Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Data
Requirement:
:
EPA
DP
Barcode
D288775
EPA
MRID
458677­
07
EPA
Guideline
70­
1(
Special
Study)

Test
material:
Purity:
98.6%
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:
April
6,
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:
Goleman,
W.
L.
and
J.
A.
Carr.
2003.
Response
of
larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology.
The
Institute
of
Environmental
and
Human
Health,
Texas
Tech
University,
Texas
Tech
University
Health
Sciences
Center,
Lubbock,
Texas.
Sponsor:
Syngenta
Crop
Protection,
Inc.,
Laboratory
Study
ID
ECORISK
Number
TTU­
01.
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
2
of
41
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
3
of
41
EXECUTIVE
SUMMARY:

In
a
78­
day
exposure,
48­
to
72­
hr
post­
hatch
African
clawed
frog
(
Xenopus
laevis)
larvae
were
exposed
to
nominal
concentrations
of
1,
10
and
25

g
atrazine/
L
in
FETAX
medium,
FETAX
medium
alone
(
negative
control),
17­
 
estradiol,
dihydrotestosterone,
or
solvent
control
(
0.0025%
ethanol)
in
a
static
renewal
system
where
50%
exposure
solution
water
changes
occurred
every
72
hours.
For
the
first
seven
days,
60
­
65
larvae
were
maintained
in
100
mL
of
exposure
solution.
On
day
14,
animals
were
transferred
to
1
L
of
exposure
solution,
and
by
Day
21,
animals
were
maintained
in
4
L
of
exposure
solution.
At
NF
Stage
66
(
forelimb
emergence
and
complete
tail
resorption)
animals
were
weighed,
measured
for
snout­
vent
length
and
examined
for
gonadal
gross
morphology.
Larynx
and
gonads
also
underwent
histological
analysis.

Mortality
over
the
study
period
ranged
from
10
to
14%
for
those
animals
that
reached
stage
66
by
80
days
post­
hatch.
Time
to
complete
metamorphosis
was
not
significantly
different
across
treatments
although
the
specific
time
was
not
reported.
In
all
treatments,
weight
and
snout­
vent
length
were
inversely
proportional
to
the
number
of
days
required
to
complete
metamorphosis,
i.
e.,
animals
completing
metamorphosis
early
tended
to
be
larger
than
animals
that
took
longer
to
complete
metamorphosis.
Sex
ratios
ranged
from
48%
to
50%
male
across
all
treatments
except
for
estradiol,
which
skewed
the
ratio
in
favor
of
females
(
67%).
While
the
incidence
of
intersex
was
correlated
with
atrazine
treatment
concentrations,
only
the
25

g
atrazine/
L
(
4.7%)
and
estradiol­
treated
(
7.4%)
males
had
incidence
rates
significantly
different
from
negative
(
0.6%)
and
solvent
(
0.0%)
controls.
Intersex
in
males
treated
with
25

g
atrazine/
L
contained
distinguishable
testicular
and
ovarian
tissue,
while
males
treated
with
estradiol
sometimes
contained
ambiguous
tissue
structures.

There
was
no
difference
in
the
cross­
sectional
area
of
larynx
dilator
muscle
in
any
atrazine­
treated
males
relative
to
negative
controls.
Dihydrotestosterone­
treated
females
had
significantly
larger
cross­
sectional
dilator
muscle
areas
than
solvent
control
females.

Although
the
report
concluded
that
atrazine
did
not
impact
length,
weight,
time
to
metamorphosis
or
dilator
muscle
area
relative
to
controls,
it
did
conclude
that
exposure
to
25

g
atrazine/
L
appeared
to
significantly
increase
the
number
of
intersex
males
and
animals
with
discontinuous
gonads.
However,
the
observation
that
both
body
weight
and
length
were
inversely
correlated
with
the
length
of
time
to
complete
metamorphosis
suggested
that
animals
in
all
treatment
groups
were
developmentally
impaired.
Also,
the
fact
that
17­
 
estradiol
treatment
only
resulted
in
67%
females,
further
suggested
that
study
animals
were
not
entirely
responsive
to
the
positive
control.
Although
dissolved
oxygen
did
not
appear
to
drop
below
3.9
mg/
L,
ammonia
levels
ranged
as
high
as
27
mg/
L
suggesting
that
the
50%
static
renewal
and
loading
rates
(
number
of
tadpoles
per
liter
of
exposure
solution)
may
have
resulted
in
poor
water
quality
that
could,
in
turn,
impair
the
development
of
test
animals.
Furthermore,
roughly
42%
of
the
animals
were
assumed
not
to
have
reached
stage
66
by
Day
78,
suggesting
that
a
large
proportion
of
the
animals
were
not
developing
at
all.
Because
of
the
declining
condition
of
the
frogs
with
increased
length
of
time
to
maturity,
it
is
uncertain
whether
these
animals
completed
metamorphosis
and/
or
survived.
Furthermore,
because
all
the
animals
in
the
study
had
not
undergone
metamorphosis,
the
percent
initiating
metamorphosis,
time
to
metamorphosis
and
percentage
of
gonadal
abnormalities
could
not
be
accurately
calculated
relative
to
the
total
animals
used
in
the
study.

This
study
indicates
that
only
one
exposure
level
(
25
ug/
L)
produced
developmental
effects
in
frogs,
but
because
of
design
problems
the
study
did
not
establish
a
dose­
response
relationship
between
atrazine
exposure
and
developmental
effects
in
frogs.
The
ability
of
this
study
to
serve
as
a
sensitive
indicator
of
developmental
effects
is
also
in
question
because
of
the
impaired
condition
of
the
animals.
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
4
of
41
I.
MATERIALS
AND
METHODS
GUIDELINE
FOLLOWED:
Nonguideline
Study
COMPLIANCE:
Not
conducted
under
full
Good
Laboratory
Practices;
however,
most
of
the
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.
°
Test
material
identity,
strength,
purity
and
composition
were
characterized.
°
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:
98.6%
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:
48
­
72
hr
larvae
Weight
at
study
initiation:
(
mean
and
range)
Not
reported
Length
at
study
initiation:
(
mean
and
range)
Not
reported
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
5
of
41
Source:
Six
breeding
pairs
of
X.
laevis
obtained
from
Xenopus
Express
(
Homosassa,
FL)
artificially
induced
to
spawn.

B.
STUDY
DESIGN:

Objective:
1.
To
determine
the
response
of
larval
Xenopus
laevis
to
atrazine
by
assessing
the
metamorphosis
and
reproductive
indices
when
animals
are
exposed
from
48
­
72
hours
after
hatching
until
the
completion
of
metamorphosis.
Indices
to
be
evaluated
include
percent
initiating
metamorphosis,
percent
completing
metamorphosis,
time
to
metamorphosis,
percentage
of
intersex
gonads,
fresh
post­
mortem
body
weight,
snout­
vent
length,
and
laryngeal
size.

1.
Experimental
Conditions
a)
Range­
finding
Study:
Trials
1
­
3
were
conducted
in
0.1x
Holtfreter's
solution
and
were
terminated
due
to
unexpectedly
high
mortality
in
all
concentrations.
In
Trial
1,
larvae
were
transferred
48
hr
post­
hatch
and
all
the
transferred
animals
died
within
24
hours
of
transfer
from
"
damaged
abdomens".
In
Trials
2
and
3,
exposures
began
when
larvae
were
less
than
24
hrs
old
resulting
in
"
unacceptably
high
mortality"
and
poor
growth
leaving
study
authors
to
conclude
that
0.1x
Holtfretter's
was
unsuitable
as
a
medium
for
raising
larvae.
FETAX
medium
was
used
in
the
fourth
and
final
attempt
at
a
definitive
study.

b)
Definitive
Study
Table
1
.
Experimental
Parameters
Parameter
Details
Acclimation:
period:
Conditions:
(
same
as
test
or
not)
Feeding:
Health:
(
any
mortality
observed)
breeding
adults
maintained
separately
for
7
days
before
breeding;
maintained
in
45­
L
glass
tanks
containing
18­
L
of
ultra­
pure
water
at
22
±
1oC.
Beginning
4
days
prior
to
breeding,
adult
tank
water
changed
to
FETAX
by
daily
50%
medium
changes.

Duration
of
the
test
78
days
or
until
frogs
reached
NF
stage
66
(
forelimb
emergence
and
complete
tail
resorption)
of
development
Test
condition
static/
flow­
through
Type
of
dilution
system
for
flowthrough
method.

Renewal
rate
for
static
renewal
static
renewal
NA
50%
exposure
solution
change
every
72
hours
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Parameter
Details
Page
6
of
41
Aeration,
if
any
not
reported
Test
vessel
Material:
(
glass/
stainless
steel)
Size:

Fill
volume:
Glass
beaker
Initially
250
mL;
on
Day
7
transferred
to
2­
L
beakers;
on
Day
21
transferred
to
10­
L
beakers
Initially
100
mL;
on
Day
7
filled
with
1­
L;
on
Day
21
filled
with
4
L
Source
of
dilution
water
Quality:
tap
water
run
through
reverse
osmosis
and
deionizer
to
convert
to
ultra­
pure
water
Water
parameters:
Hardness
pH
Dissolved
oxygen
Total
Organic
carbon
Particulate
Matter
Ammonia
Nitrite
Metals
Pesticides
Chlorine
Temperature
Salinity
Intervals
of
water
quality
measurement
not
reported
range:
6.9
to
8.3
range:
3.9
to
9.3
mg/
L;
mean
6.7
mg/
L
not
reported
not
reported
range:
0
to
27.1
mg/
L
(
unionized
0
­
0.24
mg/
L);
mean
9.4
mg/
L
not
reported
not
reported
not
reported
not
reported
range:
19
to
22.8oC
range:
0.6
to
1.2
ppt
temperature
monitored
daily;
pH,
conductivity,
salinity,
DO
and
ammonia
monitored
on
freshly
prepared
stock
solutions
and
72­
hr
exposure
solutions
(
every
72
hours).

Number
of
replicates/
groups:
negative
control:
water
treated
ones:
11
replicates
for
atrazine
and
negative
control;
6
replicates
for
ethanol,
dihydrotestosterone
and
17­
 
estradiol
(
0.1

g/
mL)
positive
controls.
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Parameter
Details
Page
7
of
41
Number
of
organisms
per
replicate
/
groups:
60
­
65
larvae
per
replicate
Biomass
loading
rate
Initially,
650/
L,
at
Day
7
65/
L
and
at
Day
21
onward
16.3/
L
Test
concentrations:
nominal:
measured:
0,
1,
10
and
25

g/
L
1.07
±
0.02

g/
L,
10.3
±
0.15

g/
L,
19.5
±
0.21

g/
L
Solvent
(
type,
percentage,
if
used)
FETAX
solution
for
atrazine
treated
and
negative
control
steroids
and
solvent
control
in
0.0025%
ethanol/
FETAX.

FETAX
medium:
NaCl
0.625
g/
L;
NaHCO3
0.096
g/
L;
KCl
0.03
g/
L;
CaCl2
0.015
g/
L,
CaSO4­
2H2O
0.06
g/
L;
MgSO4
0.075
g/
L
Lighting
12
hrs
light,
12
hrs
dark
Feeding
not
reported
Recovery
of
chemical
Level
of
Quantitation
Level
of
Detection
Measured
using
ELISA
(
RAPID
Strategic
Diagnostics,
Newark,
DE);
approximately
10%
of
samples
verified
by
second
analysis.

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

g/
L
in
0.0025%
ethanol)
17­
 
estradiol
(
0.1

g/
L
in
0.0025%
ethanol)

Other
parameters,
if
any
2.
Observations:

Table
2:
Observations
Criteria
Details
Parameters
measured
including
the
sublethal
effects/
toxicity
symptoms
water
quality
(
excess
food,
etc.);
%
mortality,
%
showing
forelimb
emergence,
%
metamorphosed
(
complete
tail
resorption)
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
8
of
41
Observation
intervals
monitored
daily
for
changes
in
general
health
and
for
parameters
described
above.

Were
raw
data
included?

Other
observations,
if
any
II.
RESULTS
and
DISCUSSION:

Atrazine
concentrations
in
both
the
freshly
prepared
stock
solutions
and
the
exposure
tanks
were
relatively
consistent
with
nominal
values
(
Table
3).

Table
3.
Nominal
and
mean­
measured
atrazine
concentrations
in
freshly
prepared
stock
solutions
and
exposure
tanks.

Nominal
Stock
Solution
Exposure
Tank
1

g/
L
1.02
±
0.03

g/
L
1.07
±
0.02

g/
L
10

g/
L
9.99
±
0.29

g/
L
10.3
±
0.15

g/
L
25

g/
L
21.3
±
0.67

g/
L
19.5
±
0.21

g/
L
Estradiol
concentrations
in
exposure
tanks
A
through
F
ranged
from
5,520
pg/
mL
to
11,700
pg/
mL
(
mean:
9,255
pg/
mL)
on
August
21.
On
October
5,
2001,
estradiol
levels
ranged
from
4,764
to
17,467
pg/
mL
(
mean:
9,897
pg/
mL).
Therefore,
average
estradiol
concentrations
were
0.009

g/
L
and
were
roughly
9%
of
the
desired
nominal
estradiol
concentration
of
0.1%.

Post­
hatch
mortality
across
all
study
groups
ranged
from
10
to
14%
(
Table
4).
There
was
no
difference
in
post­
hatch
mortality
for
atrazine
treatments
and
the
negative
control
(
ANOVA,
p
=
0.2)
or
for
hormone
treatments
and
the
ethanol
control
(
ANOVA,
p
=
0.7).
There
was
no
difference
in
body
weight
or
snout­
vent
length
among
atrazine­
treated
and
negative
controls.
Time
to
complete
metamorphosis
(
NF
stage
66)
varied
inversely
with
body
weight
and
SVL;
those
animals
reaching
NF­
stage
66
first
in
each
tank
were
significantly
larger
than
those
animals
that
were
the
last,
i.
e.,
animals
reaching
NF­
stage
66
by
Day
78,
in
every
treatment
(
paired
t­
test,
p<
0.05).
Based
on
the
report
figure
depicting
body
weights,
the
average
weight
of
animals
reaching
stage
66
first
ranged
from
0.48
to
0.62
g,
while
the
last
animals
to
reach
stage
66
had
body
weights
ranging
from
0.26
to
0.30
g.
Average
snout­
vent
length
for
animals
reaching
stage
66
first
ranged
from
16
to
17
mm
while
lengths
for
animals
reaching
stage
66
last
ranged
from
13
to
13.5
mm
roughly.
Body
weight
and
SVL
were
also
inversely
correlated
with
time
to
complete
metamorphosis
in
hormone­
treated
and
solvent
controls.
Body
weights
for
animals
reaching
stage
66
first
in
hormone
and
solvent­
treated
animals
ranged
from
0.48
to
0.54
g,
while
late
stage
66
animals
ranged
from
0.24
to
0.25
g.
Estradiol­
treated
females
had
significantly
longer
SVL
than
solvent
controls
(
ANOVA,
p
=
0.02).

The
percentage
of
animals
reaching
complete
tail
resorption
varied
between
37
­
52%
during
the
78­
day
period.
Fewer
atrazine­
treated
(
referred
to
as
a
"
weak
trend"
by
authors)
animals
reached
(
ANOVA
trend
test,
p
=
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
9
of
41
0.03)
and
completed
tail
resorption
(
ANOVA
trend
test,
p
=
0.04).
However,
the
general
ANOVA
F­
test
revealed
no
significant
effect
of
atrazine
on
either
parameter
compared
to
negative
controls.

The
incidence
of
gross
gonadal
deformities
was
less
than
5%
in
all
treatments.
The
incidence
of
edema
was
correlated
with
atrazine
concentration
(
Cochran­
Armitage
trend
test,
Z
=
­
2.3,
p=
0.02);
however,
a
chi­
square
homogeneity
test
revealed
that
none
of
the
atrazine
concentrations
significantly
increased
the
incidence
of
edema
compared
to
negative
controls
(
p
=
0.1).
Abnormal
swimming
was
also
correlated
with
atrazine
concentration
(
Cochran­
Armitage
trend
test,
Z
=
­
2.90,
p
=
0.004),
but
only
larvae
exposed
to
25

g
atrazine/
L
exhibited
significantly
greater
incidence
of
abnormal
swimming
(
chi­
square
homogeneity
test,
p
=
0.04).

There
were
no
detectable
effects
on
sex
ratio
with
ratios
ranging
from
48
to
50%
males
in
all
treatments
except
the
25

g
atrazine/
L
treatment.
There
was
a
slight
reduction
in
percentage
(
45%)
of
males
in
the
25

g
atrazine/
L.
Neither
ethanol
nor
dihyrotestosterone
significantly
affected
sex
ratios.
Estradiol
significantly
reduced
the
percentage
of
males
to
26%
and
increased
the
percentage
of
females
to
67%.
Approximately
7%
of
the
estradiol­
treated
animals
were
classified
as
intersex
based
on
gross
morphology.

Exposure
to
either
25

g
atrazine/
L
or
estradiol
significantly
increased
the
percentage
of
individuals
with
intersex
gonads.
Although
the
incidence
of
intersex
increased
with
increasing
atrazine
concentrations
(
Cochran­
Armitage,
Z=
3.6,
p
=
0.0003),
only
25

g
atrazine/
L
significantly
increased
the
average
incidence
of
intersex
animals
(
4.7%)
per
tank
compared
to
negative
control
(
0.6%)
(
p
=
0.0061)
(
Table
5).
There
was
also
a
significant
correlation
between
the
incidence
of
discontinuous
gonads
and
atrazine
concentration
(
Cochran­
Armitage
Z
=
2.9,
p
=
0.0042)
although
25

g
atrazine/
L
was
the
only
concentration
to
increase
the
incidence
of
discontinuous
gonads
compared
to
negative
controls.
The
percentage
of
intersex
gonads
was
also
significantly
higher
in
the
estradiol­
treated
group
compared
to
the
solvent
control.
(
Kruskall­
Wallis,
p
=
0.01).

Table
5.
Total
number
of
frogs
identified
as
intersex
by
treatment.

Treatment
Total
Animals
Intersex
Percentage
0
334
2
0.6%

1

g
atrazine/
L
309
3
0.97%

10

g
atrazine/
L
276
1
0.36%

25

g
atrazine/
L
296
14
4.7%

estradiol
135
10
7.4%

solvent
control
160
0
0.0%

Intersex
animals
in
the
25

g
atrazine/
L
group
tended
to
have
obvious
testicular
or
ovarian
tissues,
while
estradiol­
treated
animals
revealed
"
ambiguous"
gonadal
tissue
in
some
cases.

There
were
no
significant
differences
in
male
dilator
muscle
size
within
any
of
the
negative
control
or
atrazine
treatments
(
Table
6).
Total
muscle
cross­
sectional
area
was
20
­
25%
larger
in
males
than
females
in
all
atrazine­
treated
groups.
There
were
no
significant
effects
of
atrazine
on
total
cross­
sectional
area
in
females
(
p
=
0.5).
Exposure
to
dihydrotestosterone
increased
total
muscle
cross­
sectional
area
approximately
2­
fold
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
10
of
41
in
both
males
(
p
>
0.0001)
and
females
(
p
<
0.0001).
Total
muscle
cross­
sectional
area
was
larger
in
males
than
in
females
in
the
solvent
control
group
but
not
in
the
estradiol
and
DHT­
treated
groups.

Table
6.
Mean
(
±
standard
error)
of
total
dilator
muscle
cross­
sectional
area
(
mm2)
in
Nieuwkoop
Faber
stage
66
Xenopus
laevis
exposed
to
atrazine
or
sex
steroidsa.

Treatment
Males
Females
negative
FETAX
control
b
0.154
±
0.004
(
n
=
45)
0.117
±
0.006
(
n
=
11)

1

g
atrazine/
L
0.167
±
0.005
(
n
=
41)
0.118
±
0.006
(
n
=
10)

10

g
atrazine/
L
0.168
±
0.004
(
n
=
44)
0.129
±
0.007
(
n
=
11)

25

g
atrazine/
L
0.168
±
0.004
(
n
=
42)
0.122
±
0.006
(
n
=
12)

solvent
control
(
0.0025%
ethanol)
0.161
±
0.008
(
n
=
6)
0.121
±
0.013
(
n
=
6)

dihydrotestosterone
0.301
±
0.012*
(
n
=
6)
0.313
±
0.024*
(
n
=
6)

17­
 
estradiol
0.148
±
0.010
(
n
=
6)
0.141
±
0.009
(
n
=
6)

a
Combined
largest
cross­
sectional
area
through
the
right
and
left
laryngeal
dilator
muscles.
b
FETAX:
frog
embryo
teratogenesis
assay
 
Xenopus.
*
significantly
grater
than
ethanol
control
(
p
<
0.0001)

F.
REVIEWER'S
COMMENTS:

For
several
days,
breeding
adults
were
apparently
maintained
in
ultra­
pure
water
before
transitioning
them
into
FETAX.

Although
samples
were
collected
at
the
beginning,
middle
and
end
of
the
study
for
steroid
analyses,
the
report
also
states
though
that
estradiol
was
measured
using
RIA
kit
(
Diagnostic
Products
Corporation,
Los
Angeles,
CA;
LOD
20
pg/
mL).
DHT
values,
though,
are
reported
as
nominal,
and
it
is
unclear
whether
any
effort
was
made
to
measure
DHT.
Measured
estradiol
concentrations
were
0.009

g/
L
and
were
roughly
9%
of
nominal.
Coefficients
of
variation
for
the
means
ranged
from
46.5
to
68.9%.

Water
samples
were
collected
of
freshly
prepared
stock
solutions
and
of
exposure
solutions
just
prior
to
renewal.
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
11
of
41
Although
the
study
starts
with
60
to
65
larvae
per
replicate
with
11
replicates
in
the
atrazine­
treated
groups,
the
results
are
expressed
in
terms
of
20
­
30
larvae
per
replicate.
If
mortality
ranged
from
10
to
14%,
it
would
leave
roughly
52
larvae
per
replicate,
and
42%
of
the
animals
would
be
unaccounted..
It
is
assumed
that
the
remaining
animals
failed
to
reach
metamorphosis
by
Day
78.

The
feeding
regime
was
not
discussed;
however,
the
report
states
that
frog
brittle
feed
was
analyzed
by
immunoassay
and
"
yielded
inconclusive
results".
Because
immunoassays
are
specific
to
the
antigen
of
interest,
it
is
presumed
that
the
immunoassay
was
for
atrazine
and
that
something,
presumably
atrazine,
gave
variable
results,
i.
e.,
"
yielded
inconclusive
results."
The
report
states
that
since
a
GC/
MS
method
for
analyzing
frog
brittle
has
not
been
developed,
no
data
on
the
food
were
available
(
reported).

Body
weights
of
metamorphs
were
negatively
correlated
with
the
length
of
time
to
metamorphosis;
the
number
of
intersex
animals
was
positively
correlated
with
atrazine
concentration.

Although
not
discussed
in
the
results
section,
the
number
of
discontinuous
gonads
appeared
to
be
positively
correlated
with
atrazine
treatment.
Extrapolating
from
the
figure
depicting
the
percentage
of
discontinuous
gonads
there
appeared
to
be
3.5%
in
controls,
and
4%,
7%
and
8%
in
1,
10
and
25

g
atrazine/
L.
The
number
of
discontinuous
gonads
was
significantly
higher
than
controls
in
the
25

g
atrazine/
L
treatment
(
chi­
square,
p
<
0.05)

The
study
concluded
that
estradiol
treatment
reduced
the
number
of
phenotypic
males
to
25%;
however,
previous
literature
suggested
that
estradiol
treatment
should
result
in
100%
females.

Body
weights
and
time
to
metamorphosis
were
not
reported
in
the
study
nor
was
the
feeding
regime
mentioned.
Body
weights
and
lengths
were
extrapolated
from
figures.

Although
the
report
concluded
that
atrazine
did
not
impact
length,
weight,
time
to
metamorphosis
or
dilator
muscle
area
relative
to
controls,
it
did
conclude
that
exposure
to
25

g
atrazine/
L
appeared
to
significantly
increase
the
number
of
intersex
males
and
animals
with
discontinuous
gonads.
However,
the
observation
that
both
body
weight
and
length
were
inversely
correlated
with
the
length
of
time
to
complete
metamorphosis
suggested
that
animals
in
all
treatment
groups
were
developmentally
impaired.
In
addition,
17­
 
estradiol
treatment
only
resulted
in
67%
females,
which
further
suggested
that
study
animals
were
not
entirely
responsive
to
the
positive
control.
Although
dissolved
oxygen
did
not
appear
to
drop
below
3.9
mg/
L,
ammonia
levels
did
range
as
high
as
27
mg/
L,
suggesting
that
the
50%
static
renewal
and
loading
rates
(
number
of
tadpoles
per
liter
of
exposure
solution)
may
have
resulted
in
poor
water
quality,
which
in
turn,
accounted
for
the
slow
development
of
test
animals.

Roughly
42%
of
the
animals
were
assumed
not
to
have
reached
stage
66
by
Day
78,
suggesting
that
a
large
proportion
of
the
animals
were
not
developing
at
all.
Given
the
declining
condition
of
the
frogs
with
increased
length
of
time
to
maturity,
it
is
uncertain
whether
these
animals
would
have
undergone
metamorphosis
and/
or
survived.
Furthermore,
since
all
the
animals
in
the
study
had
not
undergone
metamorphosis,
the
percent
initiating
metamorphosis,
time
to
metamorphosis
and
percentage
of
gonadal
abnormalities
could
not
be
accurately
calculated
relative
to
the
total
animals
used
in
the
study.

After
80
days,
only
49­
61%
of
the
organisms
reached
forelimb
emergence
(
stage
58)
and
only
37­
52%
completed
metamorphosis
(
stage
66).
According
to
Nieuwkoop
and
Faber,
the
nominal
days
to
stages
58
and
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
12
of
41
66
are
approximately
44
and
58
days,
respectively.
Although
this
can
vary
between
laboratories
and
conditions,
the
developmental
rates
observed
in
this
study
are
exceedingly
slow.
(
At
the
National
Health
and
Environmental
Effects
Research
Laboratory
researchers
typically
observe
nearly
100%
tail
resorption
in
approximately
50­
54
days.)
Furthermore,
there
is
considerable
divergence
in
development
because
at
least
51­
39%
of
the
tested
organisms
are
at
or
below
stage
57,
while
some
have
completed
metamorphosis.

Taken
together,
these
observations
suggest
that
the
organisms
were
in
poor
condition.
It
is
unclear,
though,
how
such
retarded
development
could
affect
sexual
differentiation.

The
report
states
that
there
is
no
evidence
that
androgens
affected
gonadal
development
in
X.
laevis.
While
it
is
apparently
true
that
testosterone
is
incapable
of
causing
masculinization,
DHT,
the
positive
androgen
control
used
in
this
study,
has
been
shown
to
masculinize
X.
laevis.

Because
only
about
50%
of
the
tested
organisms
reached
stage
66,
and
all
of
the
analyses
were
conducted
on
stage
66
organisms,
the
sampling
strategy
was
probably
biased
and
did
not
represent
the
population
in
the
test.

The
percentage
of
tadpoles
with
abnormal
swimming
was
positively
correlated
(
r=
0.41;
p=
0.006)
with
atrazine
concentration
(
see
attached
SAS
©
[
Statistical
Analysis
System,
Release
8.01,
Cary,
North
Carolina]
output);
the
percentage
of
tadpoles
with
edema
was
also
positively
correlated
(
r=
0.40;
p=
0.007)
with
atrazine
concentration.

The
ability
of
this
study
to
serve
as
a
sensitive
indicator
of
developmental
effects
is
in
question
given
the
seemingly
impaired
condition
of
the
animals.

G.
CONCLUSIONS:
Although
the
report
concluded
that
atrazine
did
not
impact
length,
weight,
time
to
metamorphosis
or
dilator
muscle
area
relative
to
controls,
it
did
conclude
that
exposure
to
25

g
atrazine/
L
appeared
to
significantly
increase
the
number
of
intersex
males
and
animals
with
discontinuous
gonads.
However,
the
observation
that
both
body
weight
and
length
were
inversely
correlated
with
the
length
of
time
to
complete
metamorphosis
suggested
that
animals
in
all
treatment
groups
were
developmentally
impaired.
Also,
the
fact
that
17­
 
estradiol
treatment
only
resulted
in
67%
females,
further
suggested
that
study
animals
were
not
entirely
responsive
to
the
positive
control.
Although
dissolved
oxygen
did
not
appear
to
drop
below
3.9
mg/
L,
ammonia
levels
ranged
as
high
as
27
mg/
L
suggesting
that
the
50%
static
renewal
and
loading
rates
(
number
of
tadpoles
per
liter
of
exposure
solution)
may
have
resulted
in
poor
water
quality
that
could,
in
turn,
impair
the
development
of
test
animals.
Furthermore,
roughly
42%
of
the
animals
were
assumed
not
to
have
reached
stage
66
by
Day
78,
suggesting
that
a
large
proportion
of
the
animals
were
not
developing
at
all.
Because
of
the
declining
condition
of
the
frogs
with
increased
length
of
time
to
maturity,
it
is
uncertain
whether
these
animals
completed
metamorphosis
and/
or
survived.
Furthermore,
because
all
the
animals
in
the
study
had
not
undergone
metamorphosis,
the
percent
initiating
metamorphosis,
time
to
metamorphosis
and
percentage
of
gonadal
abnormalities
could
not
be
accurately
calculated
relative
to
the
total
animals
used
in
the
study.

This
study
indicates
that
only
one
exposure
level
(
25
ug/
L)
produced
developmental
effects
in
frogs,
but
because
of
design
problems
the
study
did
not
establish
a
dose­
response
relationship
between
atrazine
exposure
and
developmental
effects
in
frogs.
The
ability
of
this
study
to
serve
as
a
sensitive
indicator
of
developmental
effects
is
also
in
question
because
of
the
impaired
condition
of
the
animals.
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
13
of
41
H.
REFERENCES:

Nieuwkoop,
P.
D.
and
J.
Faber.
1967.
Normal
table
of
Xenopus
laevis
(
Daudin).
North­
Holland
Publishing
Company,
Amsterdam.
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
14
of
41
Table
4.
Hatching,
mortality,
metamorphosis,
and
gross
developmental
abnormalities
in
X.
laevis
tadpoles
exposed
to
atrazine
or
steroid
hormonesa.

Treatment
Hatching
(%)
Mortalityb
(%)
Forelimb
Emergence
(%)
Tail
Resorption
(%)
Bent
Tails
(%)
Edema
(%)
Abnormal
Swimming
(%)

Negative
94.9
±
1.02
10.4
±
1.07
60.8
±
3.00
51.5
±
2.91
0.94
±
0.43
0.15
±
0.15
1.37
±
0.44
1

g/
L
93.6
±
1.12
11.4
±
1.20
59.0
±
3.31
48.4
±
3.00
0.49
±
0.36
0.30
±
0.30
2.15
±
0.53
10

g/
L
94.5
±
0.093
14.1
±
0.99
52.2
±
3.85
42.3
±
3.42
2.13
±
0.46
0.44
±
0.23
3.12
±
0.68
25

g/
L
96.0
±
0.82
12.4
±
1.58
52.5
±
2.39
44.4
±
2.14
1.22
±
0.33
1.04
±
0.33
3.75
±
0.64*

ethanol
(
0.0025%)
93.7
±
1.47
11.7
±
1.62
53.8
±
4.93
46.1
±
3.84
0.84
±
0.56
0.57
±
0.36
1.99
±
0.73
Dihydrotestosterone
94.6
±
2.32
12.2
±
2.84
48.8
±
5.36
37.2
±
4.86
0.80
±
0.36
1.38
±
0.49
2.98
±
0.94
17­
 
estradiol
92.9
±
2.21
15.2
±
4.16
51.2
±
1.91
42.8
±
2.28
0.82
±
0.37
0.57
±
0.36
1.69
±
0.60
a
Values
are
the
mean
±
standard
error
of
eleven
(
FETAX
medium)
or
six
(
ethanol
co­
solvent)
replicates.
Sample
size
per
replicate
ranged
from
20
­
30
animals.

b
80­
day
post­
hatch
mortality.

*
significantly
different
from
FETAX
medium
(
negative)
control
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
15
of
41
PERCENT
SURVIVAL
BY
TREATMENT
GROUP
236
Obs
TREAT
_
TYPE_
_
FREQ_
SURVIVE
STD
CV
1
0.0
0
11
89.5720
3.5310
3.9420
2
1.0
0
11
88.5690
3.9837
4.4979
3
10
0
11
85.9008
3.2759
3.8136
4
25
0
11
87.6466
5.2333
5.9709
5
DHT
0
6
87.8023
6.9606
7.9276
6
ETOH
0
6
88.2824
3.9624
4.4884
7
Estr
0
6
84.8416
10.1787
11.9972
PERCENT
OF
EGGS
HATCHED
BY
TREATMENT
GROUP
237
Obs
TREAT
_
TYPE_
_
FREQ_
HATCHED
STD
CV
1
0.0
0
11
94.9294
3.38528
3.56610
2
1.0
0
11
93.6032
3.70056
3.95346
3
10
0
11
94.5459
3.09986
3.27868
4
25
0
11
95.9701
2.71563
2.82966
5
DHT
0
6
94.5905
5.68238
6.00734
6
ETOH
0
6
93.6631
3.59493
3.83815
7
Estr
0
6
92.8788
5.41676
5.83207
PERCENT
OF
TADPOLES
WITH
BENT
TAILS
BY
TREATMENT
GROUP
238
Obs
TREAT
_
TYPE_
_
FREQ_
BENT
STD
CV
1
0.0
0
11
0.93716
1.41694
151.195
2
1.0
0
11
0.48822
1.17785
241.256
3
10
0
11
2.12556
1.52127
71.571
4
25
0
11
1.21942
1.11046
91.064
5
DHT
0
6
0.80232
0.87911
109.570
6
ETOH
0
6
0.83525
1.37177
164.234
7
Estr
0
6
0.82068
0.90009
109.676
PERCENT
OF
TADPOLES
WITH
EDEMA
BY
TREATMENT
GROUP
239
Obs
TREAT
_
TYPE_
_
FREQ_
EDEMA
STD
CV
1
0.0
0
11
0.14903
0.49428
331.662
2
1.0
0
11
0.30311
0.67450
222.524
3
10
0
11
0.43770
0.74986
171.316
4
25
0
11
1.03520
1.08659
104.965
5
DHT
0
6
1.37917
1.21355
87.992
6
ETOH
0
6
0.56644
0.87922
155.219
7
Estr
0
6
0.56984
0.88284
154.928
PERCENT
OF
TADPOLES
WITH
ABNORMAL
SWIMMING
BY
TREATMENT
GROUP
240
Obs
TREAT
_
TYPE_
_
FREQ_
SWIM
STD
CV
1
0.0
0
11
1.37132
1.46806
107.055
2
1.0
0
11
2.15034
1.76982
82.304
3
10
0
11
3.11772
2.25729
72.402
4
25
0
11
3.75251
2.13240
56.826
5
DHT
0
6
2.98382
2.30207
77.152
6
ETOH
0
6
1.98706
1.77746
89.452
7
Estr
0
6
1.69367
1.46398
86.438
PERCENT
METAMORPHOSIS
BY
TREATMENT
GROUP
241
Obs
TREAT
_
TYPE_
_
FREQ_
METAMORP
STD
CV
1
0.0
0
11
51.5133
9.6405
18.7146
2
1.0
0
11
48.3625
9.9718
20.6188
3
10
0
11
42.2644
11.3256
26.7971
4
25
0
11
44.3954
7.1060
16.0061
5
DHT
0
6
37.1615
11.8957
32.0109
6
ETOH
0
6
46.0801
9.4053
20.4107
7
Estr
0
6
42.7528
5.5845
13.0622
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
16
of
41
NONPARAMETRIC
COMPARISON
OF
PERCENT
METAMORPHOSIS
ACROSS
TREATMENTS
242
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
PMET
Classified
by
Variable
TREAT
Sum
of
Expected
Std
Dev
Mean
TREAT
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
11
459.00
346.50
54.267424
41.727273
1.0
11
423.50
346.50
54.267424
38.500000
10
11
298.00
346.50
54.267424
27.090909
25
11
312.00
346.50
54.267424
28.363636
DHT
6
114.50
189.00
41.997885
19.083333
ETOH
6
199.00
189.00
41.997885
33.166667
Estr
6
147.00
189.00
41.997885
24.500000
Average
scores
were
used
for
ties.
Kruskal­
Wallis
Test
Chi­
Square
9.9774
DF
6
Pr
>
Chi­
Square
0.1256
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
PMET
Classified
by
Variable
TREAT
Sum
of
Expected
Std
Dev
Mean
TREAT
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
11
8.0
5.50
1.516305
0.727273
1.0
11
7.0
5.50
1.516305
0.636364
10
11
5.0
5.50
1.516305
0.454545
25
11
4.0
5.50
1.516305
0.363636
DHT
6
2.0
3.00
1.173477
0.333333
ETOH
6
3.0
3.00
1.173477
0.500000
Estr
6
2.0
3.00
1.173477
0.333333
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
5.2473
DF
6
Pr
>
Chi­
Square
0.5125
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
17
of
41
NONPARAMETRIC
COMPARISON
OF
PERCENT
SURVIVAL
ACROSS
TREATMENTS
244
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
SUR
Classified
by
Variable
TREAT
Sum
of
Expected
Std
Dev
Mean
TREAT
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
11
426.50
346.50
54.251705
38.772727
1.0
11
377.00
346.50
54.251705
34.272727
10
11
240.50
346.50
54.251705
21.863636
25
11
328.50
346.50
54.251705
29.863636
DHT
6
208.00
189.00
41.985719
34.666667
ETOH
6
185.50
189.00
41.985719
30.916667
Estr
6
187.00
189.00
41.985719
31.166667
Average
scores
were
used
for
ties.
Kruskal­
Wallis
Test
Chi­
Square
5.4728
DF
6
Pr
>
Chi­
Square
0.4848
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
SUR
Classified
by
Variable
TREAT
Sum
of
Expected
Std
Dev
Mean
TREAT
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
11
7.750
5.50
1.479165
0.704545
1.0
11
6.750
5.50
1.479165
0.613636
10
11
2.750
5.50
1.479165
0.250000
25
11
4.750
5.50
1.479165
0.431818
DHT
6
4.000
3.00
1.144735
0.666667
ETOH
6
2.000
3.00
1.144735
0.333333
Estr
6
3.000
3.00
1.144735
0.500000
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
6.9240
DF
6
Pr
>
Chi­
Square
0.3279
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
18
of
41
NONPARAMETRIC
COMPARISON
OF
PERCENT
OF
TADPOLES
WITH
ABNORMAL
SWIMMING
ACROSS
TREATMENTS
246
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
SWIM
Classified
by
Variable
TREAT
Sum
of
Expected
Std
Dev
Mean
TREAT
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
11
243.50
346.50
53.920541
22.136364
1.0
11
321.00
346.50
53.920541
29.181818
10
11
390.00
346.50
53.920541
35.454545
25
11
462.50
346.50
53.920541
42.045455
DHT
6
221.50
189.00
41.729430
36.916667
ETOH
6
150.50
189.00
41.729430
25.083333
Estr
6
164.00
189.00
41.729430
27.333333
Average
scores
were
used
for
ties.

Kruskal­
Wallis
Test
Chi­
Square
9.1688
DF
6
Pr
>
Chi­
Square
0.1643
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
SWIM
Classified
by
Variable
TREAT
Sum
of
Expected
Std
Dev
Mean
TREAT
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
11
4.0
5.50
1.516305
0.363636
1.0
11
5.0
5.50
1.516305
0.454545
10
11
5.0
5.50
1.516305
0.454545
25
11
9.0
5.50
1.516305
0.818182
DHT
6
4.0
3.00
1.173477
0.666667
ETOH
6
1.0
3.00
1.173477
0.166667
Estr
6
3.0
3.00
1.173477
0.500000
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
8.6461
DF
6
Pr
>
Chi­
Square
0.1945
NONPARAMETRIC
COMPARISON
OF
PERCENT
OF
TADPOLES
WITH
BENT
TAILS
ACROSS
TREATMENTS
248
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
BENT
Classified
by
Variable
TREAT
Sum
of
Expected
Std
Dev
Mean
TREAT
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
11
323.50
346.50
50.384440
29.409091
1.0
11
254.00
346.50
50.384440
23.090909
10
11
491.50
346.50
50.384440
44.681818
25
11
374.00
346.50
50.384440
34.000000
DHT
6
161.00
189.00
38.992820
26.833333
ETOH
6
174.00
189.00
38.992820
29.000000
Estr
6
175.00
189.00
38.992820
29.166667
Average
scores
were
used
for
ties.

Kruskal­
Wallis
Test
Chi­
Square
10.7175
DF
6
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
19
of
41
Pr
>
Chi­
Square
0.0975
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
BENT
Classified
by
Variable
TREAT
Sum
of
Expected
Std
Dev
Mean
TREAT
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
11
4.218750
5.50
1.468156
0.383523
1.0
11
2.281250
5.50
1.468156
0.207386
10
11
9.062500
5.50
1.468156
0.823864
25
11
7.125000
5.50
1.468156
0.647727
DHT
6
3.093750
3.00
1.136215
0.515625
ETOH
6
2.125000
3.00
1.136215
0.354167
Estr
6
3.093750
3.00
1.136215
0.515625
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
10.9792
DF
6
Pr
>
Chi­
Square
0.0890
NONPARAMETRIC
COMPARISON
OF
PERCENT
OF
TADPOLES
WITH
EDEMA
ACROSS
TREATMENTS
250
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
EDEMA
Classified
by
Variable
TREAT
Sum
of
Expected
Std
Dev
Mean
TREAT
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
11
266.50
346.50
45.035250
24.227273
1.0
11
300.00
346.50
45.035250
27.272727
10
11
314.50
346.50
45.035250
28.590909
25
11
417.00
346.50
45.035250
37.909091
DHT
6
264.50
189.00
34.853050
44.083333
ETOH
6
192.50
189.00
34.853050
32.083333
Estr
6
198.00
189.00
34.853050
33.000000
Average
scores
were
used
for
ties.

Kruskal­
Wallis
Test
Chi­
Square
10.2116
DF
6
Pr
>
Chi­
Square
0.1160
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
EDEMA
Classified
by
Variable
TREAT
Sum
of
Expected
Std
Dev
Mean
TREAT
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
11
3.619048
5.50
1.046350
0.329004
1.0
11
4.357143
5.50
1.046350
0.396104
10
11
5.095238
5.50
1.046350
0.463203
25
11
7.309524
5.50
1.046350
0.664502
DHT
6
4.523810
3.00
0.809776
0.753968
ETOH
6
3.047619
3.00
0.809776
0.507937
Estr
6
3.047619
3.00
0.809776
0.507937
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
9.4273
DF
6
Pr
>
Chi­
Square
0.1509
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
20
of
41
Plot
of
METAMORP*
CONC.
Symbol
used
is
'*'.

METAMORP
52
ê
é
é
*
é
é
é
é
50
ê
é
é
é
é
é
é
*
48
ê
é
é
é
é
é
é
46
ê
é
é
é
é
é
é
*
44
ê
é
é
é
é
é
é
*
42
ê
é
èââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââ
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
CONC
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
21
of
41
CORRELATION
ANALYSIS
OF
PERCENT
METAMORPHOSIS
OVER
CONCENTRATION
253
The
CORR
Procedure
1
With
Variables:
CONC
1
Variables:
PMET
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
CONC
44
9.00000
10.14087
396.00000
0
25.00000
PMET
44
46.63390
9.96659
2052
19.67213
66.66667
Pearson
Correlation
Coefficients,
N
=
44
Prob
>
|
r|
under
H0:
Rho=
0
PMET
CONC
­
0.24787
0.1047
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
22
of
41
Plot
of
SWIM*
CONC.
Symbol
used
is
'*'.

SWIM
4.0
é
é
é
*
é
é
é
3.5
ê
é
é
é
é
é
*
é
3.0
ê
é
é
é
é
é
é
2.5
ê
é
é
é
é
é
*
é
2.0
ê
é
é
é
é
é
é
1.5
ê
é
é
*
é
é
é
1.0
é
èââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââ
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
CONC
CORRELATION
ANALYSIS
OF
PERCENT
OF
TADPOLES
WITH
ABNORMAL
SWIMMING
OVER
CONCENTRATION
255
The
CORR
Procedure
1
With
Variables:
CONC
1
Variables:
SWIM
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
CONC
44
9.00000
10.14087
396.00000
0
25.00000
SWIM
44
2.59797
2.07806
114.31082
0
7.35294
Pearson
Correlation
Coefficients,
N
=
44
Prob
>
|
r|
under
H0:
Rho=
0
SWIM
CONC
0.40804
0.0060
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
23
of
41
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
24
of
41
Plot
of
EDEMA*
CONC.
Symbol
used
is
'*'.
EDEMA
1.2
ê
é
é
é
é
é
é
*
1.0
ê
é
é
é
é
é
é
0.8
ê
é
é
é
é
é
é
0.6
ê
é
é
é
é
é
é
*
0.4
ê
é
é
é
*
é
é
é
0.2
ê
é
é
*
é
é
é
é
0.0
ê
èââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââêââ
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
CONC
CORRELATION
ANALYSIS
OF
PERCENT
OF
TADPOLES
WITH
EDEMA
OVER
CONCENTRATION
257
The
CORR
Procedure
1
With
Variables:
CONC
1
Variables:
EDEMA
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
CONC
44
9.00000
10.14087
396.00000
0
25.00000
EDEMA
44
0.48126
0.82662
21.17552
0
3.17460
Pearson
Correlation
Coefficients,
N
=
44
Prob
>
|
r|
under
H0:
Rho=
0
EDEMA
CONC
0.40388
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
25
of
41
Mean
Body
Weight
of
Frogs
by
Treatment
1202
Obs
treat
_
TYPE_
_
FREQ_
MEAN
STDERR
1
0.0
0
335
0.32519
.005212036
2
1.0
0
312
0.31670
.005086144
3
10
0
277
0.34574
.006660663
4
25
0
299
0.32173
.005397997
5
DHT
0
135
0.32539
.008838205
6
ETOH
0
161
0.31324
.007248717
7
Estr
0
151
0.32720
.007594965
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
26
of
41
Mean
Body
Weight
of
Frogs
by
Treatment
1203
Plot
of
MEAN*
treat.
Symbol
used
is
'*'.

MEAN
é
é
0.350
ê
é
é
é
é
*
0.345
ê
é
é
é
é
0.340
ê
é
é
é
é
0.335
ê
é
é
é
é
0.330
ê
é
é
é
*
é
0.325
ê
*
*
é
é
é
*
é
0.320
ê
é
é
é
*
é
0.315
ê
é
é
*
é
é
0.310
ê
é
èââêâââââââââââêâââââââââââêâââââââââââêâââââââââââêâââââââââââêâââââââââââêââ
0.0
1.0
10
25
DHT
ETOH
Estr
treat
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
27
of
41
Analysis
of
Variance
for
Frog
Body
Weight
over
Treatment
1204
The
GLM
Procedure
Class
Level
Information
Class
Levels
Values
treat
7
0.0
1.0
10
25
DHT
ETOH
Estr
Number
of
observations
1670
NOTE:
Due
to
missing
values,
only
1661
observations
can
be
used
in
this
analysis.

Dependent
Variable:
BW
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
6
0.16575046
0.02762508
2.96
0.0071
Error
1654
15.45132260
0.00934179
Corrected
Total
1660
15.61707306
R­
Square
Coeff
Var
Root
MSE
BW
Mean
0.010613
29.69766
0.096653
0.325456
Source
DF
Type
I
SS
Mean
Square
F
Value
Pr
>
F
treat
6
0.16575046
0.02762508
2.96
0.0071
Source
DF
Type
III
SS
Mean
Square
F
Value
Pr
>
F
treat
6
0.16575046
0.02762508
2.96
0.0071
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
28
of
41
Analysis
of
Variance
for
Frog
Body
Weight
over
Treatment
1206
The
GLM
Procedure
Levene's
Test
for
Homogeneity
of
BW
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
treat
6
0.00343
0.000571
1.40
0.2098
Error
1654
0.6737
0.000407
Bartlett's
Test
for
Homogeneity
of
BW
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
treat
6
17.8554
0.0066
Analysis
of
Variance
for
Frog
Body
Weight
over
Treatment
1207
The
GLM
Procedure
Dunnett's
t
Tests
for
BW
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
1654
Error
Mean
Square
0.009342
Critical
Value
of
Dunnett's
t
2.59779
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
treat
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
10
­
0.0
0.020549
0.000124
0.040974
***
Estr
­
0.0
0.002005
­
0.022617
0.026628
DHT
­
0.0
0.000197
­
0.025410
0.025804
25
­
0.0
­
0.003465
­
0.023509
0.016578
1.0
­
0.0
­
0.008491
­
0.028309
0.011328
ETOH
­
0.0
­
0.011958
­
0.036098
0.012183
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
29
of
41
Nonparametric
comparison
of
frog
body
weight
over
treatments
1208
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
BW
Classified
by
Variable
treat
Sum
of
Expected
Std
Dev
Mean
treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
334
279296.50
277554.0
7834.89949
836.217066
1.0
309
244939.50
256779.0
7606.63049
792.684466
10
276
254956.50
229356.0
7276.19220
923.755435
25
296
242002.00
245976.0
7480.60832
817.574324
DHT
135
109378.50
112185.0
5341.56953
810.211111
ETOH
160
122301.50
132960.0
5767.33359
764.384375
Estr
151
127416.50
125481.0
5619.55184
843.817881
Average
scores
were
used
for
ties.

Kruskal­
Wallis
Test
Chi­
Square
16.0133
DF
6
Pr
>
Chi­
Square
0.0137
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
BW
Classified
by
Variable
treat
Sum
of
Expected
Std
Dev
Mean
treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
334
178.0
166.899458
8.170047
0.532934
1.0
309
143.0
154.406984
7.932014
0.462783
10
276
162.0
137.916918
7.587441
0.586957
25
296
141.0
147.910897
7.800601
0.476351
DHT
135
61.0
67.459362
5.570062
0.451852
ETOH
160
67.0
79.951836
6.014039
0.418750
Estr
151
78.0
75.454545
5.859935
0.516556
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
17.8022
DF
6
Pr
>
Chi­
Square
0.0067
Average
Snout
to
Vent
Length
of
Frogs
by
Treatment
1210
Obs
treat
_
TYPE_
_
FREQ_
MEAN
STDERR
1
0.0
0
335
13.9566
0.08149
2
1.0
0
312
13.7896
0.07939
3
10
0
277
14.0815
0.09106
4
25
0
299
13.9865
0.09052
5
DHT
0
135
13.9963
0.13501
6
ETOH
0
161
13.5938
0.10530
7
Estr
0
151
14.2914
0.11416
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
30
of
41
Average
Snout
to
Vent
Length
of
Frogs
by
Treatment
1211
Plot
of
MEAN*
treat.
Symbol
used
is
'*'.

MEAN
é
14.3
ê
é
*
é
é
é
é
14.2
ê
é
é
é
é
é
14.1
ê
é
*
é
é
é
é
14.0
ê
*
é
*
é
é
*
é
é
13.9
ê
é
é
é
é
é
13.8
ê
é
*
é
é
é
é
13.7
ê
é
é
é
é
é
13.6
ê
*
é
èââêâââââââââââêâââââââââââêâââââââââââêâââââââââââêâââââââââââêâââââââââââêââ
0.0
1.0
10
25
DHT
ETOH
Estr
treat
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
31
of
41
Analysis
of
Variance
for
Frog
Snout
to
Vent
Length
over
Treatment
1212
The
GLM
Procedure
Class
Level
Information
Class
Levels
Values
treat
7
0.0
1.0
10
25
DHT
ETOH
Estr
Number
of
observations
1670
NOTE:
Due
to
missing
values,
only
1661
observations
can
be
used
in
this
analysis.

Dependent
Variable:
SVL
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
6
51.322535
8.553756
3.94
0.0006
Error
1654
3590.329783
2.170695
Corrected
Total
1660
3641.652318
R­
Square
Coeff
Var
Root
MSE
SVL
Mean
0.014093
10.56124
1.473328
13.95033
Source
DF
Type
I
SS
Mean
Square
F
Value
Pr
>
F
treat
6
51.32253515
8.55375586
3.94
0.0006
Source
DF
Type
III
SS
Mean
Square
F
Value
Pr
>
F
treat
6
51.32253515
8.55375586
3.94
0.0006
Levene's
Test
for
Homogeneity
of
SVL
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
treat
6
81.5820
13.5970
1.10
0.3612
Error
1654
20484.3
12.3847
Bartlett's
Test
for
Homogeneity
of
SVL
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
treat
6
8.9126
0.1786
Dunnett's
t
Tests
for
SVL
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
1654
Error
Mean
Square
2.170695
Critical
Value
of
Dunnett's
t
2.59779
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
Simultaneous
treat
Between
95%
Confidence
Comparison
Means
Limits
Estr
­
0.0
0.3348
­
0.0405
0.7101
10
­
0.0
0.1249
­
0.1864
0.4363
DHT
­
0.0
0.0397
­
0.3506
0.4301
25
­
0.0
0.0299
­
0.2756
0.3354
1.0
­
0.0
­
0.1669
­
0.4690
0.1352
ETOH
­
0.0
­
0.3628
­
0.7308
0.0052
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
32
of
41
Nonparametric
comparison
of
frog
length
over
treatments
1216
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
SVL
Classified
by
Variable
treat
Sum
of
Expected
Std
Dev
Mean
treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
334
277451.50
277554.0
7761.12060
830.693114
1.0
309
243946.50
256779.0
7535.00115
789.470874
10
276
241172.50
229356.0
7207.67449
873.813406
25
296
247542.00
245976.0
7410.16568
836.290541
DHT
135
113146.50
112185.0
5291.26958
838.122222
ETOH
160
114795.50
132960.0
5713.02434
717.471875
Estr
151
142236.50
125481.0
5566.63421
941.963576
Average
scores
were
used
for
ties.

Kruskal­
Wallis
Test
Chi­
Square
22.0409
DF
6
Pr
>
Chi­
Square
0.0012
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
SVL
Classified
by
Variable
treat
Sum
of
Expected
Std
Dev
Mean
treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
334
166.322485
166.899458
7.423923
0.497972
1.0
309
141.224852
154.406984
7.207627
0.457038
10
276
150.535503
137.916918
6.894522
0.545418
25
296
150.807692
147.910897
7.088216
0.509485
DHT
135
68.059172
67.459362
5.061379
0.504142
ETOH
160
65.059172
79.951836
5.464810
0.406620
Estr
151
87.991124
75.454545
5.324780
0.582723
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
17.4213
DF
6
Pr
>
Chi­
Square
0.0079
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
33
of
41
Percentage
of
Frogs
Male
or
Female
1218
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
treat=
0.0
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
FREQ
Procedure
Cumulative
Cumulative
Sex
Frequency
Percent
Frequency
Percent
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
?
2
0.60
2
0.60
F
168
50.30
170
50.90
M
164
49.10
334
100.00
Frequency
Missing
=
1
Percentage
of
Frogs
Male
or
Female
1219
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
treat=
1.0
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
FREQ
Procedure
Cumulative
Cumulative
Sex
Frequency
Percent
Frequency
Percent
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
?
3
0.97
3
0.97
F
152
49.19
155
50.16
M
154
49.84
309
100.00
Frequency
Missing
=
3
Percentage
of
Frogs
Male
or
Female
1220
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
treat=
10
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
FREQ
Procedure
Cumulative
Cumulative
Sex
Frequency
Percent
Frequency
Percent
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
?
1
0.36
1
0.36
F
142
51.45
143
51.81
M
133
48.19
276
100.00
Frequency
Missing
=
1
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
34
of
41
Percentage
of
Frogs
Male
or
Female
1221
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
treat=
25
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
FREQ
Procedure
Cumulative
Cumulative
Sex
Frequency
Percent
Frequency
Percent
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
?
14
4.73
14
4.73
F
149
50.34
163
55.07
M
133
44.93
296
100.00
Frequency
Missing
=
3
Percentage
of
Frogs
Male
or
Female
1222
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
treat=
DHT
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
FREQ
Procedure
Cumulative
Cumulative
Sex
Frequency
Percent
Frequency
Percent
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
F
73
54.07
73
54.07
M
62
45.93
135
100.00
Percentage
of
Frogs
Male
or
Female
1223
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
treat=
ETOH
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
FREQ
Procedure
Cumulative
Cumulative
Sex
Frequency
Percent
Frequency
Percent
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
F
81
50.63
81
50.63
M
79
49.38
160
100.00
Frequency
Missing
=
1
Percentage
of
Frogs
Male
or
Female
1224
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
treat=
Estr
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
FREQ
Procedure
Cumulative
Cumulative
Sex
Frequency
Percent
Frequency
Percent
ââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
?
11
7.28
11
7.28
F
101
66.89
112
74.17
M
39
25.83
151
100.00
Average
Total
Area
of
Frog
Larynx
by
Treatment
1225
Obs
treat
_
TYPE_
_
FREQ_
MEAN
STDERR
1
0.0
0
56
0.14694
0.003893
2
1.0
0
51
0.15720
0.005215
3
10
0
55
0.15998
0.003926
4
25
0
54
0.15752
0.004347
5
DHT
0
12
0.30669
0.012958
6
ETOH
0
12
0.14100
0.009301
7
Estr
0
12
0.14480
0.006760
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
35
of
41
Plot
of
MEAN*
treat.
Symbol
used
is
'*'.

MEAN
é
é
0.325
ê
é
é
é
é
*
0.300
ê
é
é
é
é
0.275
ê
é
é
é
é
0.250
ê
é
é
é
é
0.225
ê
é
é
é
é
0.200
ê
é
é
é
é
0.175
ê
é
é
é
*
*
é
*
0.150
ê
é
*
*
é
*
é
é
0.125
ê
é
èââêâââââââââââêâââââââââââêâââââââââââêâââââââââââêâââââââââââêâââââââââââêââ
0.0
1.0
10
25
DHT
ETOH
Estr
treat
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
36
of
41
Analysis
of
Variance
for
Larynx
Total
Area
over
Treatment
1227
The
GLM
Procedure
Class
Level
Information
Class
Levels
Values
treat
7
0.0
1.0
10
25
DHT
ETOH
Estr
Number
of
observations
252
Dependent
Variable:
TOTAL
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
6
0.27515908
0.04585985
43.98
<.
0001
Error
245
0.25549096
0.00104282
Corrected
Total
251
0.53065004
R­
Square
Coeff
Var
Root
MSE
TOTAL
Mean
0.518532
20.01396
0.032293
0.161351
Source
DF
Type
I
SS
Mean
Square
F
Value
Pr
>
F
treat
6
0.27515908
0.04585985
43.98
<.
0001
Source
DF
Type
III
SS
Mean
Square
F
Value
Pr
>
F
treat
6
0.27515908
0.04585985
43.98
<.
0001
Levene's
Test
for
Homogeneity
of
TOTAL
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
treat
6
0.000021
3.543E­
6
1.82
0.0955
Error
245
0.000477
1.945E­
6
Bartlett's
Test
for
Homogeneity
of
TOTAL
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
treat
6
9.1161
0.1672
Dunnett's
t
Tests
for
TOTAL
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
245
Error
Mean
Square
0.001043
Critical
Value
of
Dunnett's
t
2.62465
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
treat
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
DHT
­
0.0
0.159753
0.132791
0.186715
***
10
­
0.0
0.013036
­
0.003054
0.029126
25
­
0.0
0.010574
­
0.005591
0.026739
1.0
­
0.0
0.010257
­
0.006148
0.026663
Estr
­
0.0
­
0.002144
­
0.029106
0.024817
ETOH
­
0.0
­
0.005940
­
0.032901
0.021022
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
37
of
41
Nonparametric
comparison
of
frog
total
larynx
area
over
treatments
1231
The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
TOTAL
Classified
by
Variable
treat
Sum
of
Expected
Std
Dev
Mean
treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
56
5953.0
7084.00
481.051626
106.303571
1.0
51
6269.0
6451.50
464.892730
122.921569
10
55
7427.0
6957.50
477.951793
135.036364
25
54
6899.0
6831.00
474.787321
127.759259
DHT
12
2956.0
1518.00
246.414285
246.333333
ETOH
12
1161.0
1518.00
246.414285
96.750000
Estr
12
1213.0
1518.00
246.414285
101.083333
Kruskal­
Wallis
Test
Chi­
Square
41.0845
DF
6
Pr
>
Chi­
Square
<.
0001
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
TOTAL
Classified
by
Variable
treat
Sum
of
Expected
Std
Dev
Mean
treat
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
0.0
56
23.0
28.00
3.306398
0.410714
1.0
51
24.0
25.50
3.195334
0.470588
10
55
32.0
27.50
3.285092
0.581818
25
54
25.0
27.00
3.263342
0.462963
DHT
12
12.0
6.00
1.693672
1.000000
ETOH
12
5.0
6.00
1.693672
0.416667
Estr
12
5.0
6.00
1.693672
0.416667
Median
One­
Way
Analysis
Chi­
Square
16.3328
DF
6
Pr
>
Chi­
Square
0.0121
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
38
of
41
Analysis
of
Variance
for
Larynx
Total
Area
over
Treatment
by
Sex
1233
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
GLM
Procedure
Class
Level
Information
Class
Levels
Values
treat
7
0.0
1.0
10
25
DHT
ETOH
Estr
Number
of
observations
62
Dependent
Variable:
TOTAL
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
6
0.19693080
0.03282180
43.04
<.
0001
Error
55
0.04194645
0.00076266
Corrected
Total
61
0.23887726
R­
Square
Coeff
Var
Root
MSE
TOTAL
Mean
0.824402
19.46572
0.027616
0.141872
Source
DF
Type
I
SS
Mean
Square
F
Value
Pr
>
F
treat
6
0.19693080
0.03282180
43.04
<.
0001
Source
DF
Type
III
SS
Mean
Square
F
Value
Pr
>
F
treat
6
0.19693080
0.03282180
43.04
<.
0001
Levene's
Test
for
Homogeneity
of
TOTAL
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
treat
6
0.000034
5.612E­
6
5.62
0.0001
Error
55
0.000055
9.985E­
7
Bartlett's
Test
for
Homogeneity
of
TOTAL
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
treat
6
16.3229
0.0121
Dunnett's
t
Tests
for
TOTAL
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
55
Error
Mean
Square
0.000763
Critical
Value
of
Dunnett's
t
2.67093
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
treat
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
DHT
­
0.0
0.19547
0.15804
0.23291
***
Estr
­
0.0
0.02421
­
0.01322
0.06165
10
­
0.0
0.01164
­
0.01981
0.04310
25
­
0.0
0.00455
­
0.02624
0.03534
ETOH
­
0.0
0.00380
­
0.03364
0.04123
1.0
­
0.0
0.00047
­
0.03176
0.03269
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
39
of
41
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
40
of
41
Analysis
of
Variance
for
Larynx
Total
Area
over
Treatment
by
Sex
1237
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
Sex=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
GLM
Procedure
Class
Level
Information
Class
Levels
Values
treat
7
0.0
1.0
10
25
DHT
ETOH
Estr
Number
of
observations
190
Dependent
Variable:
TOTAL
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
6
0.11691360
0.01948560
24.82
<.
0001
Error
183
0.14365715
0.00078501
Corrected
Total
189
0.26057076
R­
Square
Coeff
Var
Root
MSE
TOTAL
Mean
0.448683
16.70652
0.028018
0.167707
Source
DF
Type
I
SS
Mean
Square
F
Value
Pr
>
F
treat
6
0.11691360
0.01948560
24.82
<.
0001
Source
DF
Type
III
SS
Mean
Square
F
Value
Pr
>
F
treat
6
0.11691360
0.01948560
24.82
<.
0001
Levene's
Test
for
Homogeneity
of
TOTAL
Variance
ANOVA
of
Squared
Deviations
from
Group
Means
Sum
of
Mean
Source
DF
Squares
Square
F
Value
Pr
>
F
treat
6
9.284E­
6
1.547E­
6
1.20
0.3067
Error
183
0.000235
1.286E­
6
Bartlett's
Test
for
Homogeneity
of
TOTAL
Variance
Source
DF
Chi­
Square
Pr
>
ChiSq
treat
6
6.4494
0.3748
Dunnett's
t
Tests
for
TOTAL
NOTE:
This
test
controls
the
Type
I
experimentwise
error
for
comparisons
of
all
treatments
against
a
control.

Alpha
0.05
Error
Degrees
of
Freedom
183
Error
Mean
Square
0.000785
Critical
Value
of
Dunnett's
t
2.63648
Comparisons
significant
at
the
0.05
level
are
indicated
by
***.

Difference
treat
Between
Simultaneous
95%
Comparison
Means
Confidence
Limits
DHT
­
0.0
0.146489
0.114385
0.178594
***
10
­
0.0
0.013549
­
0.002112
0.029210
25
­
0.0
0.013522
­
0.002327
0.029371
1.0
­
0.0
0.012629
­
0.003319
0.028578
ETOH
­
0.0
0.006778
­
0.025327
0.038882
Data
Evaluation
Report
on
Response
of
Larval
Xenopus
laevis
to
Atrazine
Exposure:
Assessment
of
Metamorphosis
and
Gonadal
and
Laryngeal
Morphology
EPA
MRID
Number
458677­
07
Page
41
of
41
Estr
­
0.0
­
0.006049
­
0.038154
0.026055
