Document ID: EPA-HQ-OPP-2003-0024-0025
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-06-03T04:00Z

Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Data
Requirement:
:
EPA
DP
Barcode
D288775
EPA
MRID
458677­
03
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.
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
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
2
of
36
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
3
of
36
EXECUTIVE
SUMMARY:

Green
frog
(
Rana
clamitans)
tadpoles
reared
from
field­
collected
eggs
were
exposed
for
273
days,
beginning
5­
days
post­
hatch,
to
two
concentrations
(
10
and
25

g/
L)
of
atrazine.
Positive
controls,
dihydroxytestosterone
and
17­
 
estradiol
(
0.1

g/
mL
in
0.005%
ethanol),
a
negative
control
(
water)
and
a
solvent
control
(
0.005%
ethanol)
were
also
run.
Replicates
(
9)
consisted
of
30
free­
swimming
tadpoles
each.
Initially,(
exposure
days
0
­
67)
animals
were
maintained
under
static
renewal
conditions
in
4
L
of
test
solution;
50%
tank
changes
were
conducted
every
72
hours.
From
Days
68
to
Day
273,
tadpoles
were
maintained
in
tanks
containing
16L
of
test
solution
under
static
renewal.
After
273
days,
exposures
were
terminated
and
tadpoles
were
maintained
in
continuous
flow­
through
10­
L
glass
tanks
housed
in
large
acrylic
tanks
containing
80
L
of
continuously
renewing
freshwater.
At
metamorphosis
(
fore­
limb
emergence),
tadpoles
were
either
housed
individually
or
in
small
groups
in
10­
L
glass
tanks
containing
approximately
500
mL
of
freshwater.
Over
the
study
period,
mortality
across
all
treatment
groups
averaged
76.5%
and
was
attributed
to
poor
water
quality
and
overcrowding
during
the
273­
day
static­
renewal
phase
of
the
study.
While
mean­
measured
concentrations
of
atrazine
were
relatively
consistent
with
nominal
values,
measurements
were
made
on
freshly
prepared
stock
solutions;
hence
it
is
unclear
what
atrazine
concentrations
were
present
in
aged
exposure
solutions.
Additionally,
measurable
levels
of
atrazine
were
detected
in
the
negative
controls.
Although
the
concentrations
of
positive
control
hormones
were
not
measured,
the
positive
controls
using
dihyrdotestosterone
and
17­
 
estradiol
suggested
that
green
frogs
only
reacted
to
androgenic
chemicals
resulting
in
predominately
(
97.6%)
male
frogs,
while
the
frogs
were
not
affected
by
estradiol.
It
is
uncertain
whether
this
means
that
green
frogs
are
unresponsive
to
estrogenic
chemicals,
or
whether
there
was
sufficient
estradiol
in
solution
to
elicit
an
effect.
While
no
intersex
(
testicular
and
ovarian
tissue
in
the
same
animal)
was
observed
in
any
of
the
treatment
groups,
this
observation
was
based
on
gross
morphology,
and
apparently
there
were
difficulties
in
discerning
the
presence
of
gonads
using
this
process.
While
time
to
and
age
at
metamorphosis
and
the
size
of
metamorphs
were
reduced
in
frogs
treated
with
10

g/
L
atrazine,
there
was
no
difference
in
these
same
parameters
for
frogs
treated
with
25

g/
L
atrazine
relative
to
negative
controls.
Although
there
were
no
dose
dependent
effects
in
green
frogs
related
to
atrazine
treatment,
only
two
concentrations
were
monitored.
Additionally,
because
only
a
limited
number
of
frogs
survived
to
complete
metamorphosis,
the
conclusions
regarding
sex
ratio
data
are
questionable.

No
analysis
of
gonad
histology
is
provided
and
no
measurements
were
made
of
aromatase
levels.
Contrary
to
the
GLP
statement,
this
study
represents
an
interim
report
and
not
a
final
report.
.

The
high
mortality
indicative
of
poor
water
quality
and
overcrowding
and
the
lack
of
response
to
the
positive
estradiol
control
made
it
difficult
for
the
study
authors
to
test
the
hypothesis
that
atrazine
exposure
was
associated
with
developmental
effects
in
amphibians.
The
study
did
provide
the
authors
with
a
better
appreciation
for
the
conditions
under
which
green
frogs
should
be
housed,
and
it
suggests
that
the
green
frog
may
not
be
adequate
for
examining
the
effects
of
atrazine
on
amphibian
development..
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
4
of
36
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
Stanadard
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
was
archieved.

A.
MATERIALS:

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

Species:
Green
frog
(
Rana
clamitans)
.
Age
at
test
initiation:
Larvae
(
Gosner
Stage
25;
approximately
5
days
post­
hatch
Weight
at
study
initiation:
(
mean
and
range)
Length
at
study
initiation:
(
mean
and
range)

Source:
Eggs
field­
collected
as
a
single
mass
of
fertilized
eggs
from
Giesy
pond
in
Williamston,
MI
(
7/
10/
2001)
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
5
of
36
B.
STUDY
DESIGN:

Objective:
1)
To
develop
and
validate
methods
of
husbandry
and
exposure
for
conducting
laboratory
studies
with
R.
clamitans.
2)
To
determine
the
response
of
larval
R.
clamitans
to
atrazine
by
assessing
metamorphosis
and
reproduction
indices
when
animals
are
exposed
during
larval
development.
Indices
to
be
evaluated
include:
°
%
initiating
metamorphosis
°
%
completing
metamorphosis
°
time
to
metamorphosis
°
fresh
post­
mortem
body
weight
and
snout­
vent
length
°
incidence
of
gross
gonadal
abnormalities
°
histology
of
the
gonads.

1.
Experimental
Conditions
a)
Range­
finding
Study:
Current
study
represents
a
pilot
study
b.
Definitive
Study
Table
1
.
Experimental
Parameters
Parameter
Details
Acclimation:
period:
Conditions:
(
same
as
test
or
not)
Feeding:
Health:
(
any
mortality
observed)
8
days
transitioned
from
pond
to
laboratory
water
over
unspecified
time
period
not
reported
Duration
of
the
test
506­
day
study
of
which
273
days
exposed
to
test
solutions
Test
condition
static/
flow­
through
Type
of
dilution
systemfor
flow­
through
method.

Renewal
rate
for
static
renewal
static
renewal
NA
50%
test
solution
change
every
72
hours
Aeration,
if
any
NA
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Parameter
Details
Page
6
of
36
Test
vessel
Material:
(
glass/
stainless
steel)
Size:
Fill
volume:
glass
10
L
4
L
After
67
days
of
exposure,
tadpoles
transferred
from
10L
tanks
to
tanks
containing
16
L
of
test
solution.
After
273
days,
tadpoles
transferred
to
continuous
flow­
through
system
of
clean
freshwater
through
a
10­
L
glass
tank
housed
in
large
acrylic
tanks
containing
80
L
of
continuously
renewing
freshwater;
once
animals
initiated
metamorphosis
(
fore­
limb
emergence),
removed
from
flow­
through
system
and
housed
as
individuals
or
small
groups
in
10­
L
glass
tanks
containing
approximately
500
ml
of
freshwater.

Source
of
dilution
water
Quality:
Treated
well
water
(
MSU­
University
Research
Containment
Facility
Water
parameters:
Hardness
pH
Dissolved
oxygen
Total
organic
carbon
Particulate
matter
Ammonia
Nitrite
Metals
Pesticides
Chlorine
Temperature
{
Salinity
for
marine
or
estuarine
species}

Intervals
of
water
quality
measurement
426
mg/
L
as
CaCO3
(
static);
7.87
(
static);
8.0
mg/
L
(
static);
6.1
mg/
L
(
flow­
through)

0.04
mg/
L
(
static);
0.02
mg/
L
(
flow­
through)
(
see
reviewer's
comments)
0.22
mg/
L
(
static);
0.02
mg/
L
(
flow­
through)

21.3oC
(
static);
24.8oC
(
flow­
through)

NA
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Parameter
Details
Page
7
of
36
Number
of
replicates/
groups:
negative
control:
water
solvent
control:
0.005%
ethanol
treated
ones:
atrazine
at
10
and
25

g/
L
positive
controls:
dihydroxytestosterone
and
estradiol
9
9
9
+
9
9
+
9
Number
of
organisms
per
replicate
/
groups:
control:
solvent
control:
treated
ones:
(
30
tadpoles
/
rep)
x
9
reps
=
270
tadpoles
30
tadpoles
/
rep)
x
9
reps
=
270
tadpoles
30
tadpoles
/
rep)
x
9
reps
=
270
tadpoles
Biomass
loading
rate
30
tadpoles/
4
L

30
tadpoles/
10
L
Test
concentrations:
nominal:
measured:
10
and
25

g/
L
13.8
and
28.1

g
a.
i./
L
Solvent
(
type,
percentage,
if
used)
freshwater
for
atrazine;
0.005%
ethanol
for
positive
hormone
controls
Lighting
not
reported
Feeding
Appendix
reports
that
frog
brittle
was
analyzed
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}
dihydroxytestosterone
0.1

g/
L
17­
 
estradiol
0.1

g/
L
both
hormones
in
0.005%
ethanol
Other
parameters,
if
any
NA
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
8
of
36
2.
Observations:

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?

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).

II.
RESULTS
and
DISCUSSION:
[
All
results
discussed
in
this
section
and
the
next
are
those
reported
by
the
study
authors.
Although
supplemental
data
are
typically
used
in
a
qualitative
manner
only,
EFED
verified
spreadsheet
data
and
ran
basic
statistical
analyses
on
the
major
study
parameters.
See
attached
appendix.
If
results
differed
in
any
substantive
way,
the
difference
was
reported
in
the
text
below.]

Exposures
were
initiated
at
5
days
post­
hatch
when
tadpoles
were
free
swimming
and
external
gills
were
resorbed
(
Gosner
stage
25).
The
feeding
regime
for
the
tanks
was
not
discussed
Atrazine
levels
[
of
stock
solutions]
were
measured
following
static
renewal
and
therefore
represented
fresh
as
opposed
to
aged
exposure
solution
values.
Measurements
were
made
using
two
different
ELISA
kits
and
yielded
roughly
similar
exposure
estimates
(
Table
3)
over
the
course
of
the
study.
In
general,
mean­
measured
concentrations
ranged
from
112%
to
159%
of
nominal.
Atrazine
was
detected
in
the
controls
at
concentrations
that
were
within
the
LOQ
(
0.025)
for
the
assay.
Because
data
was
not
reported
for
the
solvent
control
or
either
of
the
positive
controls,
it
is
not
clear
whether
the
contamination
was
limited
to
negative
controls
or
across
all
treatments.
Triazine
ELISA
kits
did
not
arrive
within
the
first
60
days
of
exposure,
and
it
is
unclear
whether
Syngenta
was
verifying
exposure
at
this
time
or
whether
the
study
was
based
strictly
on
nominal
concentrations
during
the
first
60
days.
Dead
tadpoles
were
partially
degraded,
partially
eaten
and/
or
covered
in
fungus
when
discovered
dead
and
therefore
many
of
them
could
not
be
salvaged
for
later
analysis.
These
results
suggest
poor
tank
conditions
for
supporting
such
rapid
deterioration
of
the
tadpoles.

No
measurements
were
recorded
for
dihydroxytestosterone
or
estradiol
in
the
positive
controls.
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
9
of
36
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
0.14
(
0.07
­
0.23)
0.10
(
0.06
­
0.17)

10

g/
L
10
15.91
(
12.03
­
19.90)
11.76
(
10.21
­
13.65)

25

g/
L
25
27.95
(
24.92
­
31.24)
28.23
(
25.14
_
31.60)

Across
all
treatment
groups,
mortality
averaged
76.5%
(
Table
4).
Mortality
was
reported
to
be
greatest
during
the
first
month
of
the
exposure
period
and
decreased
as
tadpoles
grew
older.
According
to
the
report,
"
mass
mortality
events
occurred
early
in
the
study
and
usually
occurred
within
a
time
span
of
24
hours."
Mortality
rates
declined
after
273
days
when
tadpoles
were
transferred
out
of
static
renewal
into
flow­
through
water
system.
Although
there
was
no
significant
difference
in
mortality
between
atrazine­
treated
and
negative
controls,
there
was
a
difference
between
the
positive
control
treatments
and
the
ethanol
solvent
control;
the
dihydroxytestosterone
group
had
significantly
fewer
deaths.
High
mortality
rates
were
potentially
attributed
to
high
ammonia
levels
in
the
static
renewal
systems.

By
exposure
Day
58,
ammonia
(
NH3)
concentrations
were
between
0.8
­
0.9
mg/
L
and
nitrite
(
NO2)
concentrations
were
as
high
as
3.0
mg/
L
Because
of
the
loss
of
so
many
animals,
hormone
concentrations
were
not
analyzed
as
an
endpoint
in
this
study.

Table
4
.
Average
percent
mortality
for
each
treatment
group
over
506
day
study
period.

Treatment
Average
%
Mortality
Untreated
Controls
79.2
Ethanol
Control
74.8
Dihydroxytestosterone
62.7
17­
 
estradiol
85.7
10

g/
L
73.1
20

g/
L
83.3
The
first
initiation
of
metamorphosis
was
observed
on
exposure
day
99
and
the
first
completion
of
metamorphosis
was
observed
on
day
112.
As
of
day
143,
10
tadpoles
had
completed
metamorphosis.
Between
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
10
of
36
day
143
(
December
7)
and
285
(
April
28),
no
tadpoles
had
initiated
metamorphosis.
Age
at
initiation
and
completion
of
metamorphosis
was
significantly
different
among
the
atrazine­
treated
groups
and
the
untreated
controls;
frogs
treated
with
10

g/
L
atrazine
initiated
and
completed
metamorphosis
at
a
significantly
older
age
compared
to
both
untreated
control
frogs
and
frogs
exposed
to
25

g/
L
(
Table
5).
Frogs
in
the
estradiol
treatment
initiated
metamorphosis
at
a
significantly
younger
age
as
compared
with
both
the
ethanol
control
and
frogs
exposed
to
DHT.

Frogs
treated
with
10

g/
L
atrazine
were
significantly
shorter
(
SVL)
than
frogs
in
the
25

g/
L
atrazine
exposure
group;
however,
there
were
no
significant
differences
in
weight
between
any
of
the
treatment
groups
at
metamorphic
completion
(
Table
6).

The
incidence
of
gross
gonadal
deformities
ranged
from
0
to
5.9%
across
all
treatments
with
size
incongruity
between
gonad
pairs
as
the
most
commonly
observed
anomally.
No
intersex
gonads
(
testicular
and
ovarian
tissue
within
the
same
individual)
were
observed
during
gross
inspections.
In
two
frogs,
gonad
or
gonad
pairs
could
not
be
located
in
both
the
estradiol
and
DHT
treatments.

Sex
ratios
in
the
atrazine
and
untreated
controls
did
not
differ
significantly
from
the
expected
50:
50
male:
female
ratio
(
Table
7).
While
estradiol
and
ethanol
control
sex
ratios
did
not
differ
from
a
ratio
of
50:
50,
the
DHT
treated
animals
were
97.7%
male.
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
11
of
36
Table
5.
Number
of
green
frogs
surviving
to
and
completing
metamorphosis.

Treatment
Initial
N
#
Frogs
Initiating
Metamorphosis
#
Frogs
Completing
Metamorphosis
Untreated
Controls
285
58
44
Ethanol
Controls
280
69
47
Dihydroxytestosterone
291
104
75
17­
 
estradiol
282
40
33
10

g
atrazine/
L
292
77
64
25

g
atrazine/
L
292
48
37
Table
6.
Mean
ages
(
days),
lengths
(
cm),
and
weights
(
g)
at
metamorphosis
for
R.
clamitans.

Treatment
Mean
age
at
initiation
Mean
age
at
completion
Mean
Weight
(
g)
Mean
Length
(
cm)

Untreated
Controls
328.14
336.75
1.76
2.52
Ethanol
Controls
349.99
359.68
1.56
2.47
Dihydroxytestosterone
350.48
368.53
1.50
2.42
17­
 
estradiol
329.73
342.15
1.64
2.57
10

g
atrazine/
L
361.81
376.70
1.48
2.39
25

g
atrazine/
L
335.27
342.14
1.64
2.54
Table
7.
Percent
male
and
female
green
frogs
in
each
treatment.

Treatment
%
Males
%
Females
Untreated
Controls
43.1
56.9
Ethanol
Controls
47.4
50.9
Dihydroxytestosterone
97.7
2.3
17­
 
estradiol
36.8
63.2
10

g
atrazine/
L
40.3
59.7
25

g
atrazine/
L
40.9
59.1
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
12
of
36
C.
REPORTED
STATISTICS:
Kolmogrov­
Smirnov's
One
Sample
test
with
Lillifor's
transformation
was
used
to
assess
whether
or
not
the
data
sets
were
normally
distributed.
When
normally
distributed,
ANOVA
followed
by
Fisher's
LSD
was
used
to
detect
significant
differences
between
treatment
groups.
For
nonnormally
distributed
data,
non­
parametric
Kruskal­
Wallis
test/
Mann­
Whitney
U
Test
was
used.
The
Chisquare
test
was
used
to
detect
differences
in
expected
sex
ratios
and
Pearson's
Chi­
square
was
used
to
test
for
differences
in
the
incidences
of
gross
gonadal
abnormalities.

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

E.
STUDY
DEFICIENCIES:
The
feeding
regime
was
not
reported;
however,
the
animals
were
apparently
fed
frog
brittle.
The
appendix
reports
that
a
previous
analysis
of
the
food
was
"
inconclusive".
It
is
unclear
what
"
inconclusive"
refers
to;
however,
an
analysis
of
the
food
supply
was
apparently
not
run
Atrazine
was
detected
in
the
negative
control.

Water
quality
during
the
static
renewal
phase
of
the
study
was
poor.

F.
REVIEWER'S
COMMENTS:

Although
the
study
was
not
conducted
under
full
GLP,
the
report
notes
that
most
practices
were
included,
one
of
which
involved
writing
a
final
report.
The
current
study
report
does
not
constitute
a
final
report
and
therefore
a
Final
Report
was
not
written.

A
major
problem
in
this
study
is
the
low
survival
rate
which
ranged
from
37­
14%.
Although
the
report
correctly
notes
that
the
rate
of
mortality
decreased
after
the
first
30
days,
it
was
still
substantial.
For
example,
control
mortality
(
as
estimated
from
Figure
1)
at
30
days
was
about
80
individuals.
Mortality
in
the
controls
for
the
remainder
of
the
test
was
about
120
individuals.
This
high
mortality
rate
indicates
severely
inadequate
methods
and
suggests
that
the
study
may
not
be
useful..

The
high
mortality
rates
across
all
treatments
coupled
with
data
showing
high
ammonia/
nitrite
levels
suggest
that
this
study
was
probably
compromised
by
poor
water
quality
caused
by
overcrowding
in
a
static
renewal
system.
Because
only
50%
of
the
water
was
changed
every
72
hours
for
the
first
67
days
of
exposure,
there
is
a
high
potential
for
waste
products
to
accumulate.
.
The
authors
acknowledged
that
high
mortality
was
probably
caused
by
tadpole
overcrowding
in
static
tanks
and
that
poor
water
quality
(
high
ammonia
and
nitrite)
may
have
contributed
to
mortality.
The
authors
also
acknowledged
that
these
factors
may
have
delayed
growth
and
development
of
tadpoles
because
increased
rates
of
development
coincided
with
a
shift
from
static
to
flow­
through
exposure
systems.
Tadpoles
that
underwent
metamorphosis
early
tended
to
come
from
tanks
that
had
experienced
high
mortality
rates
during
the
first
month
of
exposure
and
were
therefore
subject
to
less
crowded
conditions.
Although
the
authors
stated
that
the
differences
in
time
to
complete
metamorphosis
between
treatment
groups
appeared
to
be
a
result
of
tank
effects
on
relative
growth
rates
rather
than
atrazine
treatment;
it
may
be
more
precise
to
conclude
that
tank
effects
obscured
the
study's
ability
to
detect
treatment
effects.
Given
the
confounding
tank
effects,
it
isn't
possible
for
the
authors
to
conclude
that
exposure
to
10
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
13
of
36
and
25

g/
L
atrazine
does
not
consistently
affect
age,
length,
or
weight
of
R.
clamitans
at
metamorphic
completion.

Because
only
about
24%
of
the
tested
organisms
completed
metamorphosis,
and
all
of
the
analyses
were
conducted
on
juvenile
organisms,
the
sampling
strategy
may
have
been
biased
and
did
not
represent
the
population
in
the
test.

While
dihydroxytestosterone­
treated
frogs
were
identified
as
predominately
(
97.7%)
male,
the
estradiol­
treated
frog
sex
ratio
did
not
differ
significantly
from
50%.
It
is
unclear
whether
the
estradiol
treatments,
as
a
positive
control,
should
have
skewed
sex
ratios
in
favor
of
females;
however,
it
is
clear
that
the
"
treatment"
did
not
impact
sex
ratios.
Because
hormone
levels
in
the
positive
control
were
not
measured,
it
is
uncertain
whether
the
lack
of
responsiveness
is
due
to
insufficient
stimulus,
poor
water
quality
issues,
or
insensitivity
of
green
frogs
to
estradiol
treatments
(
i.
e.,
green
frogs
represent
a
poor
species
for
testing
estrogenic
responses).
The
authors
stated
that
green
frogs
are
not
feminized
when
exposed
to
exogenous
estradiol,
but
rather
they
are
masculinized
when
exposed
to
exogenous
androgens
(
e.
g.
DHT)
and
cite
Foote
and
Witschi
1939.
The
fact
that
estradiol
did
not
affect
gonadal
differentiation
is
inconsistent
with
previous
studies,
and
it
is
not
known
if
the
frogs
in
this
study
were
exposed
to
an
efficacious
dose
of
the
hormone.
..
In
another
study
conducted
by
the
same
laboratory,
estradiol
concentrations
in
a
static
renewal
system
were
less
than
10%
of
the
nominal
target
concentration.
As
a
consequence,
they
did
not
observe
the
expected
feminizing
effects
on
X.
laevis.
This
study
with
green
frogs
did
not
analyze
estradiol
concentrations,
but
they
were
certainly
substantially
below
the
target
concentration
given
the
static­
renewal
exposure
regimen
used.

According
to
Hayes
(
1998),
estradiol
treatment
of
R.
clamitans
did
not
affect
sex
ratio
or
produce
mixed
results
(
no
effect
on
sex
ratio
to
effects
favoring
either
males
or
females);
treatment
of
Ranids
with
testosterone
produced
primarily
males.

Green
frogs
are
a
less
studied
experimental
model
than
X.
laevis.
In
X.
laevis,
the
period
of
sensitivity
toward
femininization
is
during
early
prometamorphosis.
This
study
was
conducted
in
a
manner
that
included
the
presumptive
sensitive
period
of
this
species
(
i.
e.,
prometamorphosis).

Apparently
there
was
some
difficulty
in
identifying
the
presence
of
gonads
in
some
of
the
animals,
suggesting
that
the
accuracy
in
detecting
gonadal
anomalies
based
on
visual
examinations
(
gross
morphology)
was
somewhat
limited.

The
overall
hypothesis
tested
was
that
waterborne
concentrations
of
atrazine
would
not
have
an
adverse
effect
on
the
gonads
of
the
green
frog
(
Rana
clamitans)
when
exposed
during
the
critical
phases
of
development.

Based
on
an
analysis
of
the
raw
atrazine
measured
concentration
data
(
see
attached
SAS
©
[
Statistical
Analysis
System,
Release
8.01,
Cary,
North
Carolina]
and
although
only
a
limited
number
of
analyses
are
reported
on
tank
solutions,
the
tank
atrazine
concentrations
ranged
from
116
to
347%
of
mean­
measured
concentrations
in
stock
solutions.
On
average,
mean­
measured
concentrations
(
stock
and
tank
solutions
combined)
contained
0.10
±
0.016

g/
L,
11.76
±
4.87

g/
L
and
28.23
±
8.47

g/
L
in
0,
10
and
25

g/
L
nominal
exposure
groups.
Based
on
analyses
conducted
by
Syngenta,
exposure
solutions
averaged
0.14
±
0.20

g/
L,
15.9
±
6.7

g/
L,
and
27.9
±
8.88

g/
L.
Although
both
sets
of
analyses
tended
to
agree
with
one
another,
theyindicated
that
atrazine
was
present
in
the
dilution
water
control
and
at
levels
that
other
studies
have
shown
to
result
in
gonadal
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
14
of
36
developmental
effects
(
Hayes
et
al.
2002a
and
2002b).

Previous
studies
conducted
by
Hayes
et
al.
2002a,
b
showed
effects
as
low
as
0.1

g/
L;
however,
this
study
only
used
10
and
25

g/
L
exposure
levels.
Also,
Hayes'
studies
suggest
that
the
incidence
of
gonadal
effects
was
higher
at
lower
doses.
The
effect
on
delayed
time
to
metamorphosis
and
smaller
size
of
metamorphs
treated
with
10

g/
L
relative
to
both
controls
and
animals
treated
with
25

g/
L
may
be
reflective
of
a
similar
pattern.

G.
CONCLUSIONS:
Green
frog
(
Rana
clamitans)
tadpoles
reared
from
field­
collected
eggs
were
exposed
for
273
days,
beginning
5­
days
post­
hatch,
to
two
concentrations
(
10
and
25

g/
L)
of
atrazine.
Positive
controls,
dihydroxytestosterone
and
17­
 
estradiol
(
0.1

g/
mL
in
0.005%
ethanol),
a
negative
control
(
water)
and
a
solvent
control
(
0.005%
ethanol)
were
also
run.
Replicates
(
9)
consisted
of
30
free­
swimming
tadpoles
each.
Initially
(
exposure
days
0
­
67)
animals
were
maintained
under
static
renewal
conditions
in
4
L
of
test
solution;
50%
tank
changes
were
conducted
every
72
hours.
From
Day
68
to
Day
273,
tadpoles
were
maintained
in
tanks
containing
16L
of
test
solution
under
static
renewal.
After
273
days
exposures
were
terminated
and
tadpoles
were
maintained
in
a
continuous
flow­
through
10­
L
glass
tanks
housed
in
large
acrylic
tanks
containing
80
L
of
continuously
renewing
freshwater.
At
metamorphosis
(
fore­
limb
emergence)
tadpoles
were
either
housed
individually
or
in
small
groups
in
10­
L
glass
tanks
containing
approximately
500
mL
of
freshwater.
Over
the
study
period,
mortality
across
all
treatment
groups
averaged
76.5%
and
was
attributed
to
poor
water
quality
and
overcrowding
during
the
273
day
static­
renewal
phase
of
the
study.
While
mean­
measured
concentrations
of
atrazine
were
relatively
consistent
with
nominal
values,
measurements
were
made
on
freshly
prepared
stock
solutions;
hence
it
is
unclear
what
atrazine
concentrations
were
present
in
aged
exposure
solutions.
Additionally,
measurable
levels
of
atrazine
were
detected
in
the
negative
controls.
Although
the
concentrations
of
positive
control
hormones
were
not
measured,
the
positive
controls
using
dihydrotestosterone
and
17­
 
estradiol
suggested
that
green
frogs
only
reacted
to
androgenic
chemicals
resulting
in
predominately
(
97.6%)
male
frogs,
while
the
frogs
were
not
affected
by
estradiol.
It
is
uncertain
whether
this
means
that
green
frogs
are
unresponsive
to
estrogenic
chemicals
or
whether
there
was
sufficient
estradiol
in
solution
to
elicit
an
effect.
While
no
intersex
(
testicular
and
ovarian
tissue
in
the
same
animal)
was
observed
in
any
of
the
treatment
groups,
this
observation
was
based
on
gross
morphology
and
there
were
apparently
difficulties
in
discerning
the
presence
of
gonads
at
all
using
this
process.
While
time
to
and
age
at
metamorphosis
and
the
size
of
metamorphs
were
reduced
in
frogs
treated
with
10

g/
L
atrazine,
there
was
no
difference
in
these
same
parameter
for
frogs
treated
with
25

g/
L
atrazine
relative
to
negative
controls.
Although
there
were
no
dosedependent
effects
in
green
frogs
related
to
atrazine
treatment,
only
two
concentrations
were
monitored.

Contrary
to
the
GLP
statement,
this
study
represents
an
interim
report
and
not
a
final
report.

The
high
mortality
indicative
of
poor
water
quality
and
overcrowding
and
the
lack
of
response
to
the
positive
estradiol
control
made
it
difficult
for
the
study
authors
to
test
the
hypothesis
that
atrazine
exposure
was
associated
with
developmental
effects
in
amphibians.
The
study
did
provide
the
authors
with
a
better
appreciation
for
the
conditions
under
which
green
frogs
should
be
housed,
and
it
suggests
that
the
green
frog
may
not
be
adequate
for
examining
the
effects
of
atrazine
on
amphibian
development.
The
high
mortality
indicative
of
poor
water
quality
and
overcrowding
and
the
lack
of
response
to
the
positive
estradiol
controlmake
it
difficult
to
believe
that
this
study
was
a
sensitive
indicator
of
the
potential
effects
of
atrazine
on
green
frogs.
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
15
of
36
H.
REFERENCES:

Foote,
C.
L.
and
E.
Witschi
1939.
Effect
of
sex
hormones
on
the
gonads
of
frog
larvae
(
Rana
clamitans):
sex
inversion
in
females;
stability
in
males.
The
Anatomical
Record
75(
1):
75
­
83.

Hayes,
T.
B.
1998.
Sex
determination
and
primary
sex
differentiation
in
amphibians:
genetic
and
developmental
mechanisms.
Journal
of
Experimental
Zoology
281:
373
­
399.

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.

AVERAGE
MEAN
MEASURED
CONCENTRATION
ACROSS
TREATMENTS
1265
Obs
CONC
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
0
0
30
0.1016
0.16001
157.557
2
10
0
28
11.7594
4.87077
41.420
3
25
0
27
28.2307
8.47165
30.009
PERCENT
(
PERC)
OF
ATRAZINE
IN
TANK
RELATIVE
TO
STOCK
SOLUTIONS
1266
Obs
CONC
_
TYPE_
_
FREQ_
STOCK
TANK
PERC
1
0
0
2
0.0872
0.3029
347.488
2
10
0
2
11.2688
18.1366
160.945
3
25
0
3
27.7255
32.2723
116.399
AVERAGE
SYNGENTA
MEAN
MEASURED
CONCENTRATIONS
OF
ATRAZINE
1267
Obs
CONC
_
TYPE_
_
FREQ_
MEAN2
STD
CV
1
0
0
18
0.1418
0.19528
137.680
2
10
0
10
15.9100
6.68937
42.045
3
25
0
15
27.9091
8.85104
31.714
COMPARISON
OF
MSU
VERSUS
SYNGENTA­
MEASURED
ATRAZINE
CONCENTRATIONS
AND
PERCENTAGE
RELATIVE
1268
Obs
CONC
MEAN
MEAN2
PERC
1
0
0.1016
0.1418
71.604
2
10
11.7594
15.9100
73.912
3
25
28.2307
27.9091
101.152
AVARAGE
PERCENTAGE
OF
MALES
AND
FEMALES
ACROSS
ALL
TREATMENTS
(
ACTUAL
TREATMENTS
NOT
LISTED
1269
Obs
_
TYPE_
_
FREQ_
MALES
FEMALES
STD_
M
STD_
F
1
0
54
0.50648
0.49121
0.31086
0.30996
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
16
of
36
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
17
of
36
AVERAGE
PERCENTAGE
OF
MALES
BY
TREATMENT
GROUP
1052
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
10
0
9
38.9889
14.1583
36.3138
2
25
0
9
36.9388
20.4962
55.4870
3
CONTR
0
9
44.7173
32.7762
73.2965
4
DHT
0
9
98.2194
3.5370
3.6011
5
E2
0
9
35.6803
25.4366
71.2905
6
ETOH
0
9
41.2676
24.8757
60.2791
AVERAGE
PERCENTAGE
OF
FEMALES
BY
TREATMENT
GROUP
1053
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
10
0
9
61.0111
14.1583
23.206
2
25
0
9
63.0612
20.4962
32.502
3
CONTR
0
9
55.2827
32.7762
59.288
4
DHT
0
9
1.7806
3.5370
198.635
5
E2
0
9
64.3197
25.4366
39.547
6
ETOH
0
9
57.3435
24.6788
43.037
AVERAGE
PERCENTAGE
OF
FEMALES
BY
TREATMENT
GROUP
1054
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
10
0
9
0.00000
0.00000
.
2
25
0
9
0.00000
0.00000
.
3
CONTR
0
9
0.00000
0.00000
.
4
DHT
0
9
0.00000
0.00000
.
5
E2
0
9
0.00000
0.00000
.
6
ETOH
0
9
1.38889
3.92837
282.843
AVERAGE
LENGTH
OF
FROGS
BY
GROUP
1055
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
10
0
77
2.38551
0.20537
8.6091
2
25
0
48
2.53903
0.26360
10.3818
3
Control
0
58
2.51361
0.29859
11.8789
4
DHT
0
104
2.41714
0.24883
10.2945
5
E2
0
40
2.56458
0.32778
12.7810
6
ETOH
0
69
2.46785
0.27089
10.9767
AVERAGE
WEIGHT
OF
FROGS
BY
GROUP
1056
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
10
0
77
1.48109
0.34933
23.5857
2
25
0
48
1.64568
0.40205
24.4305
3
Control
0
58
1.75386
0.54678
31.1760
4
DHT
0
104
1.50077
0.45081
30.0385
5
E2
0
40
1.63576
0.51305
31.3650
6
ETOH
0
69
1.56208
0.43097
27.5892
AVERAGE
AGE
OF
FROGS
IN
DAYS
AT
END
OF
STUDY
BY
GROUP
1057
Obs
GROUP
_
TYPE_
_
FREQ_
MEAN
STD
CV
1
10
0
77
376.703
58.6103
15.5587
2
25
0
48
342.135
47.8134
13.9750
3
Control
0
58
336.750
66.0096
19.6019
4
DHT
0
104
368.533
68.7472
18.6543
5
E2
0
40
342.152
26.5496
7.7596
6
ETOH
0
69
359.681
65.3223
18.1612
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
18
of
36
ANALYSIS
OF
VARIANCE
FOR
LENGTH
OF
FROGS
BETWEEN
GROUPS
1058
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
GLM
Procedure
Class
Level
Information
Class
Levels
Values
GROUP
6
10
25
Control
DHT
E2
ETOH
Number
of
observations
153
NOTE:
Due
to
missing
values,
only
135
observations
can
be
used
in
this
analysis.

Dependent
Variable:
LENGTH
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
5
0.90320512
0.18064102
2.47
0.0359
Error
129
9.44285747
0.07320045
Corrected
Total
134
10.34606259
R­
Square
Coeff
Var
Root
MSE
LENGTH
Mean
0.087299
10.85520
0.270556
2.492407
Source
DF
Type
I
SS
Mean
Square
F
Value
Pr
>
F
GROUP
5
0.90320512
0.18064102
2.47
0.0359
Source
DF
Type
III
SS
Mean
Square
F
Value
Pr
>
F
GROUP
5
0.90320512
0.18064102
2.47
0.0359
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
19
of
36
ANALYSIS
OF
VARIANCE
FOR
LENGTH
OF
FROGS
BETWEEN
GROUPS
1060
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
GLM
Procedure
Class
Level
Information
Class
Levels
Values
GROUP
6
10
25
Control
DHT
E2
ETOH
Number
of
observations
194
NOTE:
Due
to
missing
values,
only
162
observations
can
be
used
in
this
analysis.

Dependent
Variable:
LENGTH
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
5
0.43825553
0.08765111
1.37
0.2383
Error
156
9.97802868
0.06396172
Corrected
Total
161
10.41628421
R­
Square
Coeff
Var
Root
MSE
LENGTH
Mean
0.042074
10.40884
0.252907
2.429728
Source
DF
Type
I
SS
Mean
Square
F
Value
Pr
>
F
GROUP
5
0.43825553
0.08765111
1.37
0.2383
Source
DF
Type
III
SS
Mean
Square
F
Value
Pr
>
F
GROUP
5
0.43825553
0.08765111
1.37
0.2383
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
20
of
36
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
LENGTH
1062
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
UNIVARIATE
Procedure
Variable:
Resid
Moments
N
135
Sum
Weights
135
Mean
0
Sum
Observations
0
Std
Deviation
0.26546014
Variance
0.07046909
Skewness
0.22001009
Kurtosis
­
0.3647052
Uncorrected
SS
9.44285747
Corrected
SS
9.44285747
Coeff
Variation
.
Std
Error
Mean
0.02284717
Basic
Statistical
Measures
Location
Variability
Mean
0.00000
Std
Deviation
0.26546
Median
­
0.02028
Variance
0.07047
Mode
­
0.06828
Range
1.36128
Interquartile
Range
0.37773
NOTE:
The
mode
displayed
is
the
smallest
of
5
modes
with
a
count
of
3.

Tests
for
Location:
Mu0=
0
Test
­
Statistic­
­­­­­
p
Value­­­­­­

Student's
t
t
0
Pr
>
|
t|
1.0000
Sign
M
­
2.5
Pr
>=
|
M|
0.7308
Signed
Rank
S
­
117.5
Pr
>=
|
S|
0.7974
Tests
for
Normality
Test
­­
Statistic­­­
­­­­­
p
Value­­­­­­

Shapiro­
Wilk
W
0.989195
Pr
<
W
0.3771
Kolmogorov­
Smirnov
D
0.053022
Pr
>
D
>
0.1500
Cramer­
von
Mises
W­
Sq
0.068716
Pr
>
W­
Sq
>
0.2500
Anderson­
Darling
A­
Sq
0.452489
Pr
>
A­
Sq
>
0.2500
Quantiles
(
Definition
5)

Quantile
Estimate
100%
Max
0.720692
99%
0.631720
95%
0.420692
90%
0.363100
75%
Q3
0.198425
50%
Median
­
0.020280
25%
Q1
­
0.179308
10%
­
0.354575
5%
­
0.382900
1%
­
0.533280
0%
Min
­
0.640591
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
21
of
36
Extreme
Observations
­­­­­­
Lowest­­­­­­
­­­­­­
Highest­­­­­

Value
Obs
Value
Obs
­
0.640591
58
0.453425
28
­
0.533280
84
0.531720
79
­
0.467280
85
0.620692
136
­
0.454900
120
0.631720
87
­
0.388280
98
0.720692
135
Missing
Values
­­­­­
Percent
Of­­­­­
Missing
Missing
Value
Count
All
Obs
Obs
.
18
11.76
100.00
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
LENGTH
1064
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
UNIVARIATE
Procedure
Variable:
Resid
Stem
Leaf
#
Boxplot
7
2
1
|
6
23
2
|
5
3
1
|
4
222235
6
|
3
12222336777
11
|
2
0022223457779
13
+­­­­­+
1
02223355778899
14
|
|
0
12222222245667777
17
|
+
|
­
0
988777766443332222
18
*­­­­­*
­
1
9988887777766654433110
22
+­­­­­+
­
2
88876555320
11
|
­
3
988887775544443
15
|
­
4
75
2
|
­
5
3
1
|
­
6
4
1
|
­­­­+­­­­+­­­­+­­­­+­­
Multiply
Stem.
Leaf
by
10**­
1
Normal
Probability
Plot
0.75+
*
|
*
*++
|
*+++
|
****
|
*****+
|
****+
|
****
0.05+
+****
|
****
|
******
|
****
|
********
|
*
*+++
|
*++++
­
0.65+*++
+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+
­
2
­
1
0
+
1
+
2
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
22
of
36
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
LENGTH
1065
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
UNIVARIATE
Procedure
Variable:
Resid
Moments
N
162
Sum
Weights
162
Mean
0
Sum
Observations
0
Std
Deviation
0.24894846
Variance
0.06197533
Skewness
0.31544092
Kurtosis
0.99990473
Uncorrected
SS
9.97802868
Corrected
SS
9.97802868
Coeff
Variation
.
Std
Error
Mean
0.01955924
Basic
Statistical
Measures
Location
Variability
Mean
0.000000
Std
Deviation
0.24895
Median
0.003363
Variance
0.06198
Mode
0.103363
Range
1.46290
Interquartile
Range
0.32346
Tests
for
Location:
Mu0=
0
Test
­
Statistic­
­­­­­
p
Value­­­­­­

Student's
t
t
0
Pr
>
|
t|
1.0000
Sign
M
2
Pr
>=
|
M|
0.8138
Signed
Rank
S
­
12.5
Pr
>=
|
S|
0.9834
Tests
for
Normality
Test
­­
Statistic­­­
­­­­­
p
Value­­­­­­

Shapiro­
Wilk
W
0.979442
Pr
<
W
0.0164
Kolmogorov­
Smirnov
D
0.037429
Pr
>
D
>
0.1500
Cramer­
von
Mises
W­
Sq
0.038106
Pr
>
W­
Sq
>
0.2500
Anderson­
Darling
A­
Sq
0.389134
Pr
>
A­
Sq
>
0.2500
Quantiles
(
Definition
5)

Quantile
Estimate
100%
Max
0.9033625
99%
0.8605833
95%
0.3669048
90%
0.3000417
75%
Q3
0.1603625
50%
Median
0.0033625
25%
Q1
­
0.1630952
10%
­
0.3090952
5%
­
0.3996375
1%
­
0.5374167
0%
Min
­
0.5595333
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
23
of
36
Extreme
Observations
­­­­­­
Lowest­­­­­­
­­­­­­
Highest­­­­­

Value
Obs
Value
Obs
­
0.559533
39
0.396042
22
­
0.537417
155
0.403363
134
­
0.533095
179
0.648363
121
­
0.513222
63
0.860583
166
­
0.489417
167
0.903363
101
Missing
Values
­­­­­
Percent
Of­­­­­
Missing
Missing
Value
Count
All
Obs
Obs
.
32
16.49
100.00
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
LENGTH
1067
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
UNIVARIATE
Procedure
Variable:
Resid
Stem
Leaf
#
Boxplot
9
0
1
0
8
6
1
0
7
6
5
1
0
5
4
00
2
|
3
000002557778
12
|
2
012235555667788
15
|
1
0000000222334455666678889
25
+­­­­­+
0
00012222335555666778888999
26
*­­+­­*
­
0
999998776654444332222111
24
|
|
­
1
87655433221111000
17
+­­­­­+
­
2
9887755554444332110
19
|
­
3
776543100
9
|
­
4
972000
6
|
­
5
6431
4
|
­­­­+­­­­+­­­­+­­­­+­­­­+­
Multiply
Stem.
Leaf
by
10**­
1
Normal
Probability
Plot
0.95+
*
|
*
|
0.65+
*
+
|
++++
|
+++*
0.35+
*******
|
****
|
*****
0.05+
*****
|
******
|
+***
­
0.25+
*****
|
****
|
+***
­
0.55+*+*+**
+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+
­
2
­
1
0
+
1
+
2
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
24
of
36
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
LENGTH
1068
The
UNIVARIATE
Procedure
Variable:
Resid
Schematic
Plots
|
1
+
|
|
0
|
0
0.8
+
|
|
|
|
|
0
0.6
+
|
|
|
|
|
|
|
0.4
+
|
|
|
|
|
|
|
|
|
|
|
0.2
+
+­­­­­+
|
|
|
|
+­­­­­+
|
|
|
|
|
|
|
|
|
|
0
+
*­­+­­*
*­­+­­*
|
|
|
|
|
|
|
|
|
|
|
|
|
+­­­­­+
­
0.2
+
+­­­­­+
|
|
|
|
|
|
|
|
|
|
­
0.4
+
|
|
|
|
|
|
|
|
|
|
|
­
0.6
+
|
|
|
|
|
­
0.8
+
­­­­­­­­­­­­+­­­­­­­­­­­+­­­­­­­­­­­
SEX
F
M
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
25
of
36
NONPARAMETRIC
COMPARISON
OF
FROG
LENGTH
ACROSS
GROUPS
1069
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
LENGTH
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
10
40
2141.00
2720.0
207.438789
53.525000
25
22
1699.50
1496.0
167.783300
77.250000
Control
25
1923.50
1700.0
176.467412
76.940000
DHT
2
178.50
136.0
54.883145
89.250000
E2
20
1549.00
1360.0
161.384599
77.450000
ETOH
26
1688.50
1768.0
179.142278
64.942308
Average
scores
were
used
for
ties.

Kruskal­
Wallis
Test
Chi­
Square
9.9388
DF
5
Pr
>
Chi­
Square
0.0770
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
LENGTH
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
10
40
14.0
19.851852
2.662548
0.350000
25
22
13.0
10.918519
2.153556
0.590909
Control
25
15.0
12.407407
2.265020
0.600000
DHT
2
1.0
0.992593
0.704444
0.500000
E2
20
11.0
9.925926
2.071427
0.550000
ETOH
26
13.0
12.903704
2.299353
0.500000
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
5.4793
DF
5
Pr
>
Chi­
Square
0.3602
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
26
of
36
NONPARAMETRIC
COMPARISON
OF
FROG
LENGTH
ACROSS
GROUPS
1071
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
LENGTH
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
10
24
1881.00
1956.00
211.993261
78.375000
25
15
1582.00
1222.50
172.974144
105.466667
Control
18
1456.00
1467.00
187.540207
80.888889
DHT
72
5359.50
5868.00
296.527103
74.437500
E2
12
1157.00
978.00
156.283506
96.416667
ETOH
21
1767.50
1711.50
200.445401
84.166667
Average
scores
were
used
for
ties.

Kruskal­
Wallis
Test
Chi­
Square
6.9456
DF
5
Pr
>
Chi­
Square
0.2247
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
LENGTH
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
10
24
12.0
12.00
2.267787
0.500000
25
15
11.0
7.50
1.850382
0.733333
Control
18
8.0
9.00
2.006202
0.444444
DHT
72
31.0
36.00
3.172083
0.430556
E2
12
9.0
6.00
1.671835
0.750000
ETOH
21
10.0
10.50
2.144254
0.476190
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
7.8765
DF
5
Pr
>
Chi­
Square
0.1632
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
27
of
36
ANALYSIS
OF
VARIANCE
FOR
WEIGHT
OF
FROGS
BETWEEN
GROUPS
1073
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
GLM
Procedure
Class
Level
Information
Class
Levels
Values
GROUP
6
10
25
Control
DHT
E2
ETOH
Number
of
observations
153
NOTE:
Due
to
missing
values,
only
134
observations
can
be
used
in
this
analysis.

Dependent
Variable:
WEIGHT
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
5
2.71751620
0.54350324
2.51
0.0330
Error
128
27.66481514
0.21613137
Corrected
Total
133
30.38233134
R­
Square
Coeff
Var
Root
MSE
WEIGHT
Mean
0.089444
28.95896
0.464899
1.605373
Source
DF
Type
I
SS
Mean
Square
F
Value
Pr
>
F
GROUP
5
2.71751620
0.54350324
2.51
0.0330
Source
DF
Type
III
SS
Mean
Square
F
Value
Pr
>
F
GROUP
5
2.71751620
0.54350324
2.51
0.0330
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
28
of
36
ANALYSIS
OF
VARIANCE
FOR
WEIGHT
OF
FROGS
BETWEEN
GROUPS
1075
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
GLM
Procedure
Class
Level
Information
Class
Levels
Values
GROUP
6
10
25
Control
DHT
E2
ETOH
Number
of
observations
194
NOTE:
Due
to
missing
values,
only
162
observations
can
be
used
in
this
analysis.

Dependent
Variable:
WEIGHT
Sum
of
Source
DF
Squares
Mean
Square
F
Value
Pr
>
F
Model
5
0.79520647
0.15904129
0.86
0.5087
Error
156
28.80682131
0.18465911
Corrected
Total
161
29.60202778
R­
Square
Coeff
Var
Root
MSE
WEIGHT
Mean
0.026863
27.92739
0.429720
1.538704
Source
DF
Type
I
SS
Mean
Square
F
Value
Pr
>
F
GROUP
5
0.79520647
0.15904129
0.86
0.5087
Source
DF
Type
III
SS
Mean
Square
F
Value
Pr
>
F
GROUP
5
0.79520647
0.15904129
0.86
0.5087
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
29
of
36
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
WEIGHT
1077
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
UNIVARIATE
Procedure
Variable:
Resid
Moments
N
134
Sum
Weights
134
Mean
0
Sum
Observations
0
Std
Deviation
0.45607689
Variance
0.20800613
Skewness
0.78265338
Kurtosis
0.6746575
Uncorrected
SS
27.6648151
Corrected
SS
27.6648151
Coeff
Variation
.
Std
Error
Mean
0.03939904
Basic
Statistical
Measures
Location
Variability
Mean
0.00000
Std
Deviation
0.45608
Median
­
0.07348
Variance
0.20801
Mode
0.30667
Range
2.42000
Interquartile
Range
0.60333
Tests
for
Location:
Mu0=
0
Test
­
Statistic­
­­­­­
p
Value­­­­­­

Student's
t
t
0
Pr
>
|
t|
1.0000
Sign
M
­
12
Pr
>=
|
M|
0.0465
Signed
Rank
S
­
382
Pr
>=
|
S|
0.3983
Tests
for
Normality
Test
­­
Statistic­­­
­­­­­
p
Value­­­­­­

Shapiro­
Wilk
W
0.962472
Pr
<
W
0.0010
Kolmogorov­
Smirnov
D
0.092468
Pr
>
D
<
0.0100
Cramer­
von
Mises
W­
Sq
0.224995
Pr
>
W­
Sq
<
0.0050
Anderson­
Darling
A­
Sq
1.348236
Pr
>
A­
Sq
<
0.0050
Quantiles
(
Definition
5)

Quantile
Estimate
100%
Max
1.5448000
99%
1.2600000
95%
0.8948000
90%
0.5866667
75%
Q3
0.2700000
50%
Median
­
0.0734848
25%
Q1
­
0.3333333
10%
­
0.4933333
5%
­
0.6261538
1%
­
0.8336364
0%
Min
­
0.8752000
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
30
of
36
Extreme
Observations
­­­­­­
Lowest­­­­­­
­­­­­­
Highest­­­­­

Value
Obs
Value
Obs
­
0.875200
84
0.993846
135
­
0.833636
58
1.114800
79
­
0.785200
98
1.206667
14
­
0.765200
77
1.260000
124
­
0.665200
88
1.544800
87
Missing
Values
­­­­­
Percent
Of­­­­­
Missing
Missing
Value
Count
All
Obs
Obs
.
19
12.42
100.00
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
WEIGHT
1079
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
UNIVARIATE
Procedure
Variable:
Resid
Stem
Leaf
#
Boxplot
15
4
1
0
14
13
12
16
2
0
11
1
1
|
10
|
9
69
2
|
8
79
2
|
7
13
2
|
6
68
2
|
5
13588899
8
|
4
014
3
|
3
111115777
9
|
2
124466678
9
+­­­­­+
1
223369
6
|
|
0
12456789
8
|
+
|
­
0
9997774322111000
16
*­­­­­*
­
1
99765432211
11
|
|
­
2
977666554222100
15
|
|
­
3
887755433331
12
+­­­­­+
­
4
9999655432110
13
|
­
5
733
3
|
­
6
73322
5
|
­
7
97
2
|
­
8
83
2
|
­­­­+­­­­+­­­­+­­­­+
Multiply
Stem.
Leaf
by
10**­
1
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
31
of
36
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
WEIGHT
1080
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
UNIVARIATE
Procedure
Variable:
Resid
Normal
Probability
Plot
1.55+
*
|
|
|
*
*
|
*
+
|
+++
|
**
++
|
**++
|
*++
|
*+
|
***
|
+*
0.35+
***
|
***
|
+**
|
++***
|
+****
|
+***
|
****
|
****
|
*****
|
**+++
|
****+
|
*
*
++
­
0.85+*
*
++
+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+
­
2
­
1
0
+
1
+
2
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
32
of
36
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
WEIGHT
1081
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
UNIVARIATE
Procedure
Variable:
Resid
Moments
N
162
Sum
Weights
162
Mean
0
Sum
Observations
0
Std
Deviation
0.42299451
Variance
0.17892436
Skewness
1.66369399
Kurtosis
5.77981391
Uncorrected
SS
28.8068213
Corrected
SS
28.8068213
Coeff
Variation
.
Std
Error
Mean
0.03323359
Basic
Statistical
Measures
Location
Variability
Mean
0.00000
Std
Deviation
0.42299
Median
­
0.03063
Variance
0.17892
Mode
­
0.22875
Range
2.79958
Interquartile
Range
0.44792
NOTE:
The
mode
displayed
is
the
smallest
of
2
modes
with
a
count
of
4.

Tests
for
Location:
Mu0=
0
Test
­
Statistic­
­­­­­
p
Value­­­­­­

Student's
t
t
0
Pr
>
|
t|
1.0000
Sign
M
­
6
Pr
>=
|
M|
0.3875
Signed
Rank
S
­
649.5
Pr
>=
|
S|
0.2788
Tests
for
Normality
Test
­­
Statistic­­­
­­­­­
p
Value­­­­­­

Shapiro­
Wilk
W
0.882296
Pr
<
W
<
0.0001
Kolmogorov­
Smirnov
D
0.095985
Pr
>
D
<
0.0100
Cramer­
von
Mises
W­
Sq
0.354957
Pr
>
W­
Sq
<
0.0050
Anderson­
Darling
A­
Sq
2.887157
Pr
>
A­
Sq
<
0.0050
Quantiles
(
Definition
5)

Quantile
Estimate
100%
Max
2.051250
99%
1.881250
95%
0.491250
90%
0.377333
75%
Q3
0.211250
50%
Median
­
0.030625
25%
Q1
­
0.236667
10%
­
0.462500
5%
­
0.572667
1%
­
0.744762
0%
Min
­
0.748333
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
33
of
36
Extreme
Observations
­­­­­­
Lowest­­­­­­
­­­­­
Highest­­­­­

Value
Obs
Value
Obs
­
0.748333
167
1.20125
142
­
0.744762
179
1.28750
22
­
0.718750
108
1.40167
166
­
0.688333
155
1.88125
121
­
0.628750
147
2.05125
101
Missing
Values
­­­­­
Percent
Of­­­­­
Missing
Missing
Value
Count
All
Obs
Obs
.
32
16.49
100.00
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
WEIGHT
1083
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
UNIVARIATE
Procedure
Variable:
Resid
Stem
Leaf
#
Boxplot
20
5
1
*
18
8
1
*
16
14
0
1
0
12
09
2
0
10
8
1
0
8
6
1
1
|
4
0223996
7
|
2
0001112333455566779001455577888
31
+­­­­­+
0
012334455567899991122234456689
30
|
+
|
­
0
988776543322220009976665543322210
33
*­­­­­*
­
2
977763220086665443333322221100
30
+­­­­­+
­
4
87755110866633210
17
|
­
6
5429300
7
|
­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­
Multiply
Stem.
Leaf
by
10**­
1
Normal
Probability
Plot
2.1+
*
|
*
|
|
*
|
**
|
*
++
|
+++++
0.7+
++*+
|
++++****
|
+********
|
+******
|
*******
|
*******
|
********+
­
0.7+*
*
***++++
+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+­­­­+
­
2
­
1
0
+
1
+
2
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
34
of
36
PROC
UNIVARIATE
OUTPUT
FOR
RESIDUALS
FROM
GLM
PROCEDURE
USING
WEIGHT
1084
The
UNIVARIATE
Procedure
Variable:
Resid
Schematic
Plots
|
2
+
*
|
*
|
|
1.5
+
0
|
0
|
0
0
|
|
0
1
+
|
|
|
|
|
|
|
|
|
0.5
+
|
|
|
|
|
|
+­­­­­+
+­­­­­+
|
|
|
|
|
0
+
|
+
|
*­­+­­*
|
*­­­­­*
|
|
|
|
|
+­­­­­+
|
+­­­­­+
|
­
0.5
+
|
|
|
|
|
|
|
|
|
|
­
1
+
­­­­­­­­­­­­+­­­­­­­­­­­+­­­­­­­­­­­
SEX
F
M
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
35
of
36
NONPARAMETRIC
COMPARISON
OF
FROG
WEIGHT
ACROSS
GROUPS
1085
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
F
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
WEIGHT
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
10
39
2190.50
2632.50
204.134406
56.166667
25
22
1584.00
1485.00
166.472482
72.000000
Control
25
2031.00
1687.50
175.067430
81.240000
DHT
2
195.00
135.00
54.490911
97.500000
E2
20
1388.00
1350.00
160.136203
69.400000
ETOH
26
1656.50
1755.00
177.713596
63.711538
Average
scores
were
used
for
ties.

Kruskal­
Wallis
Test
Chi­
Square
8.2408
DF
5
Pr
>
Chi­
Square
0.1435
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
WEIGHT
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
10
39
12.0
19.50
2.638993
0.307692
25
22
12.0
11.00
2.152110
0.545455
Control
25
17.0
12.50
2.263223
0.680000
DHT
2
2.0
1.00
0.704443
1.000000
E2
20
11.0
10.00
2.070197
0.550000
ETOH
26
13.0
13.00
2.297432
0.500000
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
11.3060
DF
5
Pr
>
Chi­
Square
0.0456
Data
Evaluation
Report
on
a
Pilot
Study
of
Larval
R.
clamitans
Response
to
Atrazine
Exposure
in
Terms
of
Metamorphosis,
Gonadal
and
Laryngeal
Morphology
and
Selected
Hormonal
and
Enzymatic
Activities.
EPA
MRID
Number
458677­
03
Page
36
of
36
NONPARAMETRIC
COMPARISON
OF
FROG
WEIGHT
ACROSS
GROUPS
1087
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
SEX=
M
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The
NPAR1WAY
Procedure
Wilcoxon
Scores
(
Rank
Sums)
for
Variable
WEIGHT
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
10
24
1944.50
1956.00
212.080099
81.020833
25
15
1551.50
1222.50
173.044998
103.433333
Control
18
1709.00
1467.00
187.617028
94.944444
DHT
72
5186.00
5868.00
296.648568
72.027778
E2
12
1105.00
978.00
156.347523
92.083333
ETOH
21
1707.00
1711.50
200.527508
81.285714
Average
scores
were
used
for
ties.

Kruskal­
Wallis
Test
Chi­
Square
8.3092
DF
5
Pr
>
Chi­
Square
0.1400
Median
Scores
(
Number
of
Points
Above
Median)
for
Variable
WEIGHT
Classified
by
Variable
GROUP
Sum
of
Expected
Std
Dev
Mean
GROUP
N
Scores
Under
H0
Under
H0
Score
âââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââââ
10
24
13.0
12.00
2.267787
0.541667
25
15
10.0
7.50
1.850382
0.666667
Control
18
13.0
9.00
2.006202
0.722222
DHT
72
29.0
36.00
3.172083
0.402778
E2
12
7.0
6.00
1.671835
0.583333
ETOH
21
9.0
10.50
2.144254
0.428571
Average
scores
were
used
for
ties.

Median
One­
Way
Analysis
Chi­
Square
8.8182
DF
5
Pr
>
Chi­
Square
0.1165