlett's
Test
for
Homogeneity
of
MEAN
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
GROUP
1
0.2867
0.5923
Dunnett's
t
Tests
for
MEAN
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
19
Error
Mean
Square
7.461316
Critical
Value
of
Dunnett's
t
2.09309
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
GROUP
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
CONTROL
­
ATRAZINE
0.9272
­
1.5709
3.4253
Data
Evaluation
Report
on
Methods
Development
for
the
Study
of
Mechanism
of
Action
of
Atrazine
in
Adult
and
Metamorphosing
Xenopus
laevis
and
Rana
clamitans:
Aromatase
Induction
EPA
MRID
Number
458677­
08
Page
28
of
28
NONPARAMETRIC
COMPARISON
OF
GONADAL
AROMATASE
ACTIVITY
BETWEEN
CONTROL
AND
ATRAZINE­
TREATED
317
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
MEAN
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
ATRAZINE
11
113.0
121.0
14.200939
10.272727
CONTROL
10
118.0
110.0
14.200939
11.800000
Wilcoxon
Two­
Sample
Test
Statistic
118.0000
Normal
Approximation
Z
0.5281
One­
Sided
Pr
>
Z
0.2987
Two­
Sided
Pr
>
|
Z|
0.5974
t
Approximation
One­
Sided
Pr
>
Z
0.3016
Two­
Sided
Pr
>
|
Z|
0.6032
Z
includes
a
continuity
correction
of
0.5.

Kruskal­
Wallis
Test
Chi­
Square
0.3174
DF
1
Pr
>
Chi­
Square
0.5732
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
MEAN
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
ATRAZINE
11
5.0
5.238095
1.171274
0.454545
CONTROL
10
5.0
4.761905
1.171274
0.500000
Median
Two­
Sample
Test
Statistic
5.0000
Z
0.2033
One­
Sided
Pr
>
Z
0.4195
Two­
Sided
Pr
>
|
Z|
0.8389
Median
One­
Way
Analysis
Chi­
Square
0.0413
DF
1
Pr
>
Chi­
Square
0.8389