Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Data
Requirement:
:
EPA
DP
Barcode
D288775
Abcdef8
EPA
MRID
458677­
04
EPA
Guideline
70­
1(
Special
Study)

Test
material:
Purity:
97.1%
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
27,
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:
Hecker,
M.,
K.
K.
Coady,
D.
L.
Villeneuve,
M.
B.
Murphy,
P.
D.
Jones
and
J.
P.
Giesy.
2003.
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
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
04.
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
2
of
48
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
3
of
48
EXECUTIVE
SUMMARY:

African
clawed
frogs
(
Xenopus
laevis)
were
exposed
(
approximately
30
tadpoles/
replicate
with
8
replicates/
treatment)
to
atrazine
at
0.1,
1.0,
10,
and
25

g/
L
in
FETAX
media
in
a
static
renewal
system
where
50%
of
the
exposure
solutions
were
changed
every
72
hours..
Exposures
were
also
conducted
using
negative
and
positive
(
17­
 
estradiol
and
dihydrotestosterone)
and
a
solvent
(
0.005%
ethanol)
control.
Larvae
were
exposed
from
72­
hours
post­
hatch
through
metamorphosis
(
fore­
limb
emergence
and
tail
resorption;
NF
stage
66).
At
metamorphosis,
a
subset
(
number
not
recorded)
was
euthanized;
gonads
were
examined
for
gross
morphology
and
gonad/
larynx
prepared
for
histology.
The
remaining
animals
were
exposed
until
2­
3
months
post­
metamorphosis.
Afterwards,
half
the
frogs
were
used
for
gross
morphology
and
histology
of
the
gonads.
All
frogs
were
examined
for
gross
morphology,
and
50
frogs
per
treatment
were
serially
sectioned
for
gonad
histology.
One
frog
from
each
replicate
tank
(
64
frogs
total)
was
randomly
selected,
and
blood
(
drawn
by
cardiac
puncture),
brain
and
gonads
were
collected
for
sex
steroid
hormone
and
aromatase
activity
assays.
Plasma
concentrations
of
testosterone
and
estradiol
were
measured
by
ELISA,
while
tritium­
labeled
water
release
assay
was
used
to
measure
aromatase
in
brain
and
gonad
tissue.

The
study
authors
concluded
that
atrazine
treatment
did
not
affect
mortality,
time
to
metamorphosis,
sex
ratio,
gonadal
development,
aromatase
activity
or
steroid
hormone
plasma
concentrations
in
a
dose­
dependent
fashion.
Also,
estradiol
(
positive
control)
treatment
only
appeared
to
increase
estradiol
plasma
concentrations.
Dihyrotestosterone
(
positive
control)
increased
larynx
dilator
muscle
area
in
females,
and
neither
positive
control
influenced
sex
ratios.

Although
the
most
frequent
gonadal
abnormality
based
on
gross
morphology
was
discontinuous
gonads,
histology
indicated
that
mixed
sex/
intersex
(
ovarian
and
testicular
tissue
in
the
same
frog)
was
much
more
common
than
indicated
by
gross
morphology.
Since
histology
is
still
being
conducted,
it
is
premature
to
conclude
that
gonadal
abnormalities
were
not
treatment­
related.

Poor
water
quality
(
elevated
ammonia
and
nitrite
with
low
dissolved
oxygen)
resulting
from
relatively
high
loading
rates
(
30
tadpoles/
4
liters
of
exposure
solution)
under
static
conditions
may
have
compromised
the
growth
and
development
of
the
test
animals.
On
average,
it
took
73
days
for
frogs
to
complete
metamorphosis
and
17
(<
2%)
frogs
in
the
study
never
underwent
metamorphosis.
Furthermore,
the
negative
controls
were
contaminated
with
atrazine
at
levels
comparable
to
those
in
the
0.1

g/
L
atrazine
treatment.
High
variability
(
coefficients
of
variability
ranging
as
high
as
524%)
associated
with
gonadal
aromatase
activity
and
with
plasma
steroid
hormone
levels
made
it
difficult
to
differentiate
treatment
effects.
Also,
it
is
unclear
why
estradiol
treatment
failed
to
skew
sex
ratios
significantly
in
favor
of
females
when
other
studies
have
demonstrated
this
effect.
In
summary,
a
combination
of
tank
effects,
contaminated
controls,
high
variability
and
an
apparent
lack
of
responsiveness
to
estradiol
made
it
difficult
for
the
study
authors
to
test
their
hypothesis
and
to
differentiate
treatment
effects.
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
4
of
48
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
QA
Inspections
were
conducted.
°
Final
Report
was
written
°
Raw
data,
documentation,
records,
protocols,
and
final
report
were
archieved.

A.
MATERIALS:

1.
Test
Material
Atrazine
Description:
Not
reported
Lot
No./
Batch
No.
:
Not
reported
Purity:
97.1%
Stability
of
compound
undertest
conditions:
Not
reported
Storage
conditions
of
test
chemicals:
Not
reported
2.
Test
organism:

Species:
African
clawed
frog
(
Xenopus
laevis)
.
Age
at
test
initiation:
Larvae
(
72­
hours
post­
hatch)
Weight
at
study
initiation:
(
mean
and
range)
Not
reported
Length
at
study
initiation:
(
mean
and
range)
Not
reported
Source:
Sexually
mature
X.
laevis
obtained
from
Xenopus
Express
®
(
Homossa,
FL)
induced
with
human
chorionic
gonadotropin;
fertilized
eggs
dejellied
in
2%
L­
cysteine
in
FETAX
medium
checked
for
viability,
divided
into
groups
of
35
fertilized
eggs,
then
distributed
into
exposure
solutions
at
72­
hours
post­
hatch.
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
5
of
48
B.
STUDY
DESIGN:

Objective:
1.
To
determine
the
effects
of
atrazine
on
metamorphosis
and
reproductive
indices
of
larval,
Xenopus
laevis
were
exposed
from
72
hours
after
hatching
until
the
completion
of
metamorphosis.
Indices
evaluated
at
the
time
of
metamorphic
completion
included
%
initiation
of
metamorphosis,
%
completion
of
metamorphosis,
time
to
metamorphosis,
fresh
body
weight,
snout­
vent
length,
size
of
the
laryngeal
dilator
muscle
and
gonad
development.

2.
To
determine
the
concentration
of
circulating
hormones,
including
testosterone
and
estradiol
in
control
and
atrazine­
treated
X.
laevis
3.
To
investigate
aromatase
activity
in
the
gonads
and
brain
tissue
of
control
and
atrazine­
exposed
X.
laevis.

1.
Experimental
Conditions
a)
Range­
finding
Study:
Exposure
concentrations
based
on
previous
work
b.
Definitive
Study
Table
1
.
Experimental
Parameters
Parameter
Details
Acclimation:
period:
Conditions:
(
same
as
test
or
not)
Feeding:
Health:
(
any
mortality
observed)
72
hours
FETAX
solution
not
reported
not
reported
Duration
of
the
test
185­
day
study
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
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Parameter
Details
Page
6
of
48
Test
vessel
Material:
(
glass/
stainless
steel)
Size:
Fill
volume:
glass
10
L
4
L
When
frogs
began
to
undergo
metamorphosis,
they
were
transferred
to
40­
L
aquariums
containing
10
L
of
test
solution.
According
to
the
Protocol
Changes/
Revisions
section
of
the
report,
frogs
were
maintained
in
4­
L
of
test
solution
until
approximately
one
month
post­
metamorphosis,
at
which
point
they
were
transferred
to
larger
aquariums.

Source
of
dilution
water
Quality:
Treated
well
water
(
MSU­
University
Research
Containment
Facility;

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

Intervals
of
water
quality
measurement
140
mg/
L
as
CaCO3
7.7
(
range
6.3
­
8.1)
median
DO
7.4
mg/
L
(
low
range:
2.5
­
8.8
mg/
L)

median
total
ammonia:
0.02
mg/
L
(
range:
0.02
­
1.6
mg/
L)
median
nitrite
conc.
0.06
mg/
L
(
range:
0.02
­
4.0
mg/
L)

17
­
23oC
(
median
temperature
20oC)

NA
not
reported
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Parameter
Details
Page
7
of
48
Number
of
replicates/
groups:
negative
control:
FETAX
media
solvent
control:
0.005%
ethanol
treated
ones:
atrazine
at
0.1,
1.0,
10
and
25

g/
L
in
FETAX
positive
controls:
dihydrotestosterone
and
estradiol
in
0.005%
EtOH.
8
8
8
8
Number
of
organisms
per
replicate
/
groups:
control:
solvent
control:
treated
ones:
(
30
tadpoles
/
rep)
x
8
reps
=
240
tadpoles
30
tadpoles
/
rep)
x
8
reps
=
240
tadpoles
30
tadpoles
/
rep)
x
8
reps
=
240
tadpoles
Biomass
loading
rate
30
tadpoles/
4
L
Test
concentrations:
nominal:
measured:
0.1,
1.0,
10
and
25

g/
L
0.2,
1.0,
16,
and
29

g
a.
i./
L
Solvent
(
type,
percentage,
if
used)
FETAX
for
atrazine;
0.005%
ethanol/
FETAX
for
positive
hormone
controls
FETAX:
0.625
g/
L
NaCl;
0.030
g/
L
KCl;
0.015
g/
L
CaCl2;
0.096
g/
L
NaHCO3;
0.06
g/
L
CaSO4*
2H2O;
and
0.075
g/
L
MgSO4)

Lighting
not
reported
Feeding
Appendix
reports
that
frog
brittle
was
previously
analyzed
(
TTU­
10/
Syngenta
Number
1833­
01)
by
immunoassay
yielding
inconclusive
results.
Feeding
regime
is
not
reported
Recovery
of
chemical
Level
of
Quantitation
Level
of
Detection
ELISA
(
Envirogard
Triazine
®
;
Strategic
Diagnostics
Newark,
DE)/
Beacon
Analytical
triazine
plate
(
Beacon
Analytical
Systems,
Portland,
ME)

LOD
0.025

g/
L
(
Envirogard);
0.05

g/
L
(
Beacon)

Positive
control
{
if
used,
indicate
the
chemical
and
concentrations}
dihydrotestosterone
0.1

g/
L
17­
 
estradiol
0.1

g/
L
both
hormones
in
0.005%
ethanol
Other
parameters,
if
any
NA
2.
Observations:
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
8
of
48
Table
2:
Observations
Criteria
Details
Parameters
measured
including
the
sublethal
effects/
toxicity
symptoms
mortality;
time
to
metamorphosis,
number
completing
metamorphosis,
age
(
days)
at
metamorphosis,
length,
weight,
gonadal
abnormalities,
sex
Observation
intervals
daily
Were
raw
data
included?
Yes
Other
observations,
if
any
Animals
not
reaching
metamorphosis
by
506
days
were
sacrificed.

All
frogs
completing
metamorphosis
were
analyzed
for
gross
morphology
and
histology
of
the
gonads
(
no
mention
of
kidneys).

At
metamorphosis,
a
subset
(
number
not
recorded)
was
euthanized
at
metamorphic
completion.
Gonads
were
examined
for
gross
morphology,
and
gonad/
larynx
was
prepared
for
histology.
Remaining
tadpoles/
metamorphs
were
transferred
to
40­
L
aquariums
containing
10
L
of
test
solution
where
frogs
were
exposed
until
2­
3
months
post­
metamorphosis.
Afterwards,
half
the
frogs
were
fixed
in
Bouin's
solution
and
set
aside
for
gross
morphology
and
histology
of
the
gonads.
All
frogs
were
examined
for
gross
morphology,
and
50
frogs
per
treatment
were
serially
sectioned
for
gonad
histology.
The
remaining
half
of
the
"
grow­
out"
frogs
were
killed
and
necropsied
from
June
11
to
June
24,
2002.
One
frog
from
each
replicate
tank
(
64
frogs
total)
was
randomly
selected
and
blood
(
drawn
by
cardiac
puncture),
brain
and
gonads
were
collected
for
sex
steroid
hormone
and
aromatase
activity
assays.
Plasma
concentrations
of
testosterone
and
estradiol
were
measured
by
ELISA.
Tritium­
labeled
androstenedione
water
release
assay
was
used
to
measure
aromatase
in
brain
and
gonad
tissue.

II.
RESULTS
and
DISCUSSION:

Water
Quality
No
ammonia
or
nitrite
levels
are
reported
for
February
6
through
21,
2002.
Total
ammonia
nitrogen
(
range
0.02
­
1.6
mg/
L)
and
nitrite
(
range:
0.02
­
3.0
mg/
L)
appeared
to
be
highest
during
the
January
10
through
April
1
,
2002.
This
period
corresponds
to
roughly
exposure
days
21
through
80
and
suggests
that
water
quality
may
have
influenced
this
study.
Also,
pH
during
the
first
week
of
the
study
(
December
21
­
27,
2001)
was
unusually
low
(
pH
range:
6.3
­
6.9).
The
lower
range
of
dissolved
oxygen
was
consistently
low
from
February
21
through
March
20,
2002,
averaging
4.9
mg/
L
and
dropped
as
low
as
2.5
mg/
L.

Mean­
measured
concentrations
of
atrazine
(
Table
3)
in
the
0.1,
10,
and
25

g/
L
treatments
ranged
from
1.2
to
2.0
times
higher
than
nominal
concentrations,
while
the
1.0

g/
L
group
measured
values
were
consistent
with
nominal.
Atrazine
was
however
detected
at
measurable
levels
in
the
dilution
water
control.
Atrazine
concentrations
in
the
dilution
water
control
ranged
as
high
as
those
detected
in
the
lowest
(
0.1

g/
L)
atrazine
Data
Evaluation
Report
on
Response
of
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laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
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9
of
48
treatment.
While
atrazine
was
detected
in
positive
controls
and
the
solvent
control,
the
levels
reported
are
at
and/
or
below
the
detection
limit
of
the
ELISA
assay
used
for
analysis.

Mortality
According
to
the
study,
total
average
observed
mortality
across
all
treatments
was
16.1%
(
Table
4);
however,
there
were
many
unaccounted
animals
and
if
they
were
dead,
then
the
average
mortality
across
treatments
would
be
20.2%.

A
total
of
17
surviving
tadpoles
did
not
initiate
metamorphosis;
therefore,
98.9%
of
surviving
frogs
initiated
metamorphosis
during
the
study
period.
There
were
no
significant
differences
in
age
at
completion
of
metamorphosis
among
treatment
groups
when
treatments
were
compared
to
the
appropriate
controls
(
ANOVA
atrazine
data
set,
p
=
0.986;
ANOVA
positive
control
data
set,
p
=
0.703).
The
average
age
at
metamorphic
completion
across
all
treatment
groups
was
72.8
days.

Growth
Average
snout­
vent
length
at
completion
of
metamorphosis
across
all
treatments
was
1.85
cm;
there
was
no
difference
in
length
between
atrazine­
treated
and
controls
(
ANOVA
p=
0.066),
nor
was
there
a
difference
in
weights
compared
to
the
positive
controls
(
ANOVA
p
=
0.512).
However,
frogs
in
the
solvent
control
were
significantly
(
p
=
0.032)
longer
(
1.89
cm)
than
negative
control
frogs
(
1.75
cm).

Average
weight
of
frogs
at
completion
of
metamorphosis
across
all
treatments
was
0.78
g.
There
was
no
significant
difference
in
weight
at
completion
of
metamorphosis
between
treated
and
control
animals
(
P
=
0.22)
or
between
treated
and
positive
controls
(
p
=
0.311).
However,
frogs
in
the
solvent
control
(
0.85
gm)
were
significantly
different
(
p
=
0.046)
than
negative
controls
(
0.70
g).

Gonadal
Abnormalities
Frogs
were
examined
for
gonadal
deformities
at
two
points
along
the
course
of
the
study;
one
subset
was
examined
upon
completion
of
metamorphosis
(
NF
Stage
66),
while
the
other
set
was
examined
2
to
3
months
after
metamorphic
completion
(
referred
to
as
the
"
grow­
out"
frogs).
Four
types
of
gross
gonadal
abnormalities
were
observed:
discontinuous
gonad
(
abnormal
segmentation
of
the
gonad),
intersex
gonad
(
ovarian
and
testicular
tissue
separated
left/
right
or
rostral/
caudal
in
a
single
individual),
mixed
sex
gonad
(
co­
occurrence
of
both
ovarian
and
testicular
tissue
in
a
single
gonad)
and
size
irregularity
(
large
size
discrepancy
between
gonad
pairs).
Other
abnormalities
included
small
or
underdeveloped
ovaries
(
relatively
few
or
no
eggs).
Although
these
effects
were
observed,
no
statistically
significant
treatment
effects
on
the
occurrence
of
gross
gonadal
abnormalities
were
found
among
NF
Stage
66
frogs
or
among
"
grow­
out"
frogs.

The
most
common
gross
gonadal
abnormality
was
discontinuous
gonads
for
both
NF
Stage
66
(
Tables
5)
and
grow­
out
frogs
(
Table
6).
Table
7
shows
percentages
of
gross
gonadal
abnormalities
for
Stage
66
and
growout
frogs.
However,
the
most
common
gonadal
abnormality
at
a
tissue
level
at
Stage
66
was
intersex.
Since
these
evaluations
are
still
underway,
the
actual
percentage
has
yet
to
be
determined.
While
there
were
no
observations
of
intersex
based
on
gross
gonadal
morphology
of
grow­
out
frogs,
histology
revealed
a
higher
percentage
of
both
mixed
and
intersex
gonads,
especially
among
males
(
Tables
8
and
9).
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Sex
Ratios
There
were
no
consistent
deviations
from
the
expected
50:
50
sex
ratio
(
Table
10).
While
several
tanks
had
statistically
significant
deviations
from
the
50:
50
ratio,
"
the
ratio
was
not
consistently
skewed
in
favor
of
one
sex
over
the
other,
but
varied
from
tank
to
tank."
One
ethanol
and
one
dihydrotestosterone
exposed
tank
and
two
0.1

g
atrazine/
L
tanks
had
sex
ratios
in
favor
of
more
males,
while
one
ethanol
and
one
estradiol
treated
tank
had
skewed
sex
ratios
in
favor
of
more
females.
There
was
however
no
statistical
difference
between
the
percent
males
(
ANOVA,
p=
0.108)
or
percent
females
(
ANOVA,
p=
0.137)
in
atrazine­
treated
and
negative
controls.
Also,
there
was
no
statistical
difference
between
the
percent
of
males
(
ANOVA,
p=
0.111)
or
females
(
ANOVA,
p=
0.232)
in
positive
controls
versus
the
solvent
control.
The
estradiol
group
did
have
a
greater
percentage
of
"
unknown"
(
sexually
unidentifiable)
frogs
as
compared
to
all
other
treatment
groups
except
the
10

g
atrazine/
L
group.

Larynx
Muscle
Overall,
male
frogs
had
laryngeal
dilator
muscle
cross­
sectional
areas
that
were
significantly
greater
than
female
muscle
areas
(
Mann0Whitney
U,
p=
0.0001);
there
were
no
significant
differences
between
male
atrazine­
treated
frogs
and
negative
controls
(
Kruskal­
Wallis,
p
=
0.476).
Male
frogs
exposed
to
DHT
had
greater
laryngeal
muscle
area
than
"
all
other
treatment
groups"
(
Table
11).
Female
atrazine­
treated
frog
laryngeal
muscle
area
did
not
differ
significantly
from
negative
controls
(
Kruskal­
Wallis,
p
=
0.181);
however,
females
treated
with
DHT
had
greater
laryngeal
muscle
area
compared
to
"
all
other
treatment
groups"
(
Kruskal­
Wallis,
p
=
0.0001)
(
Table
12).

Aromatase
Activity
Aromatase
activity
in
female
gonads
of
juvenile
X.
laevis
were
significantly
greater
than
in
males
(
Mann0Whitney,
p
=
0.0001).
There
was
no
difference
in
activity
of
males
(
Table
13)
treated
with
atrazine
and
controls
(
Kruskal­
Wallis,
p
=
0.075),
nor
was
there
any
difference
between
positive
control
males
and
solvent
controls
(
Kruskal­
Wallis,
p
=
0.382).
There
was
no
difference
in
female
gonadal
aromatase
(
Table
14)
activity
in
atrazine­
treated
and
control
animals
(
Kruskal­
Wallis,
p
=
0.821);
however,
females
treated
with
estradiol
had
statistically
less
gonadal
aromatase
activity
than
solvent
controls
(
Mann
Whitney
U,
p
=
0.0003).

Similarly
brain
aromatase
activity
was
significantly
greater
(
Mann­
Whitney
U,
p=
0.024)
in
females
(
mean
=
8.9
x
102
fmol/
h/
mg
protein)
than
in
males
(
7.2
x
102
fmol/
h/
mg
protein).
There
were
no
significant
differences
in
male
(
Table
15)
brain
aromatase
activity
between
atrazine­
treated
and
control
frogs
(
Kruskall­
Wallis,
p
=
0.410).
Estradiol­
treated
males
had
significantly
higher
activity
than
DHT­
treated
(
Mann­
Whitney,
p
=
0.012);
however,
neither
positive
control
differed
significantly
from
the
solvent
control.
Additionally,
there
was
no
difference
in
activity
between
atrazine­
treated
females
(
Table
16)
and
controls
(
Kruskal
Wallis,
p
=
0.885)
nor
among
positive
control
females
and
solvent
control
(
Kruskal­
Wallis,
p=
0.597).

Steroid
Hormone
Levels
While
testosterone
and
estradiol
were
measurable
in
plasma,
estradiol
concentration
in
both
male
(
Table
17)
females
(
Table
18)
were
often
less
than
the
assay
detection
limit.
Estradiol
was
significantly
(
Kruskal­
Wallis,
p=
0.02)
higher
in
females
(
mean
4.2
ng/
L)
than
in
males
(
2.7
ng/
L).
Frogs
treated
with
1

g/
L
atrazine
had
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significantly
less
estradiol
than
controls,
0.1
and
25

g/
L­
treated
frogs
(
Mann­
Whitney,
p
<
0.015);
however,
males
exposed
to
0.1,
10
and
25

g/
L
were
not
different
than
untreated
controls.
Plasma
estradiol
in
males
treated
with
estradiol
were
higher
than
those
in
the
solvent
control
(
Mann­
Whitney
U,
p
=
0.008)

Among
female
there
was
no
difference
in
atrazine­
treated
and
control
frogs
nor
between
positive
control
and
solvent
controls.

There
were
no
differences
between
male
and
female
testosterone
levels
(
Kruskal­
Wallis,
p=
0.170);
there
was
no
significant
difference
between
atrazine­
treated
males
(
Table
19)
and
untreated
control
males
(
Kruskal­
Wallis,
p
=
0,270)
nor
between
positive
controls
and
solvent
control
(
Kruskal­
Wallis,
p
=
0.187).
Likewise,
there
was
no
difference
in
plasma
testosterone
levels
in
atrazine­
treated
females
(
Table
20)
and
controls
(
Kruskal­
Wallis,
p
=
0.179)
or
between
positive
controls
and
solvent
controls
(
Kruskal­
Wallis,
p=
0.363).

C.
REPORTED
STATISTICS:
Table
3.
Nominal
versus
mean­
measured
atrazine
concentrations.

Treatment
Atrazine
(
nominal)


g/
L
Syngenta
mean­
measured

g/
L
MSU
mean­
measured

g/
L
Control
­­
0.16
(
0.11
­
0.20)
0.11
(
0.07
­
0.15)

Solvent
Control
­­
0.010
(
0.01
­
0.01)
<
0.05
Dihydrotestosterone
(
0.1

g/
L)
­­
0.020
(
0.01
­
0.01)
<
0.05
17­
 
estradiol
­­
0.04
(
0.03
­
0.05)
<
0.05
0.1

g/
L
0.1
0.22
(
0.17
­
0.26)
0.23
(
0.16
­
0.31)

1.0

g/
L
1.0
1.0
(
0.93)
1.4
(
1.2
­
1.7)

10

g/
L
10
16
(
14
­
19)
11
(
9.7
­
13)

25

g/
L
25
29(
24
­
35)
25
(
22
­
28)
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Table
4.
Percent
observed
natural
mortality,
percent
unaccounted
for
X.
laevis,
and
total
percent
mortality
(
observed
+
unaccounted).

Treatment
Average
%
Natural
Mortality
(
observed)
%
Unaccounted
Animals
Total
%
Mortality
Control
11.3
6.6
17.9
Solvent
Control
14.4
3.4
17.8
Dihydrotestosterone
(
0.1

g/
L)
21.3
1.7
23.0
17­
 
estradiol
8.3
2.6
10.9
0.1

g/
L
20.4
3.4
23.8
1.0

g/
L
11.5
5.3
16.8
10

g/
L
18.7
3.1
21.8
25

g/
L
23.0
6.4
29.4
Table
5.
Percent
gross
gonadal
abnormalities
in
NF
Stage
66
X.
laevis
exposed
to
negative
control,
solvent
control,
positive
controls
or
various
concentrations
of
atrazine.

Treatment
N
%
Discontinuou
s
Gonads
%
Mixed
Gonads
%
Size
Irregularitie
s
%
Intersex
%
other
Control
45
2.2
0.0
2.2
0.0
2.2
Solvent
Control
45
0.0
0.0
0.0
0.0
2.2
Dihydrotestosterone
(
0.1

g/
L)
42
4.8
2.4
2.4
0.0
4.78
17­
 
estradiol
46
6.5
4.4
0.0
2.2
2.2
0.1

g/
L
40
5.0
0.0
0.0
0.0
7.5
1.0

g/
L
46
2.2
0.0
2.2
0.0
0.0
10

g/
L
43
7.0
0.0
0.0
0.0
4.7
25

g/
L
39
5.1
2.6
0.0
0.0
0.0
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Table
6.
Percent
gross
gonadal
abnormalities
in
male
and
female
grow­
out
X.
laevis
exposed
to
negative
control,
solvent
control,
positive
controls
or
various
concentrations
of
atrazine.

Treatment
N
%
Discontinuous
Gonads
%
Mixed
Gonads
%
Size
Irregularities
Control
75
1.4
0.0
2.7
Solvent
Control
75
2.7
0.0
1.3
Dihydrotestosterone
(
0.1

g/
L)
72
1.4
0.0
0.0
17­
 
estradiol
77
2.6
0.0
0.0
0.1

g/
L
71
4.2
0.0
0.0
1.0

g/
L
79
1.3
0.0
2.5
10

g/
L
73
4.1
2.7
0.0
25

g/
L
67
3.0
0.0
0.0
Table
7.
Percent
gross
gonadal
abnormalities
in
Stage
66
and
male
and
female
grow­
out
X.
laevis
exposed
to
negative
control,
solvent
control,
positive
controls
or
various
concentrations
of
atrazine.

Treatment
N
%
Discontinuous
Gonads
%
Mixed
Gonads
%
Size
Irregularities
Control
120
3.6
0.0
4.9
Solvent
Control
120
2.7
0.0
1.3
Dihydrotestosterone
(
0.1

g/
L)
114
6.2
2.4
2.4
17­
 
estradiol
123
9.1
6.6
0.0
0.1

g/
L
111
9.2
0.0
0.0
1.0

g/
L
125
3.5
0.0
4.7
10

g/
L
116
11.1
2.7
0.0
25

g/
L
106
8.1
2.6
0.0
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Table
8.
Percent
gonadal
abnormalities
at
the
tissue
level
(
based
on
histology)
in
grow­
out
male
X.
laevis
exposed
to
negative
control,
solvent
control,
positive
controls
or
various
concentrations
of
atrazine.

Treatment
N
%
Mixed
Sex
%
Intersex
%
Other
Abnormalities
Control
25
8.0
16.0
0.0
Solvent
Control
25
20.0
4.0
0.0
Dihydrotestosterone
(
0.1

g/
L)
25
4.0
0.0
0.0
17­
 
estradiol
25
32.0
0.0
0.0
0.1

g/
L
25
16.0
0.0
0.0
1.0

g/
L
25
8.0
4.0
0.0
10

g/
L
25
12.0
0.0
0.0
25

g/
L
25
8.0
0.0
0.0
Table
9.
Percent
gonadal
abnormalities
at
the
tissue
level
(
based
on
histology)
in
grow­
out
female
X.
laevis
exposed
to
negative
control,
solvent
control,
positive
controls
or
various
concentrations
of
atrazine.

Treatment
N
%
Mixed
Sex
%
Intersex
%
Other
Abnormalities
Control
25
0.0
0.0
0.0
Solvent
Control
25
0.0
0.0
4.0
Dihydrotestosterone
(
0.1

g/
L)
25
0.0
0.0
0.0
17­
 
estradiol
25
8.0
0.0
0.0
0.1

g/
L
24
0.0
0.0
0.0
1.0

g/
L
25
0.0
0.0
8.0
10

g/
L
25
0.0
0.0
0.0
25

g/
L
25
0.0
0.0
4.0
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Table
10.
Percent
male
and
female
grow­
out
X..
laevis
in
each
treatment.

Treatment
%
Males
%
Females
%
Unkown
Control
48.2
51.8
0.0
Solvent
Control
45.2
54.8
0.0
Dihydrotestosterone
(
0.1

g/
L)
50.5
49.5
0.0
17­
 
estradiol
36.2
61.3
2.5
0.1

g/
L
57.9
42.1
0.0
1.0

g/
L
50.7
49.3
0.0
10

g/
L
49.2
49.7
1.1
25

g/
L
42.5
56.9
0.6
Table
11.
Layrngeal
dilator
muscle
areas
for
male
X.
laevis
exposed
to
positive
controls
and
various
concentrations
of
atrazine.

Treatment
Mean
Left
Laryngeal
Muscle
Area
(
mm2)
Standard
Error
Mean
Right
Laryngeal
Muscle
Area
(
mm2)
Standard
Error
Control
2.1
x
10­
1
9.0
x
10­
3
2.1
x
10­
1
1.0
x
10­
2
Solvent
Control
1.9
x
10­
1
2.0
x
10­
2
1.7
x
10­
1
1.6
x
10­
2
Dihydrotestosterone
(
0.1

g/
L)
3.0
x
10­
1*
2.2
x
10­
2
3.1
x
10­
1*
2.1
x
10­
2
17­
 
estradiol
2.2
x
10­
1
2.3
x
10­
2
2.1
x
10­
1
2.0
x
10­
2
0.1

g/
L
2.1
x
10­
1
2.4
x
10­
2
2.2
x
10­
1
2.8
x
10­
2
1.0

g/
L
2.3
x
10­
1
1.1
x
10­
2
2.2
x
10­
1
8.0
x
10­
3
10

g/
L
2.2
x
10­
1
1.3
x
10­
2
2.2
x
10­
1
1.8
x
10­
2
25

g/
L
2.0
x
10­
1
1.3
x
10­
2
1.9
x
10­
1
3.0
x
10­
2
*
significantly
different
(
Kruskal­
Wallis,
p
=
0.0001)
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
16
of
48
Table
12.
Layrngeal
dilator
muscle
areas
for
female
X.
laevis
exposed
to
positive
controls
and
various
concentrations
of
atrazine.

Treatment
Mean
Left
Laryngeal
Muscle
Area
(
mm2)
Standard
Error
Mean
Right
Laryngeal
Muscle
Area
(
mm2)
Standard
Error
Control
1.5
x
10­
1
1.0
x
10­
2
1.5
x
10­
1
1.0
x
10­
2
Solvent
Control
1.9
x
10­
1
2.8
x
10­
2
1.9
x
10­
1
3.6
x
10­
2
Dihydrotestosterone
(
0.1

g/
L)
3.5
x
10­
1*
6.8
x
10­
2
3.6
x
10­
1*
7.2
x
10­
2
17­
 
estradiol
1.5
x
10­
1**
8.0
x
10­
3
1.5
x
10­
1
1.3
x
10­
2
0.1

g/
L
2.1
x
10­
1
2.5
x
10­
2
2.1
x
10­
1
2.8
x
10­
2
1.0

g/
L
1.7
x
10­
1
1.2
x
10­
2
1.6
x
10­
1
1.4
x
10­
2
10

g/
L
1.5
x
10­
1
1.3
x
10­
2
1.7
x
10­
1
3.6
x
10­
2
25

g/
L
1.5
x
10­
1
7.0
x
10­
3
1.5
x
10­
1
8.0
x
10­
2
*
significantly
different
(
Kruskal­
Wallis,
p
=
0.0001)
**
value
corrected
to
1.5
x
10­
1
rather
than
15
x
10­
1
as
reported
in
study;
assumed
to
be
a
typo.
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
17
of
48
Table
13.
Gonadal
aromatase
activity
in
male
juvenile
X.
laevis
exposed
to
positive
controls
and
various
concentrations
of
atrazine.

Treatment
N
Male
Mean
Gonad
Aromatase
Activity
(
fmol/
h/
mg
protein)
Male
Median
Gonad
Aromatase
Activity
(
fmol/
h/
mg
protein)
Standa
rd
Error
Coefficie
nt
of
Variatio
n
Control
19
8.9
0.0
4.2
206%

Solvent
Control
21
5.6
0.0
2.7
221%

Dihydrotestosterone
(
0.1

g/
L)
21
7.2
0.0
3.2
204%

17­
 
estradiol
25
1.3
x
10­
1
0.0
4.2
161%

0.1

g/
L
24
8.7
0.0
4.7
264%

1.0

g/
L
23
3.6
x
10­
1
0.0
2.9
386%

10

g/
L
18
1.9
0.0
1.9
424%

25

g/
L
21
0.9
0.0
0.9
457%
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
18
of
48
Table
14.
Gonadal
aromatase
activity
in
female
juvenile
X.
laevis
exposed
to
positive
controls
and
various
concentrations
of
atrazine.

Treatment
N
Female
Mean
Gonad
Aromatase
Activity
(
fmol/
h/
mg
protein)
Female
Median
Gonad
Aromatase
Activity
(
fmol/
h/
mg
protein)
Standa
rd
Error
Coefficie
nt
of
Variatio
n
Control
16
530
270
160
121%

Solvent
Control
17
610
400
130
88%

Dihydrotestosterone
(
0.1

g/
L)
15
200
110
52
101%

17­
 
estradiol
15
110*
380
45
158%

0.1

g/
L
10
330
230
120
115%

1.0

g/
L
15
490
210
160
127%

10

g/
L
18
510
330
120
100%

25

g/
L
16
500
350
110
88%

*
significantly
different
(
Kruskal­
Wallis,
p
=
0.0003)
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
19
of
48
Table
15.
Brain
aromatase
activity
in
male
juvenile
X.
laevis
exposed
to
positive
controls
and
various
concentrations
of
atrazine.

Treatment
N
Male
Mean
Brain
Aromatase
Activity
(
fmol/
h/
mg
protein)
Male
Median
Brain
Aromatase
Activity
(
fmol/
h/
mg
protein)
Standa
rd
Error
Coefficie
nt
of
Variatio
n
Control
18
0.71
0.54
0.12
72%

Solvent
Control
21
0.93
0.89
0.17
84%

Dihydrotestosterone
(
0.1

g/
L)
19
0.54
0.47
0.09
73%

17­
 
estradiol
24
1.1
0.91
0.16
71%

0.1

g/
L
24
0.58
0.55
0.09
76%

1.0

g/
L
22
0.71
0.47
0.15
99%

10

g/
L
18
0.43
0.27
0.10
99%

25

g/
L
21
0.80
0.81
0.16
92%

Table
16.
Brain
aromatase
activity
in
female
juvenile
X.
laevis
exposed
to
positive
controls
and
various
concentrations
of
atrazine.

Treatment
N
Female
Mean
Brain
Aromatase
Activity
(
fmol/
h/
mg
protein)
Female
Median
Brain
Aromatase
Activity
(
fmol/
h/
mg
protein)
Standa
rd
Error
Coefficie
nt
of
Variatio
n
Control
15
1.1
0.81
0.22
77%

Solvent
Control
15
0.81
0.70
0.12
57%

Dihydrotestosterone
(
0.1

g/
L)
15
0.63
0.46
0.11
68%

17­
 
estradiol
15
0.86
0.48
0.20
90%

0.1

g/
L
10
0..
93
0.85
0.22
75%

1.0

g/
L
16
0.95
0.86
0.17
72%

10

g/
L
18
0.98
0.90
0.16
69%

25

g/
L
16
0.94
0.59
0.23
98%
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
20
of
48
Table
17.
Plasma
estradiol
concentrations
in
male
juvenile
X.
laevis
exposed
to
positive
controls
and
various
concentrations
of
atrazine.

Treatment
N
Male
Mean
Estradiol
(
ng/
mL)
Male
Median
Estradiol
(
ng/
mL)
Standa
rd
Error
Coefficie
nt
of
Variatio
n
Control
20
3.5
0.4
1.5
192%

Solvent
Control
21
0.6
0.03
0.5
382%

Dihydrotestosterone
(
0.1

g/
L)
21
1.9
0.2
0.6
145%

17­
 
estradiol
25
6.9**
0.4
5.8
420%

0.1

g/
L
26
1.3
0.1
0.4
157%

1.0

g/
L
24
0.029*
0.024
3.1
524%

10

g/
L
20
5.8
0.1
4.0
308%

25

g/
L
23
1.8
0.4
0.5
133%

*
significantly
different
(
Mann­
Whitney
U,
p
=
0..
015)
**
significantly
different
(
Mann­
Whitney
U,
p
=
0.008)
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
21
of
48
Table
18.
Plasma
estradiol
concentrations
in
female
juvenile
X.
laevis
exposed
to
positive
controls
and
various
concentrations
of
atrazine.

Treatment
N
Female
Mean
Estradiol
(
ng/
mL)
Female
Median
Estradiol
(
ng/
mL)
Standa
rd
Error
Coefficie
nt
of
Variatio
n
Control
15
9.8
0.2
6.0
237%

Solvent
Control
18
0.066
0.024
0.022
141%

Dihydrotestosterone
(
0.1

g/
L)
16
15
0.035
15
400%

17­
 
estradiol
15
2.2
0.2
0.9
158%

0.1

g/
L
12
4.2
0.017
2.6
214%

1.0

g/
L
16
0.5
0.027
0.3
240%

10

g/
L
17
0.9
0.018
0.9
412%

25

g/
L
17
1.2
0.032
0.6
206%

Table
19.
Plasma
testosterone
concentrations
in
male
juvenile
X.
laevis
exposed
to
positive
controls
and
various
concentrations
of
atrazine.

Treatment
N
Male
Mean
Testosterone
(
ng/
mL)
Male
Median
Testosterone
(
ng/
mL)
Standa
rd
Error
Coefficie
nt
of
Variatio
n
Control
20
1.7
0.1
0.8
210%

Solvent
Control
21
0.6
0.1
0.4
306%

Dihydrotestosterone
(
0.1

g/
L)
21
0.3
0.2
0.1
153%

17­
 
estradiol
25
1.2
0.2
0.8
333%

0.1

g/
L
26
0.4
0.1
0.1
127%

1.0

g/
L
24
0.1
0.1
0.018
88%

10

g/
L
20
1.6
0.2
1.1
307%

25

g/
L
23
0.3
0.1
0.1
160%
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
22
of
48
Table
20.
Plasma
testosterone
concentrations
in
female
juvenile
X.
laevis
exposed
to
positive
controls
and
various
concentrations
of
atrazine.

Treatment
N
Female
Mean
Testosterone
(
ng/
mL)
Female
Median
Testosterone
(
ng/
mL)
Standa
rd
Error
Coefficie
nt
of
Variatio
n
Control
15
2.5
0.1
1.3
201%

Solvent
Control
18
0.1
0.1
0.03
127%

Dihydrotestosterone
(
0.1

g/
L)
16
2.6
0.1
2.4
369%

17­
 
estradiol
15
0.7
0.1
0.4
221%

0.1

g/
L
12
2.4
0.1
1.7
245%

1.0

g/
L
16
0.1
0.1
0.023
92%

10

g/
L
17
0.3
0.1
0.2
275%

25

g/
L
17
0.2
0.1
0.1
206%

D.
VERIFICATION
OF
STATISTICAL
RESULTS:
Basic
analyses
run
using
SAS
®
(
Statistical
Analysis
System,
Release
8.01,
Cary,
North
Carolina);
see
attached
printout.

E.
STUDY
DEFICIENCIES:
The
feeding
regime
is
not
reported;
however,
the
animals
were
apparently
fed
frog
brittle.
The
appendix
reports
that
a
previous
immunoassay
of
the
food
was
"
inconclusive".
It
is
unclear
what
"
inconclusive"
refers
to;
however,
an
analysis
of
the
current
study's
food
supply
was
apparently
not
run.
Also,
an
immunoassay
would
not
provide
information
on
a
broad
range
of
contaminants
and
suggests
that
the
food
analysis
may
have
only
looked
for
atrazine
residues.

Atrazine
was
detected
in
the
negative
control.
Apparently
tanks
were
not
covered
and
animals
may
have
hopped
between
treatments.

Water
quality
parameters
in
terms
of
total
ammonia
and
nitrite
were
unusually
high,
while
dissolved
oxygen
dropped
very
low.

F.
REVIEWER'S
COMMENTS:

From
January
10
through
April
1,
2002,
water
quality
parameters,
i.
e.,
total
ammonia
nitrogen,
nitrite
and
dissolved
oxygen,
suggest
that
water
quality
may
have
been
unreasonably
poor.
Total
ammonia
nitrogen
and
nitrite
rose
as
high
as
1.6
mg/
L
and
3
mg/
L,
respectively,
while
dissolved
oxygen
dropped
as
low
as
2.5
mg/
L.
In
some
cases,
i.
e.,
February
6,
2002,
dissolved
oxygen
concentrations
were
low
across
all
replicate
tanks
over
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
23
of
48
5
out
of
the
8
treatments.
At
a
median
temperature
of
20oC,
the
solubility
of
oxygen
is
8.84
mg/
L
(
Boyd
1984);
thus,
at
dissolved
oxygen
concentrations
of
2.5
mg/
L,
the
water
is
roughly
28%
of
saturation
In
a
study
of
Rana
clamitans
initiated
in
August
2001
(
ECORISK
Number
MSU­
03)
by
the
same
group
of
researchers,
a
similar
stocking
rate
of
30
tadpoles
per
4
L
of
exposure
solution
resulted
in
sufficiently
poor
water
quality
to
impair
the
survival/
development
of
the
tadpoles;
the
researchers
concluded
that
"
to
limit
mortality
and
maximize
development
in
future
laboratory
exposures
with
R.
clamitans,
it
is
recommended
that
tadpoles
be
reared
at
low
densities
in
static
tanks
(<
1
tadpole/
L)
or
in
a
continuous
flow­
through
system."
Mortality
in
the
current
study
averaged
20%
across
all
treatments;
however,
no
data
are
provided
to
determine
whether
mortality
was
associated
with
the
period
of
poor
water
quality.
The
similar
stocking
rate
for
X.
laevis
is
probably
responsible
for
the
poor
water
quality
during
a
considerable
portion
(
80
days)
of
the
developmental/
growth
period
for
these
tadpoles.

When
frogs
began
to
undergo
metamorphosis,
they
were
transferred
to
40­
L
aquariums
containing
10
L
of
test
solution.
According
to
the
Protocol
Changes/
Revisions
section
of
the
report,
frogs
were
maintained
in
4­
L
of
test
solution
until
approximately
one
month
post­
metamorphosis,
at
which
point
they
were
transferred
to
larger
aquariums.
However,
when
the
frogs
were
in
various
stages
of
metamorphosis,
the
10­
L
tanks
containing
4
L
of
test
solution
was
partitioned
into
three
sections.
One
section
housed
pre­
metamorphic
tadpoles,
one
section
held
metamorphosing
tadpoles
and
froglets,
and
the
third
section
contained
post­
metamorphic
frogs.
This
was
done
to
prevent
predation
on
smaller­
sized
tadpoles
and
to
facilitate
enumeration
of
individuals
in
each
stage
of
development.
While
the
tanks
may
have
been
larger,
the
partitioning
may
have
crowded
the
animals.

Atrazine
was
apparently
present
in
negative
control
samples
at
levels
comparable
to
the
0.1

g/
L
atrazine
treatment
concentration
level.
Additionally,
although
mean­
measured
concentrations
indicate
that
atrazine
treatment
levels
were
similar
or
even
higher
than
nominal,
the
data
represent
an
average
of
freshly
prepared
stock
solutions
and
72­
hour
aged
exposure
solutions.
It
would
have
been
useful
to
know
atrazine
levels
in
the
exposure
solutions
at
72
hours
before
the
solutions
were
renewed.

The
authors
dismissed
the
apparent
atrazine
contamination
of
the
controls.
However,
21
of
49
measurements
exceeded
0.1
ug/
L,
the
nominal
concentration
of
the
lowest
test
concentration
tested.
And
although
the
actual
concentrations
in
the
0.1
ug/
L
treatments
were
on
average
higher
than
nominal,
10
of
the
control
concentrations
exceeded
the
concentrations
measured
on
the
same
day
in
the
0.1
ug/
L
treatment.
This
suggests
that
these
exposures
were
probably
not
different.
The
author
notes
in
the
discussion
that
contaminants
in
surface
waters
can
give
false
positives
due
to
cross
reactivity
with
the
antibody
used
in
the
analysis.
However,
it
should
also
be
noted
that
they
used
well
water
that
was
treated
with
reverse
osmosis.
While
it
is
technically
true
that
the
controls
are
indistinguishable
from
the
low
test
concentration,
it
is
also
evident
that
the
animals
did
not
show
any
dose
dependent
effects
from
atrazine
over
the
concentrations
tested.

In
terms
of
exposure,
in
general
the
50%
renewal
every
72
hours
is
not
optimal,
and
probably
explains
the
lack
of
effects
in
the
"
positive
controls."
Exposure
to
these
compounds
may
not
be
efficacious
based
on
mass
limitations
of
the
compound
and
sorption
to
organic
matter
and
system
surfaces.
This
exposure
protocol
is
probably
also
responsible
for
the
mortality
rates
of
approximately
20%
and
may
explain
the
relatively
long
time
to
metamorphosis.

The
percentage
of
grow­
out
frogs
with
gonadal
deformities
based
on
gross
morphology
was
considerably
different
than
estimates
based
on
histology.
Since
the
histological
analysis
is
still
being
conducted
on
both
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
24
of
48
groups
of
frog
gonad
samples,
the
data
presented
in
this
report
are
preliminary
and
inconclusive.
Based
on
preliminary
data,
it
appears
that
the
incidence
of
mixed/
intersex
animals
was
highest
in
the
estradiol
positive
control
(
32%);
however,
both
the
negative
and
solvent
controls
had
relatively
high
incidence
(
24%)
of
mixed/
intersex
animals.
Because
negative
control
animals
were
apparently
exposed
to
levels
of
atrazine
consistent
with
those
in
the
0.1

g/
L
atrazine
treatment,
the
relatively
high
incidence
of
mixed/
intersex
tissue
in
male
gonads
(
negative
control
=
24%)
cannot
be
considered
reflective
of
background.
The
ability
of
this
study
to
discriminate
treatment
effects
appears
compromised.
Additionally,
males
were
clearly
more
prone
to
mixed/
intersex
phenomena
because
only
females
treated
with
estradiol
demonstrated
the
effect
(
8%)
based
on
histological
analysis.

The
utility
of
the
positive
controls
is
uncertain.
Previous
studies
(
Chang
and
Witschi
1955a,
b,
Gallien
1953,
Gallien
1954,
Gallien
1957,
Hayes
et
al.
2002a,
b
)
suggested
that
treatment
with
estradiol
would
skew
X.
laevis
sex
ratios
in
favor
of
females
at
estradiol
concentrations
of
0.1

g/
L;
however,
at
lower
estradiol
concentrations,
i.
e.,
0.04

g/
L
estradiol
may
not
impact
Xenopus
sex
ratios
(
Chang
and
Witschi
1955a,
b).
In
this
study,
the
authors
report
that
there
"
were
no
significant
differences
in
the
%
females
or
%
males
among
the
positive
controls
(
ANOVA,
p=
0.111
and
0.232,
respectively"
and
therefore,
0.1

g/
L
of
estradiol
had
no
apparent
affect
on
sex
ratios.
It
is
unclear
whether
the
unresponsiveness
of
X.
laevis
to
this
steroid
is
reflective
of
the
frog's
genuine
lack
of
sensitivity
(
contrary
to
the
Hayes'
study)
or
if
the
hormone
levels
were
not
sufficiently
high
enough
to
result
in
a
significant
effect
on
sex
ratios.
Dihydrotestosterone
only
appeared
to
affect
laryngeal
dilator
muscle
areas
.
Although
water
samples
were
collected
from
positive
controls,
hormone
concentrations
were
not
reported.

Gonad
aromatase
levels
in
females
were
generally
two
orders
of
magnitude
greater
than
in
males.
Although
gonad
aromatase
activity
in
both
atrazine­
treated
males
and
females
was
not
significantly
different
from
controls,
it
is
clear
from
Tables
13
and
14
that
there
was
considerable
variability
associated
with
these
estimates.
The
median
value
for
males
across
all
treatments
was
0.0
fmol/
h/
mg
protein,
while
mean
values
ranged
from
0.9
to
36
fmol/
h/
mg
protein.
Coefficients
of
variability
(
CV
=
[
standard
deviation
÷
mean]*
100)
ranged
from
264%
to
457%.
It
is
interesting
to
note
that
the
CV
for
atrazine­
treated
males
is
positively
correlated
(
r
=
0.97)
with
the
concentration
of
atrazine.

Brain
aromatase
levels,
while
statistically
higher
in
females
than
in
males,
were
within
the
same
order
of
magnitude
and
were
generally
less
variable
(
CV
range:
69
­
99%)
than
gonadal
aromatase
levels.
Once
again,
the
highest
variability
in
male
aromatase
activity
was
associated
with
atrazine­
treated
animals.

The
measurement
of
aromatase
activity
is
conducted
at
a
lifestage
that
is
not
relevant
to
gonadal
differentiation,
so
these
measurements
are
not
very
useful.
Furthermore,
it
is
still
an
open
question
as
to
whether
atrazine
can,
in
fact,
induce
aromatase
activity
because
there
are
no
efforts
to
evaluate
the
time
course
of
activity
in
the
presence
of
atrazine
exposure.
Since
aromatase
activity
is
part
of
a
homeostatically
controlled
system,
it
is
possible
that
there
could
be
transient
perturbation
in
activity
that
subsides
upon
the
system
reaching
homeostasis.
Because
there
are
no
apparent
changes
in
gonadal
differentiation,
though,
this
is
probably
a
moot
point.

The
high
variability
of
the
aromatase
activity
in
male
gonads
may
be
caused
by
the
relatively
high
rate
of
"
mixed
sex"
and
"
intersex"
tissues
as
determined
by
histological
measurements.
Ovaries
and
brains
from
both
sexes
had
substantially
lower
variances
associated
with
the
aromatase
measurements.
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
25
of
48
This
study
does
include
exposure
to
the
appropriate
life
stages
to
evaluate
the
effects
of
atrazine
on
gonadal
differentiation,
notably
stages
44­
54,
which
are
apparently
the
most
sensitive
to
femininization
by
exogenous
estrogenic
chemicals.

Plasma
steroid
hormone
levels
showed
significantly
less
estradiol
in
males
treated
with
1

g/
L
atrazine;
however,
none
of
the
other
atrazine
treated
animals
were
significantly
different
than
controls.
Estradiol
treated
frogs
had
significantly
higher
estradiol
than
the
solvent
controls.
Similar
to
the
gonad
aromatase
assay,
the
variability
associated
with
the
plasma
estradiol
and
testosterone
were
considerable
with
CVs
ranging
from
133%
to
524%
and
88%
to
369%,
respectively.
Given
this
level
of
variability
and
the
fact
that
many
of
the
frogs
tested
had
plasma
steroid
levels
at
or
near
the
detection
limit
of
the
assay,
jevenile
frog
plasma
steroid
levels
may
not
be
a
reliable
measure
of
atrazine­
treatment
effects.
Although
an
effort
was
made
to
draw
blood
samples
within
a
3­
hour
window
during
the
night,
the
fact
that
this
process
continued
over
a
number
of
days
may
have
confounded
the
study.

In
the
entire
experiment,
17
surviving
tadpoles
did
not
initiate
metamorphosis
by
506
days
and
were
apparently
terminated.
It
is
unclear
whether
these
animals
were
necropsied
or
what
the
status
of
their
gonads
was.

G.
CONCLUSIONS:

Atrazine
contamination
of
the
negative
controls
and
the
lack
of
responsiveness
to
the
positive
estradiol
control
limited
the
value
of
this
study
in
differentiatingtreatment
effects.
In
addition,
high
variability
in
gonad
aromatase
activity
and
plasma
activity
and
plasma
estradiol
and
testosterone
concentrations
made
it
difficult
to
test
the
hypothesis
of
this
study.
Mortality
was
relatively
consistent
(
mean
=
20%)
across
all
exposure
groups
with
approximately
5%
of
the
mortality
presumably
due
to
cannabalism.
Poor
water
quality
(
high
ammonia/
nitrite
and
low
dissolved
oxygen)
probably
resulted
from
relatively
high
stocking
rates
(
30
tadpoles
in
4
L
of
static
exposure
solution)
in
which
only
50%
of
the
exposure
solution
was
changed
every
72
hours.
More
rigorous
laboratory
testing
is
needed
to
characterize
the
effects
of
atrazine
on
amphibian
development.

H.
REFERENCES:

Boyd,
C.
E.
1984.
Water
quality
in
warmwater
fish
ponds.
Auburn
University
Agricultural
Experiment
Station,
Auburn,
Al.

Chang,
D.,­
Y
and
E.
Witschi
1955a.
Breeding
of
sex­
reversed
males
of
Xenopus
laevis.
Daudin.
Soc.
Exp.
Biol.
89:
150
­
151.

Chang,
D.,­
Y
and
E.
Witschi.
1955b.
Genic
and
hormonal
reversal
of
sex
differentiation
in
Xenopus.
Proc.
Soc.
Exp.
Biol.
Med.,
93:
140
­
144.

Gallien,
L.
1953.
Inversion
totale
du
sexe
chez
Xenopus
laevis
Daud.
à
la
suite
d'un
traitment
gynoène
par
le
benzoate
du
oestroadiol,
administré
pendant
la
vie
larvaire.
C.
R.
Acad.
Sci
237:
1565
­
1566.

Hayes,
T.
B.,
A.
Collins,
M.
Lee,
M.
Mendoza,
N.
Noriega,
A.
S.
Stuart,
and
A.
Vonk.
2002a.
Hermaphroditic,
demasculinized
frogs
after
exposure
to
the
herbicide
atrazine
at
low
ecologically
relevant
doses.
Proceedings
of
the
National
Academy
of
Sciences
of
the
United
States
of
America
99(
8):
5476
­
5480.
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
26
of
48
Hayes,
T.
B.,
K.
Haston,
M.
Tsui,
A.
Hoang,
C.
Haeffele,
and
A.
Vonk.
2002b.
Atrazine­
induced
hermaphroditism
at
0.1
ppb
in
American
leopard
frogs
(
Rana
pipiens):
laboratory
and
field
evidence.
Environmental
Health
Perspectives.

H.
REFERENCES:

Boyd,
C.
E.
1984.
Water
quality
in
warmwater
fish
ponds.
Auburn
University
Agricultural
Experiment
Station,
Auburn,
Al.

Chang,
D.,­
Y
and
E.
Witschi
1955a.
Breeding
of
ses­
reversed
males
of
Xenopus
laevis.
Daudin.
Soc.
Exp.
Biol.
89:
150
­
151.

Chang,
D.,­
Y
and
E.
Witschi.
1955b.
Genic
and
hormonal
reversal
of
sex
differentiation
in
Xenopus.
Proc.
Soc.
Exp.
biol.
Med.,
93:
140
­
144.

Gallien,
L.
1953.
Inversion
totale
du
sexe
chez
Xenopus
laevis
Daud.
à
la
suite
d'un
traitment
gynoène
par
le
benzoate
du
oestroadiol,
administré
pendant
la
vie
larvaire.
C.
R.
Acad.
Sci
237:
1565
­
1566.

Hayes,
T.
B.,
A.
Collins,
M.
Lee,
M.
Mendoza,
N.
Noriega,
A.
S.
Stuart,
and
A.
Vonk.
2002a.
Hermaphroditic,
demasculinized
frogs
after
exposure
to
the
herbicide
atrazine
at
low
ecologically
relevant
doses.
Proceedings
of
the
National
Academy
of
Sciences
of
the
United
States
of
America
99(
8):
5476
­
5480.

Hayes,
T.
B.,
K.
Haston,
M.
Tsui,
A.
Hoang,
C.
Haeffele,
and
A.
Vonk.
2002b.
Atrazine­
induced
hermaphroditism
at
0.1
ppb
in
American
leopard
frogs
(
Rana
pipiens):
laboratory
and
field
evidence.
Environmental
Health
Perspectives.

Hecker,
M.,
K.
K.
Coady,
D.
L.
Villeneuve,
M.
B.
Murphy,
P.
D.
Jones
and
J.
P.
Giesy.
2003.
A
pilot
study
of
response
of
larval
Rana
clamitans
to
atrazine
exposure:
assessment
of
metamorphosis
and
gonadal
and
laryngeal
morphology
and
selected
hormones
and
enzyme
activities.
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
03.
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
27
of
48
AVERAGE
LENGTH
AND
WEIGHT
OF
MALE
AND
FEMALE
FROGS
BY
TREATMENT
128
Obs
Treat
Sex
_
TYPE_
_
FREQ_
WEIGHT
LENGTH
W_
SD
L_
SD
1
0
F
0
73
3.21890
3.00730
0.79658
0.27341
2
0
M
0
73
3.04475
2.95132
0.82616
0.25585
3
0.1
F
0
56
3.40339
3.12521
1.10743
0.73651
4
0.1
M
0
82
3.40073
3.03395
0.91877
0.27963
5
1
F
0
77
2.83974
2.85431
0.91108
0.30657
6
1
M
0
80
2.86188
2.86024
0.75054
0.23584
7
10
F
0
70
3.26000
2.96167
1.10588
0.30883
8
10
M
0
68
3.10881
2.94512
0.75772
0.24669
9
10
U
0
2
3.43000
3.02150
1.10309
0.29345
10
25
F
0
74
3.61644
3.10945
1.15980
0.32159
11
25
M
0
55
3.16273
2.98069
0.91015
0.31387
12
DHT
F
0
69
3.22638
2.94362
1.72071
0.41002
13
DHT
M
0
72
3.25056
2.98472
1.31500
0.37757
14
E2
F
0
89
3.05114
3.03123
0.76144
0.26143
15
E2
M
0
62
2.76694
2.93268
0.65439
0.27893
16
E2
U
0
2
2.75500
2.97600
0.81317
0.28567
17
ETOH
F
0
80
3.26163
2.99979
0.95336
0.31710
18
ETOH
M
0
63
3.26365
3.01614
0.92449
0.31516
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
28
of
48
ANOVA
FOR
WEIGHT
OF
FROGS
ACROSS
TREATMENT
BY
SEX
129
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
Treat
5
0
0.1
1
10
25
Number
of
observations
40
Dependent
Variable:
WEIGHT
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
4
3.66900699
0.91725175
2.22
0.0863
Error
35
14.43121687
0.41232048
Corrected
Total
39
18.10022385
R­
Square
Coeff
Var
Root
MSE
WEIGHT
Mean
0.202705
19.12619
0.642122
3.357291
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
Treat
4
3.66900699
0.91725175
2.22
0.0863
Levene's
Test
for
Homogeneity
of
WEIGHT
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
Treat
4
1.7887
0.4472
1.57
0.2033
Error
35
9.9528
0.2844
Bartlett's
Test
for
Homogeneity
of
WEIGHT
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
Treat
4
6.1708
0.1868
Dunnett's
t
Tests
for
WEIGHT
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
35
Error
Mean
Square
0.41232
Critical
Value
of
Dunnett's
t
2.55790
Minimum
Significant
Difference
0.8212
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Simultaneous
Treat
Between
95%
Confidence
Comparison
Means
Limits
25
­
0
0.5080
­
0.3133
1.3292
0.1
­
0
0.4326
­
0.3886
1.2539
10
­
0
0.0530
­
0.7682
0.8743
1
­
0
­
0.3188
­
1.1401
0.5024
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
29
of
48
ANOVA
FOR
WEIGHT
OF
FROGS
ACROSS
TREATMENT
BY
SEX
133
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
Treat
5
0
0.1
1
10
25
Number
of
observations
40
Dependent
Variable:
WEIGHT
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
4
1.48024068
0.37006017
1.07
0.3857
Error
35
12.09767780
0.34564794
Corrected
Total
39
13.57791848
R­
Square
Coeff
Var
Root
MSE
WEIGHT
Mean
0.109018
18.29415
0.587918
3.213696
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
Treat
4
1.48024068
0.37006017
1.07
0.3857
Levene's
Test
for
Homogeneity
of
WEIGHT
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
Treat
4
0.5338
0.1334
0.35
0.8426
Error
35
13.3648
0.3819
Bartlett's
Test
for
Homogeneity
of
WEIGHT
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
Treat
4
2.9535
0.5656
Dunnett's
t
Tests
for
WEIGHT
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
35
Error
Mean
Square
0.345648
Critical
Value
of
Dunnett's
t
2.55790
Minimum
Significant
Difference
0.7519
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Simultaneous
Treat
Between
95%
Confidence
Comparison
Means
Limits
0.1
­
0
0.3171
­
0.4348
1.0691
25
­
0
0.1513
­
0.6006
0.9033
10
­
0
­
0.0259
­
0.7779
0.7260
1
­
0
­
0.2599
­
1.0118
0.4920
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
30
of
48
NONPARAMETRIC
COMPARISON
OF
FROG
WEIGHT
ACROSS
TREATMENTS
BY
SEX
137
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
WEIGHT
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0
8
157.0
164.0
29.574764
19.6250
0.1
8
196.0
164.0
29.574764
24.5000
1
8
98.0
164.0
29.574764
12.2500
10
8
162.0
164.0
29.574764
20.2500
25
8
207.0
164.0
29.574764
25.8750
Kruskal­
Wallis
Test
Chi­
Square
6.6604
DF
4
Pr
>
Chi­
Square
0.1550
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
WEIGHT
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0
8
4.0
4.0
1.281025
0.5000
0.1
8
5.0
4.0
1.281025
0.6250
1
8
2.0
4.0
1.281025
0.2500
10
8
4.0
4.0
1.281025
0.5000
25
8
5.0
4.0
1.281025
0.6250
Median
One­
Way
Analysis
Chi­
Square
2.9250
DF
4
Pr
>
Chi­
Square
0.5705
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
31
of
48
NONPARAMETRIC
COMPARISON
OF
FROG
WEIGHT
ACROSS
TREATMENTS
BY
SEX
139
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
WEIGHT
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0
8
145.0
164.0
29.574764
18.1250
0.1
8
219.0
164.0
29.574764
27.3750
1
8
110.0
164.0
29.574764
13.7500
10
8
170.0
164.0
29.574764
21.2500
25
8
176.0
164.0
29.574764
22.0000
Kruskal­
Wallis
Test
Chi­
Square
5.9287
DF
4
Pr
>
Chi­
Square
0.2045
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
WEIGHT
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0
8
2.0
4.0
1.281025
0.2500
0.1
8
6.0
4.0
1.281025
0.7500
1
8
3.0
4.0
1.281025
0.3750
10
8
5.0
4.0
1.281025
0.6250
25
8
4.0
4.0
1.281025
0.5000
Median
One­
Way
Analysis
Chi­
Square
4.8750
DF
4
Pr
>
Chi­
Square
0.3004
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
32
of
48
ANOVA
FOR
LENGTH
OF
FROGS
ACROSS
TREATMENT
BY
SEX
141
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
Treat
5
0
0.1
1
10
25
Number
of
observations
40
Dependent
Variable:
LENGTH
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
4
0.46918445
0.11729611
4.21
0.0069
Error
35
0.97530236
0.02786578
Corrected
Total
39
1.44448681
R­
Square
Coeff
Var
Root
MSE
LENGTH
Mean
0.324810
5.507625
0.166930
3.030898
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
Treat
4
0.46918445
0.11729611
4.21
0.0069
Levene's
Test
for
Homogeneity
of
LENGTH
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
Treat
4
0.00398
0.000996
1.29
0.2939
Error
35
0.0271
0.000774
Bartlett's
Test
for
Homogeneity
of
LENGTH
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
Treat
4
2.7074
0.6079
Dunnett's
t
Tests
for
LENGTH
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.
Alpha
0.05
Error
Degrees
of
Freedom
35
Error
Mean
Square
0.027866
Critical
Value
of
Dunnett's
t
2.55790
Minimum
Significant
Difference
0.2135
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Treat
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
0.1
­
0
0.14893
­
0.06457
0.36242
25
­
0
0.13545
­
0.07804
0.34895
10
­
0
­
0.04026
­
0.25375
0.17324
1
­
0
­
0.13709
­
0.35058
0.07641
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
33
of
48
ANOVA
FOR
LENGTH
OF
FROGS
ACROSS
TREATMENT
BY
SEX
145
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
Treat
5
0
0.1
1
10
25
Number
of
observations
40
Dependent
Variable:
LENGTH
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
4
0.15470697
0.03867674
1.38
0.2623
Error
35
0.98388783
0.02811108
Corrected
Total
39
1.13859480
R­
Square
Coeff
Var
Root
MSE
LENGTH
Mean
0.135875
5.628738
0.167664
2.978707
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
Treat
4
0.15470697
0.03867674
1.38
0.2623
Levene's
Test
for
Homogeneity
of
LENGTH
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
Treat
4
0.00309
0.000772
0.36
0.8370
Error
35
0.0756
0.00216
Bartlett's
Test
for
Homogeneity
of
LENGTH
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
Treat
4
2.3528
0.6712
Dunnett's
t
Tests
for
LENGTH
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
35
Error
Mean
Square
0.028111
Critical
Value
of
Dunnett's
t
2.55790
Minimum
Significant
Difference
0.2144
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.
Difference
Treat
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
0.1
­
0
0.07055
­
0.14388
0.28498
25
­
0
0.03457
­
0.17986
0.24901
10
­
0
­
0.03291
­
0.24735
0.18152
1
­
0
­
0.11130
­
0.32573
0.10313
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
34
of
48
NONPARAMETRIC
COMPARISON
OF
FROG
LENGTHS
ACROSS
TREATMENTS
BY
SEX
149
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
LENGTH
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0
8
159.0
164.0
29.574764
19.8750
0.1
8
224.0
164.0
29.574764
28.0000
1
8
89.0
164.0
29.574764
11.1250
10
8
133.0
164.0
29.574764
16.6250
25
8
215.0
164.0
29.574764
26.8750
Kruskal­
Wallis
Test
Chi­
Square
11.7183
DF
4
Pr
>
Chi­
Square
0.0196
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
LENGTH
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0
8
3.0
4.0
1.281025
0.3750
0.1
8
6.0
4.0
1.281025
0.7500
1
8
2.0
4.0
1.281025
0.2500
10
8
3.0
4.0
1.281025
0.3750
25
8
6.0
4.0
1.281025
0.7500
Median
One­
Way
Analysis
Chi­
Square
6.8250
DF
4
Pr
>
Chi­
Square
0.1454
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
35
of
48
NONPARAMETRIC
COMPARISON
OF
FROG
LENGTHS
ACROSS
TREATMENTS
BY
SEX
151
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
LENGTH
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0
8
154.0
164.0
29.574764
19.2500
0.1
8
217.0
164.0
29.574764
27.1250
1
8
106.0
164.0
29.574764
13.2500
10
8
162.0
164.0
29.574764
20.2500
25
8
181.0
164.0
29.574764
22.6250
Kruskal­
Wallis
Test
Chi­
Square
6.0055
DF
4
Pr
>
Chi­
Square
0.1987
The
NPAR1WAY
Procedure
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
LENGTH
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0
8
3.0
4.0
1.281025
0.3750
0.1
8
6.0
4.0
1.281025
0.7500
1
8
3.0
4.0
1.281025
0.3750
10
8
4.0
4.0
1.281025
0.5000
25
8
4.0
4.0
1.281025
0.5000
Median
One­
Way
Analysis
Chi­
Square
2.9250
DF
4
Pr
>
Chi­
Square
0.5705
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
36
of
48
ANOVA
FOR
WEIGHT
OF
FROGS
ACROSS
POSITIVE
CONTROLS
BY
SEX
153
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
Treat
3
DHT
E2
ETOH
Number
of
observations
24
Dependent
Variable:
WEIGHT
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
2
3.17425911
1.58712955
0.86
0.4380
Error
21
38.80382271
1.84780108
Corrected
Total
23
41.97808182
R­
Square
Coeff
Var
Root
MSE
WEIGHT
Mean
0.075617
39.27037
1.359338
3.461487
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
Treat
2
3.17425911
1.58712955
0.86
0.4380
Levene's
Test
for
Homogeneity
of
WEIGHT
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
Treat
2
111.3
55.6423
1.88
0.1779
Error
21
622.7
29.6509
Bartlett's
Test
for
Homogeneity
of
WEIGHT
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
Treat
2
32.4133
<.
0001
Dunnett's
t
Tests
for
WEIGHT
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
21
Error
Mean
Square
1.847801
Critical
Value
of
Dunnett's
t
2.37033
Minimum
Significant
Difference
1.611
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Simultaneous
Treat
Between
95%
Confidence
Comparison
Means
Limits
ETOH
­
DHT
­
0.5422
­
2.1532
1.0688
E2
­
DHT
­
0.8832
­
2.4943
0.7278
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
37
of
48
ANOVA
FOR
WEIGHT
OF
FROGS
ACROSS
POSITIVE
CONTROLS
BY
SEX
157
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
Treat
3
DHT
E2
ETOH
Number
of
observations
24
Dependent
Variable:
WEIGHT
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
2
4.32184582
2.16092291
1.94
0.1680
Error
21
23.33843834
1.11135421
Corrected
Total
23
27.66028416
R­
Square
Coeff
Var
Root
MSE
WEIGHT
Mean
0.156247
32.05405
1.054208
3.288845
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
Treat
2
4.32184582
2.16092291
1.94
0.1680
Levene's
Test
for
Homogeneity
of
WEIGHT
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
Treat
2
32.0265
16.0133
4.05
0.0327
Error
21
83.1297
3.9586
Bartlett's
Test
for
Homogeneity
of
WEIGHT
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
Treat
2
20.9573
<.
0001
Dunnett's
t
Tests
for
WEIGHT
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
21
Error
Mean
Square
1.111354
Critical
Value
of
Dunnett's
t
2.37033
Minimum
Significant
Difference
1.2494
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Treat
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
ETOH
­
DHT
­
0.6254
­
1.8748
0.6240
E2
­
DHT
­
1.0317
­
2.2811
0.2177
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
38
of
48
NONPARAMETRIC
COMPARISON
OF
FROG
WEIGHT
ACROSS
POSITIVE
CONTROLS
161
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
WEIGHT
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
DHT
8
91.0
100.0
16.329932
11.3750
E2
8
85.0
100.0
16.329932
10.6250
ETOH
8
124.0
100.0
16.329932
15.5000
Kruskal­
Wallis
Test
Chi­
Square
2.2050
DF
2
Pr
>
Chi­
Square
0.3320
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
WEIGHT
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
DHT
8
4.0
4.0
1.179536
0.5000
E2
8
3.0
4.0
1.179536
0.3750
ETOH
8
5.0
4.0
1.179536
0.6250
Median
One­
Way
Analysis
Chi­
Square
0.9583
DF
2
Pr
>
Chi­
Square
0.6193
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
39
of
48
NONPARAMETRIC
COMPARISON
OF
FROG
WEIGHT
ACROSS
POSITIVE
CONTROLS
163
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
WEIGHT
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
DHT
8
115.0
100.0
16.329932
14.3750
E2
8
69.0
100.0
16.329932
8.6250
ETOH
8
116.0
100.0
16.329932
14.5000
Kruskal­
Wallis
Test
Chi­
Square
3.6050
DF
2
Pr
>
Chi­
Square
0.1649
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
WEIGHT
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
DHT
8
5.0
4.0
1.179536
0.6250
E2
8
2.0
4.0
1.179536
0.2500
ETOH
8
5.0
4.0
1.179536
0.6250
Median
One­
Way
Analysis
Chi­
Square
2.8750
DF
2
Pr
>
Chi­
Square
0.2375
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
40
of
48
ANOVA
FOR
LENGTH
OF
FROGS
ACROSS
POSITIVE
CONTROLS
BY
SEX
165
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
Treat
3
DHT
E2
ETOH
Number
of
observations
24
Dependent
Variable:
LENGTH
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
2
0.02901222
0.01450611
0.16
0.8568
Error
21
1.95623777
0.09315418
Corrected
Total
23
1.98524999
R­
Square
Coeff
Var
Root
MSE
LENGTH
Mean
0.014614
9.974886
0.305212
3.059801
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
Treat
2
0.02901222
0.01450611
0.16
0.8568
Levene's
Test
for
Homogeneity
of
LENGTH
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
Treat
2
0.2482
0.1241
2.06
0.1530
Error
21
1.2675
0.0604
Bartlett's
Test
for
Homogeneity
of
LENGTH
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
Treat
2
21.9346
<.
0001
Dunnett's
t
Tests
for
LENGTH
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
21
Error
Mean
Square
0.093154
Critical
Value
of
Dunnett's
t
2.37033
Minimum
Significant
Difference
0.3617
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Treat
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
ETOH
­
DHT
­
0.06883
­
0.43056
0.29290
E2
­
DHT
­
0.07785
­
0.43958
0.28388
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
41
of
48
ANOVA
FOR
LENGTH
OF
FROGS
ACROSS
POSITIVE
CONTROLS
BY
SEX
169
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
ANOVA
Procedure
Class
Level
Information
Class
Levels
Values
Treat
3
DHT
E2
ETOH
Number
of
observations
24
Dependent
Variable:
LENGTH
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
2
0.15041338
0.07520669
0.91
0.4167
Error
21
1.72987563
0.08237503
Corrected
Total
23
1.88028900
R­
Square
Coeff
Var
Root
MSE
LENGTH
Mean
0.079995
9.484117
0.287011
3.026223
Source
DF
Anova
SS
Mean
Square
F
Value
Pr
>
F
Treat
2
0.15041338
0.07520669
0.91
0.4167
Levene's
Test
for
Homogeneity
of
LENGTH
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
Treat
2
0.1367
0.0684
3.85
0.0375
Error
21
0.3725
0.0177
Bartlett's
Test
for
Homogeneity
of
LENGTH
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
Treat
2
13.2247
0.0013
Dunnett's
t
Tests
for
LENGTH
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
21
Error
Mean
Square
0.082375
Critical
Value
of
Dunnett's
t
2.37033
Minimum
Significant
Difference
0.3402
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Simultaneous
Treat
Between
95%
Confidence
Comparison
Means
Limits
ETOH
­
DHT
­
0.1378
­
0.4780
0.2023
E2
­
DHT
­
0.1871
­
0.5272
0.1531
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
42
of
48
NONPARAMETRIC
COMPARISON
OF
FROG
LENGTHS
ACROSS
POSITIVE
CONTROLS
BY
SEX
173
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
LENGTH
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
DHT
8
78.0
100.0
16.329932
9.750
E2
8
110.0
100.0
16.329932
13.750
ETOH
8
112.0
100.0
16.329932
14.000
Kruskal­
Wallis
Test
Chi­
Square
1.8200
DF
2
Pr
>
Chi­
Square
0.4025
The
NPAR1WAY
Procedure
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
LENGTH
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
DHT
8
2.0
4.0
1.179536
0.2500
E2
8
5.0
4.0
1.179536
0.6250
ETOH
8
5.0
4.0
1.179536
0.6250
Median
One­
Way
Analysis
Chi­
Square
2.8750
DF
2
Pr
>
Chi­
Square
0.2375
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
43
of
48
NONPARAMETRIC
COMPARISON
OF
FROG
LENGTHS
ACROSS
POSITIVE
CONTROLS
BY
SEX
175
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
LENGTH
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
DHT
8
103.0
100.0
16.329932
12.8750
E2
8
92.0
100.0
16.329932
11.5000
ETOH
8
105.0
100.0
16.329932
13.1250
Kruskal­
Wallis
Test
Chi­
Square
0.2450
DF
2
Pr
>
Chi­
Square
0.8847
The
NPAR1WAY
Procedure
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
LENGTH
Classified
by
Variable
Treat
Sum
of
Expected
Std
Dev
Mean
Treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
DHT
8
4.0
4.0
1.179536
0.50
E2
8
4.0
4.0
1.179536
0.50
ETOH
8
4.0
4.0
1.179536
0.50
Median
One­
Way
Analysis
Chi­
Square
0.0000
DF
2
Pr
>
Chi­
Square
1.0000
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
44
of
48
AVERAGE
LENGTH
OF
FROGS
OVER
TREATMENTS
BY
SEX
177
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

Plot
of
LENGTH*
CONC.
Symbol
used
is
'*'.

LENGTH
é
é
3.15
ê
é
é
é
*
é
é
*
3.10
ê
é
é
é
é
é
3.05
ê
é
é
é
é
é
*
3.00
ê
é
é
é
é
é
*
2.95
ê
é
é
é
é
é
2.90
ê
é
é
é
é
é
*
2.85
ê
é
èââêâââââââââââââêâââââââââââââêâââââââââââââêâââââââââââââêâââââââââââââêââ
0
5
10
15
20
25
CONC
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
45
of
48
AVERAGE
LENGTH
OF
FROGS
OVER
TREATMENTS
BY
SEX
178
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

Plot
of
LENGTH*
CONC.
Symbol
used
is
'*'.

LENGTH
é
3.050
ê
é
é
é
*
é
3.025
ê
é
é
é
é
3.000
ê
é
é
é
é
*
2.975
ê
é
é
é
é
2.950
ê
*
é
*
é
é
é
2.925
ê
é
é
é
é
2.900
ê
é
é
é
é
2.875
ê
é
é
é
*
é
2.850
ê
èââêâââââââââââââêâââââââââââââêâââââââââââââêâââââââââââââêâââââââââââââêââ
0
5
10
15
20
25
CONC
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
46
of
48
AVERAGE
WEIGHT
OF
FROGS
OVER
TREATMENTS
BY
SEX
179
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

Plot
of
WEIGHT*
CONC.
Symbol
used
is
'*'.

WEIGHT
é
é
3.7
ê
é
é
é
*
3.6
ê
é
é
é
3.5
ê
é
é
é
3.4
ê
*
é
é
é
3.3
ê
é
é
*
é
*
3.2
ê
é
é
é
3.1
ê
é
é
é
3.0
ê
é
é
é
2.9
ê
é
é
*
é
2.8
ê
é
èââêâââââââââââââêâââââââââââââêâââââââââââââêâââââââââââââêâââââââââââââêââ
0
5
10
15
20
25
CONC
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
47
of
48
AVERAGE
WEIGHT
OF
FROGS
OVER
TREATMENTS
BY
SEX
180
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

Plot
of
WEIGHT*
CONC.
Symbol
used
is
'*'.

WEIGHT
é
é
3.4
ê
*
é
é
é
é
é
3.3
ê
é
é
é
é
é
3.2
ê
é
é
*
é
é
é
*
3.1
ê
é
é
é
*
é
é
3.0
ê
é
é
é
é
é
2.9
ê
é
é
*
é
é
é
2.8
ê
é
èââêâââââââââââââêâââââââââââââêâââââââââââââêâââââââââââââêâââââââââââââêââ
0
5
10
15
20
25
CONC
Data
Evaluation
Report
on
Response
of
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
the
Mechanism
of
Action
of
Atrazine.
EPA
MRID
Number
458677­
04
Page
48
of
48
