Document ID: EPA-HQ-OPP-2003-0024-0032
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-06-03T04:00Z

Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Data
Requirement:
EPA
DP
Barcode
None
EPA
MRID
Not
Assigned
EPA
Guideline
Open
Literature
Test
material:
Purity:
98%
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
29,
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
Frankenberry,
Senior
Statistician
Date:
Environmental
Fate
and
Effects
Division,
ERB
3,
U.
S.
Environmental
Protection
Agency
EPA
PC
Code
080803
Date
Evaluation
Completed:
04/
29/
2003
CITATION:
Hayes,
T.
B.,
K.
Haston,
M.
Tsui,
A.
Hoang,
C.
Haeffele
and
A.
Vonk.
2002.
Atrazine­
induced
hermaphroditism
at
0.1
ppb
in
American
leopard
frogs
(
Rana
pipiens):
laboratory
and
field
evidence.
Environmental
Health
Perspectives
Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Page
2
of
11
EXECUTIVE
SUMMARY:

The
objective
of
this
study
was
to
examine
the
effects
of
atrazine
on
leopard
frogs
(
Rana
pipiens)
under
controlled
laboratory
conditions,
and
once
the
effects
were
identified
to
examine
wild
R.
pipiens
from
a
variety
of
habitats
in
areas
with
reportedly
low
atrazine
use
and
high
atrazine
use.
Water
samples
were
also
collected
at
each
field
site
to
determine
atrazine
exposure.
The
combination
of
both
laboratory
and
field
studies
was
intended
to
address
the
ecological
significance
and
relevance
of
the
initial
laboratory
studies.

In
the
laboratory,
leopard
frog
larvae
were
exposed
from
48­
hrs
post­
hatch
through
complete
tail
resorption
(
NF­
Stage
66)
to
atrazine
at
0.1
and
25

g/
L
in
0.0036%
ethanol
in
10%
Holtfreter's
solution.
Animals
were
sacrificed;
gross
morphology
and
histological
analysis
of
gonads
revealed
that
36%
and
12%
of
the
males
treated
with
atrazine
at
0.1
and
25

g/
L,
respectively,
suffered
form
gonadal
dysgenesis
(
under­
developed
testes
with
poorly
structured,
closed
lobules
and
low
to
absent
germ
cells).
Further,
29%
of
the
0.1

g/
L
and
8%
of
the
25

g/
L
animals
displayed
varying
degrees
of
sex
reversal;
testicular
lobules
of
sex­
reversed
males
contained
oocytes,
and
males
that
metamorphosed
later
contained
large
numbers
of
oocytes.
In
a
few
cases,
testicular
oocytes
were
reported
to
be
vitellogenic,
i.
e.,
contained
yolk.

In
a
field
reconnaissance
survey
of
leopard
frogs
in
four
low
atrazine­
use
and
four
high
atrazine­
use
sites,
testicular
oocytes
were
identified
in
males
from
seven
of
the
eight
collection
sites.
All
sites
with
atrazine
levels
exceeding
0.2

g/
L
had
males
that
displayed
sex­
reversal
similar
to
those
abnormalities
induced
by
atrazine
in
the
lab.
The
highest
incidence
(
92%)
and
most
advanced
cases
of
hermaphroditism
were
observed
in
animals
collected
from
the
North
Platte
River
in
Wyoming
where
atrazine
residues
found
in
water
samples
were
among
the
lowest
recorded.
At
sites
with
similar
atrazine
residues,
the
incidence
of
gonadal
abnormalities
varied
considerably
suggesting
that
there
was
not
a
clear
pattern
of
response.
Even
at
sites
where
no
residues
were
reported,
the
incidence
of
testicular
oogenesis
appeared
to
be
as
high
as
18%.
Additionally,
atrazine
residues
reported
for
each
of
the
sampling
sites
may
not
be
reflective
of
actual
exposure
conditions
during
larval
development.
Although
young
frogs
were
reportedly
sampled,
there
is
no
information
to
support
when
the
animals
may
have
undergone
metamorphosis
relative
to
the
atrazine
residue
analysis.

Although
the
researchers
conducted
pesticide
residue
analysis
for
chemicals
that
were
reportedly
used
in
the
watershed,
the
scope
of
chemical
analyses
was
on
a
site­
by­
site
basis.
As
the
authors
noted,
even
atrazine
was
present
where
it
was
not
used,
and
it
is
likely
that
other
chemical
contaminants
may
have
been
present.
Chemical
profiles
of
the
sites
and
information
on
the
morphoedapic
characteristics
of
the
sites
would
have
shed
light
on
the
comparability
of
the
study
sites.

In
their
paper,
the
study
authors
suggested
that
the
enhanced
response
at
lower
doses
was
consistent
with
low­
dose
effects
reported
for
other
endocrine­
disrupting
chemicals;
however,
the
data
did
not
show
a
clear
dose
response
pattern.
In
a
previous
study
using
Xenopus
(
Hayes
et
al.
2002),
there
appeared
to
be
a
threshold
effect
for
testicular
abnormalities
at
0.1

g/
L
but
the
response
appeared
to
remain
steady
across
increasing
concentrations
of
atrazine
with
16
­
20%
of
the
males
exhibiting
gonadal
abnormalities.
In
the
laboratory
study,
only
two
doses
of
atrazine
were
tested,
and
the
field
study
did
not
indicate
a
clear
trend.
From
these
two
studies,
the
study
authors
concluded
there
is
an
"
inverted­
U"
(
parabolic)
dose
response
curve.
Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Page
3
of
11
I.
MATERIALS
AND
METHODS
GUIDELINE
FOLLOWED:
Nonguideline
Study
COMPLIANCE:
Not
conducted
under
full
Good
Laboratory
Practices
A.
MATERIALS:

1.
Test
Material
Atrazine
Description:
Chemservice,
Chester,
PA
Lot
No./
Batch
No.
:
Not
reported
Purity:
98%
Stability
of
compound
under
test
conditions:
Not
reported
Storage
conditions
of
test
chemicals:
Not
reported
2.
Test
organism:

Species:
American
leopard
frog
(
Rana
pipiens)
.
Age
at
test
initiation:
48­
hrs
post­
hatch
Weight
at
study
initiation:
(
mean
and
range)
not
reported
Length
at
study
initiation:
(
mean
and
range)
not
reported
Source:
Leopard
frogs
[
eggs]
obtained
from
Sensiba
Marsh,
Brown
County,
Wisconsin.

B.
STUDY
DESIGN:

Objective:
To
examine
the
effects
of
atrazine
on
leopard
frogs
(
Rana
pipiens)
under
controlled
laboratory
conditions
and
once
the
effects
were
identified
to
examine
wild
Rana
pipiens
from
a
variety
of
habitats
in
areas
with
reportedly
low
atrazine
use
and
high
atrazine
use.
Water
samples
were
also
collected
at
each
field
site
to
determine
level
of
atrazine.
The
combination
of
both
laboratory
and
field
studies
was
intended
to
address
the
ecological
significance
and
relevance
of
the
initial
laboratory
studies.

1.
Experimental
Conditions
a)
Range­
finding
Study:
Not
reported
b)
Definitive
Study
Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Page
4
of
11
Table
1
.
Experimental
Parameters
Parameter
Details
Acclimation:
period:
Conditions:
(
same
as
test
or
not)
Feeding:
Health:
(
any
mortality
observed)
Leopard
frog
eggs
were
allowed
to
hatch
(
conditions
not
stated)
and
then
apportioned
to
rearing
tanks.

Duration
of
the
test
48­
hrs
post­
hatch
through
Nieuwkoop­
Faber
(
NF)
Stage
66
Test
condition
static/
flow­
through
Type
of
dilution
system
for
flowthrough
method.

Renewal
rate
for
static
renewal
static
renewal
NA
complete
exposure
solution
change
every
72
hours
Aeration,
if
any
exposure
tanks
aerated
Test
vessel
Material:
(
glass/
stainless
steel)
Size:

Fill
volume:
plastic
cages
(
personal
communication,
T.
Hayes,
2002)

4
L
Source
of
dilution
water
quality:
Deionized,
distilled
water
(
personal
communication,
T.
Hayes,
2002)
Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Parameter
Details
Page
5
of
11
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
not
reported
not
reported
not
reported
not
reported
not
reported
not
reported
not
reported
not
reported
not
reported
22oC
not
reported
not
reported
Number
of
replicates/
groups:
negative
control:
0.004%
ethanol
treated
ones:
3
replicates
Number
of
organisms
per
replicate
/
groups:
30
larvae
per
replicate
Biomass
loading
rate
30
larvae/
4
L
Test
concentrations:
nominal:
0.1
and
25

g/
L
Solvent
(
type,
percentage,
if
used)
0.0036%
ethanol
in
10%
Holtfreter's
solution
Holtfreter's
medium:

Lighting
12
hrs
light,
12
hrs
dark
Feeding
Purina
rabbit
chow
ad
libitum
(
Purina
Mills,
St.
Louis,
MO)
Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Parameter
Details
Page
6
of
11
Recovery
of
chemical
Level
of
Quantitation
Level
of
Detection
detection
limit:
0.1

g/
L
Positive
control
{
if
used,
indicate
the
chemical
and
concentrations}
not
reported
Other
parameters,
if
any
2.
Observations:

Table
2:
Observations
Criteria
Details
Parameters
measured
including
the
sublethal
effects/
toxicity
symptoms
mortality,
time
to
metamorphosis,
weight
and
length
at
metamorphosis,
sex
based
on
gross
morphology
(
all
animals)
and
histology
(
9
females
per
treatment
and
on
all
males).

Observation
intervals
not
reported
Were
raw
data
included?
No
Other
observations,
if
any
Field
study
sites
were
initially
selected
based
on
atrazine
sales
data
and
were
located
between
39oN
and
43oN
latitude.
Counties
with
less
than
0.4
kg/
km2
atrazine
use
were
chosen
as
potential
control
sites,
while
areas
with
>
9.3
kg/
km2
atrazine
use
were
chosen
as
potential
atrazine­
exposed
sites.
Sampling
began
in
Utah
on
July
15,
2001
and
moved
eastward.
Sampling
stopped
at
the
Iowa­
Illinois
border
because
R.
pipiens
populations
were
reportedly
low
or
threatened
in
Illinois
and
Indiana.

A
total
of
8
sites
(
4
control
and
4
"
atrazine­
contaminated")
were
sampled
with
100
frogs
collected
at
each
site.
Small
frogs
were
intentionally
collected
in
order
to
sample
newly
metamorphosed
animals.
Animals
were
immediately
euthanized
and
fixed
in
Bouin's
for
48
hours
and
preserved
in
70%
ethanol.

Back
in
the
lab
animals
were
measured,
sex
determined
and
histological
analysis
conducted
on
the
gonads
of
20
males
from
each
site
and
a
subset
of
females
from
each
site.

Water
samples
(
100
ml)
were
collected
at
each
site
in
chemical­
free
glass
jars
(
Fisher
Scientific
Co.,
Houston,
TX)
and
frozen
on
dry
ice
immediately
upon
collection.
Atrazine
levels
were
determined
by
liquid
chromatography/
mass
spectrophotometry
(
PTRL
West,
Inc.,
Hercules,
CA);
duplicate
samples
were
analyzed
using
gas
chromatography
with
a
nitrogen
phosphorous
detectro
(
EPA
method
507;
Hygenic
Laboratory
Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Page
7
of
11
University
of
Iowa,
Iowa
City,
Iowa).
In
addition
to
parent
atrazine,
diaminochlorotriazine
(
DAC),
deisopropylatrazine
(
DIA),
deethylatrazine
(
DEA),
two
other
triazines
(
simazine
and
hexazinone)
and
two
other
herbicides
(
diuron
and
norfluraone)
were
analyzed
at
all
sites.
The
following
pesticides
were
analyzed
for
water
collected
in
Utah,
Wyoming
and
Nebraska::
metolachlor,
alachlor,
glyphosate,
metalaxyl,
nicosulfuron,
propiconazole,
 ­
cyfluthrin,
 ­
cyhalothrin,
and
tebupirimphos.

II.
RESULTS
and
DISCUSSION:

Laboratory
Study
Control
and
atrazine­
treated
animals
were
sexually
differentiated
at
metamorphosis,
but
36%
and
12%
of
the
males
treated
with
atrazine
at
0.1
and
25

g/
L,
respectively,
suffered
form
gonadal
dysgenesis
(
underdeveloped
testes
with
poorly
structured,
closed
lobules
and
low
to
absent
germ
cells.)
Further,
29%
of
the
0.1

g/
L
and
8%
of
the
25

g/
L
animals
displayed
varying
degrees
of
sex
reversal.
Testicular
lobules
of
sexreversed
males
contained
oocytes,
and
males
that
metamorphosed
later
contained
large
numbers
of
oocytes.
Males
that
appear
to
have
undergone
complete
sex
reversal
had
gonads
completely
filled
with
oocytes.
In
2
males,
oocytes
were
vitellogenic
making
the
oocytes
observable
by
gross
morphology.
Control
males
"
never"
contained
testicular
oocytes,
although
2
control
males
contained
2
­
3
degenerating
extragonadal
oocytes
(
not
in
lobule)
and
a
single
male
showed
gonadal
dysgenesis.

Field
Study
None
of
the
collection
sites
were
atrazine­
free
(
Table
3);
however,
atrazine
residues
were
not
reported
at
site
7.
Except
for
metalochlor
at
site
5
(
York
County
NE),
pesticides
were
not
found
at
any
of
the
sites.
Testicular
oocytes
were
identified
in
males
from
7
out
of
8
collection
sites.
All
sites
with
atrazine
levels
exceeding
0.2

g/
L
had
males
that
displayed
sex­
reversal
similar
to
those
abnormalities
induced
by
atrazine
in
the
lab.
The
highest
incidence
(
92%)
and
most
advanced
cases
of
hermaphroditism
were
observed
in
animals
collected
from
the
North
Platte
River
in
Wyoming
(
site
3).
Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Page
8
of
11
Table
3.
Sample
site,
date,
locality,
description
and
atrazine
residues
for
American
leopard
frog
(
Rana
pipien)
collections
(
source:
Hayes
et
al.
2002).

Site
Date
State
County
Altitude
(
m)
Source
Habitat
Atrazine

g/
L
1
7/
15/
01
Utah
Juab
1500
Pond
Grazeland
0.1
2
7/
17/
01
Utah
Cache
1555
Pond
Golf
Course
0.2
3
7/
19/
01
Wyoming
Carbon
1952
river
Wildlife
Area
0.2
4
7/
23/
01
Nebraska
Cherry
1031
Pond
Prarie
0.3
5
7/
22/
01
Nebraska
York
480
Ditch
Corn
Field
0.81
6
7/
26/
01
Iowa
Polk
252
Ditch
Corn
Field
6.7
7
7/
28/
01
Iowa
Polk
246
Marsh
Wildlife
Area
NA
2
8
7/
28/
01
Iowa
Clinton
211
Stream
River
Valley
0.5
1
according
to
report
figure,
this
value
is
more
likely
8.0

g/
L.
2
inconsistent
results,
one
lab
reported
below
level
of
detection
According
to
the
authors,
atrazine
exposure
disrupted
gonadal
development
in
exposed
larvae
as
evidenced
by
poorly
developed
testicular
tubules
and
reduced
germ
cells
(
gonadal
dysgenesis)
and
oocytes
developing
in
testes
(
testicular
oogenesis);
in
a
few
cases,
oocytes
were
vitellogenic.

F.
REVIEWER'S
COMMENTS:

This
study
is
useful
in
identifying
a
potential
hazard
to
amphibians
and
presents
information
on
measurement
endpoints,
such
as
gonadal
deformities.
The
study,
however,
does
not
show
a
clear
dose
response
that
demonstrates
a
causal
relationship
between
atrazine
exposure
and
developmental
effects.
The
study
authors
suggested
that
the
enhanced
response
at
lower
doses
was
consistent
with
low­
dose
effects
reported
for
other
endocrine­
disrupting
chemicals,
but
the
data
did
not
show
a
clear
pattern
of
response.
In
a
previous
study
using
Xenopus
(
Hayes
et
al.
2002),
there
appeared
to
be
a
threshold
effect
for
testicular
abnormalities
at
0.1

g/
L.
The
response,
though,
appeared
to
remain
steady
across
increasing
concentrations
of
atrazine
with
16
­
20%
of
the
males
exhibiting
gonadal
abnormalities.
Additional
information
is
needed
to
support
the
authors
conclusion
that
of
an
"
inverted­
U"
(
parabolic)
dose
response
curve.

Access
to
the
raw
data
supporting
this
study
would
also
help
the
Agency
verify
the
study's
conclusions
regarding
time
to
metamorphosis,
growth,
and
gonadal
development.
Although
the
study
authors
report
that
in
some
cases
animals
exhibited
a
mix
of
testicular
and
ovarian
tissue
and
that
the
oocytes
were
vitellogenic,
Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Page
9
of
11
i.
e.,
contained
yolk,
they
provide
no
evidence
that
any
differential
staining
was
done
to
verify
that
the
oocytes
were
indeed
vitellogenic.
To
better
support
their
argument,
a
stain
capable
of
differentiating
phospholipids
or
glycolipids
should
have
been
used.
The
authors
go
on
to
state
that
induction
of
vitellogenesis
in
males
would
be
consistent
with
upregulation
of
aromatase
resulting
in
the
increased
production
of
endogenous
estrogen.
Upregulation
of
aromatase,
according
to
the
authors
would
also
account
for
the
failure
to
induce
spermatogenesis
(
demasculinization)
and
induction
of
oocyte
growth
(
feminization).
The
authors
note
that
testicular
oogenesis
was
not
observed
in
any
of
the
control
animals
nor
in
any
of
the
R.
pipiens
routinely
studied
in
their
laboratories.

In
this
study,
total
atrazine
residues
(
including
atrazine
degradates)
are
presented
in
graphical
form
and
required
extrapolation.
According
to
this
graph,
parent
atrazine
residues
(
0.2

g/
L)
at
site
3
(
highest
incidence
and
most
advanced
cases
of
hermaphroditism)
were
similar
to
atrazine
residues
(
0.2

g/
L)
at
site
2
(
second
lowest
incidence
of
hermaphroditism.
At
site
4
(
second
highest
incidence
of
testicular
oogenesis),
atrazine
residues
(
0.3

g/
L)
were
relatively
similar
to
site
3;
however
the
atrazine
degradates
DEA,
DIA
and
DAC
resulted
in
total
residues
of
approximately
1.2

g/
L.
The
highest
residues
of
atrazine
and
its
degradates
occurred
at
site
5
[
the
study
reports
residues
at
0.8

g/
L,
but
,
the
figure
suggests
that
the
reported
value
is
actually
8

g/
L].
This
site
had
the
third
highest
rate
of
gonadal
abnormalities,
and
the
abnormalities
were
primarily
gonadal
dysgenesis
rather
than
hermaphroditism.
The
second
highest
site
(
site
6)
for
atrazine
residues
(
6.7

g/
L)
corresponded
to
the
third
lowest
incidence
of
gonadal
abnormalities.
Although
the
study
showed
gonadal
effects
in
frogs,
there
was
no
clear
pattern
of
response.
Atrazine
residues
were
not
reported
at
Site
7
,
and
at
least
one
of
the
two
labs
measuring
atrazine
reported
that
residues
were
below
the
level
of
detection;
however,
testicular
oogenesis
was
reported
in
roughly
18%
of
the
males
examined.

In
its
evaluation,
the
Agency
had
questions
concerning
the
use
of
ethanol
as
a
co­
solvent
Atrazine
concentrations
used
in
the
exposures
are
less
than
the
30
mg/
L
solubility
limit
of
atrazine,
and
a
co­
solvent
should
not
have
been
necessary
.
Previous
studies
conducted
by
the
authors
(
personal
communication:
Tyrone
Hayes
2002)
have
used
dihydrotestosterone
and
17­
 
estradiol
as
positive
controls
and
required
ethanol
as
a
co­
solvent.
The
current
study,
though,
did
not
use
these
steroids,
and
a
co­
solvent
should
not
have
been
needed.

EPA
also
needs
more
specific
information
concerning
the
methods
used
to
determine
the
presence
of
vitillogenic
oocytes.
To
better
support
their
argument,
a
stain
capable
of
differentiating
phospholipids
or
glycolipids
should
be
be
used.
In
this
study,
the
authors
state
that
induction
of
vitellogenesis
in
males
would
be
consistent
with
upregulation
of
aromatase
resulting
in
the
increased
production
of
endogenous
estrogen.
Upregulation
of
aromatase,
according
to
the
authors
would
also
account
for
the
failure
to
induce
spermatogenesis
(
demasculinization)
and
induction
of
oocyte
growth
(
feminization).
At
the
same
time,
the
authors
note
that
testicular
oogenesis
has
not
been
observed
in
any
of
the
control
animals
nor
in
any
of
the
R.
pipiens
routinely
studied
in
their
laboratories.

Small
frogs
were
intentionally
sampled
to
assure
that
animals
had
recently
metamorphosed
implying
that
measured
atrazine
residues
may
have
been
reflective
of
exposure
conditions
during
larval
development.
Additional
information
concerning
the
weights
and
age
of
the
animals
is
needed
to
support
this
contention.
Actual
exposure
conditions
may
have
been
considerably
different
than
those
reported
in
the
study.

Although
the
researchers
conducted
pesticide
residue
analysis
for
chemicals
that
were
reportedly
used
in
the
watershed,
the
scope
of
chemical
analyses
was
on
a
site­
by­
site
basis.
As
the
authors
noted,
even
atrazine
Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Page
10
of
11
Atrazine
Concentration
(
ug/
L)
0
2
4
6
8
Prevalence
of
Male
Gonadal
Abnormalities
(%)

0
20
40
60
80
100
Figure
1.
The
prevalence
of
gonadal
abnormalities
in
seven
sites
plotted
against
measured
atrazine
concentrations.
One
site
is
omitted
because
of
a
lack
of
atrazine
analysis.
Prevalence
data
are
estimated
from
graphical
representations
of
the
data.
This
analysis
also
assumes
that
atrazine
concentrations
at
site
5
are
0.8
ug/
L,
although
figure
11A
of
the
publication
indicates
that
it
was
actually
8.0
ug/
L.
was
present
where
it
was
not
used
,
and
it
is
likely
that
other
chemical
contaminants
may
have
been
present.
Additional
information
on
the
chemical
profile
and
morphoedapic
characteristics
of
the
sites
is
needed
in
order
to
compare
the
study
sites.
The
relationship
of
the
potential
atrazine
exposure
at
the
critical
developmental
stages
is
unknown
because
the
surface
water
sampling
was
conducted
during
the
postmetamorphic
period.
When
the
prevalence
of
male
gonadal
abnormalities
is
plotted
as
a
function
of
atrazine
concentration,
there
is
no
clear
relationship
(
Figure
1).
Additional
data
would
help
clarify
this
problem.

The
Agency
also
needs
clarifying
data
concerning
the
number
of
males
and
females
collected
at
each
site.
Presumably
at
least
20
males
were
analyzed
histologically,
but
it
is
unclear
how
these
organisms
were
selected.
If
they
were
selected
based
on
gross
observations,
then
there
is
a
strong
possibility
that
the
sample
set
was
biased.
Because
a
subset
of
organisms
was
selected
and
prevalence
calculations
were
based
on
this
selection,
the
subset
should
have
been
selected
randomly.

In
the
laboratory
study,
30
organisms
per
replicate
with
3
replicates
were
exposed
per
treatment
level.
The
prevalence
of
gonadal
abnormalities
was
evidently
based
on
combining
the
data
of
the
replicates.
It
would
be
useful
to
understand
the
variance
among
the
replicates
and
whether
responses
in
individual
tanks
are
driving
the
analysis.
The
Agency
also
needs
data
that
indicates
the
distribution
of
males
and
females
in
the
study
and
the
sample
sizes
to
gauge
the
robustness
of
the
analyses.
A
further
note
concerning
the
laboratory
study
is
the
use
of
30
organisms
with
an
estimated
maximal
weight
of
2.5
g
in
4
liters
of
test
solution.
These
conditions
would
result
in
a
loading
rate
of
18.8
g/
L,
which
is
37
times
the
recommended
loading
rate
for
static
renewal
toxicity
tests.

The
highest
prevalence
of
gonadal
abnormalities
was
observed
at
the
site
in
Wyoming.
Is
it
possible
that
there
is
a
genetic
component
to
this
phenomenon?
The
authors
state
that
in
7,000
organisms
collected
from
four
different
states,
no
gonadal
abnormalities
[
of
the
type
described
in
the
study]
have
ever
been
noted.
Given
the
ubiquitous
nature
of
atrazine
contamination
suggested
in
the
paper,
it
is
hard
to
imagine
that
these
were
all
from
atrazine­
free
environments,
particularly
in
Wisconsin.
If
it
is,
in
fact,
true,
then
these
data
should
have
been
published,
as
they
would
have
provided
great
weight
to
the
analysis.
Data
Evaluation
Report
on
Atrazine­
Induced
Hermaphroditism
at
0.1
ppb
in
American
Leopard
Frogs
(
Rana
pipiens):
Laboratory
and
Field
Evidence
EPA
MRID
Number:
None
Page
11
of
11
As
the
study
authors
noted,
the
ecological
relevancy
of
these
findings
is
unclear
because
the
researchers
did
not
have
problems
in
locating
frogs.
Further
information
is
needed
to
determine
if
gonadal
abnormalities
impair
the
reproductive
success,
growth,
and
survival
of
amphibians.

H.
REFERENCES:

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

Nieuwkoop,
P.
D.
and
J.
Faber.
1994.
Normal
table
of
Xenopus
laevis
(
Daudin).
North­
Holland
Publishing
Company,
Amsterdam.