Data
Evaluation
Report
on
Forms
and
Prevalence
of
Intersexuality
and
Effects
of
Environmental
Contaminants
on
Sexuality
in
Cricket
Frogs
(
Acris
crepitans)
EPA
MRID
Number:
None
Data
Requirement:
:
EPA
DP
Barcode
None
EPA
MRID
Not
Assigned
EPA
Guideline
Open
Literature
Test
material:
Purity:
n
o
t
reported
Common
name
Atrazine
chemical
name:
IUPAC
CAS
name
6­
chloro­
N­
ethyl­
N'­(
1­
methylethyl)­
1,3,5­
triazine­
2,4­
diamine
CAS
No.
1912­
24­
9
synonyms
EPA
PC
Code:
80803
Primary
Reviewer:
Thomas
M.
Steeger,
Ph.
D.,
Senior
Biologist
Dat
e
:
May
1,
2003
Environmental
Fate
and
Effects
Division,
ERB
4,
U.
S.
Environmental
Protection
Agency
Secondary
Reviewer(
s):
Joseph
E.
Tietge,
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:
05/
01/
03
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/
01/
2003
CITATION:
Reeder,
A.
L.,
G.
L.
Foley,
D.
K.
Nichols,
L.
G.
Hansen,
B.
Wikoff,
S.
Faeh,
J.
Eisold,
M.
B.
Wheeler,
R.
Warner,
J.
E.
Murphy,
and
V.
R.
Beasley
1998.
Forms
and
prevalence
of
intersexuality
and
effects
of
environmental
contaminants
on
sexuality
in
cricket
frogs
(
Acris
crepitans).
Environmental
Health
Perspectives
106
(
5):
Data
Evaluation
Report
on
Forms
and
Prevalence
of
Intersexuality
and
Effects
of
Environmental
Contaminants
on
Sexuality
in
Cricket
Frogs
(
Acris
crepitans)
EPA
MRID
Number:
None
Page
2
of
10
EXECUTIVE
SUMMARY:

In
an
effort
to
assess
the
prevalence
of
gonadal
intersexuality
in
adult
and
juvenile
cricket
frogs
(
Acris
crepitans)
and
to
determine
whether
sexuality
is
altered
in
response
to
exposure
to
environmental
contaminants,
cricket
frogs
were
collected
over
a
three
year
period
(
1993
­
1995)
in
various
locations
throughout
the
state
of
Illinois.
Additionally,
water/
sediment
samples
were
collected
at
sampling
sites
in
1994
and
1995
to
determine
whether
the
prevalence
of
intersex
could
be
related
to
chemical
residues.
In
a
separate
study,
cricket
frogs
were
also
collected
at
a
site
known
to
be
contaminated
with
PCBs
and
PCDFs
and
the
prevalence
of
intersex
was
determined
relative
to
control
sites.

Of
the
55
adult
and
juvenile
male
and
female
frogs
collected
in
1993,
2
(
3.6%)
had
both
an
ovary
and
testis.
In
the
testis
of
one,
spermatogenesis
was
normal;
in
the
other,
an
immature
ovary
was
present
as
well
as
a
testis
with
no
active
spermatogenesis.
Of
the
243
frogs
examined
in
1994,
6
(
2.5%)
contained
both
ovary
and
testis;
five
of
the
affected
animals
had
areas
of
normal
spermatogenesis
in
the
testis
interspersed
with
oocytes.
One
animal
had
a
mature
ovary
and
mature
testis
with
normal
spermatogenesis.
Of
the
43
frogs
examined
in
1995,
only
one
(
2.3%)
had
an
ovotestis.
Across
all
three
sampling
years
the
prevalence
of
intersex
was
2.8%.
In
specimens
with
an
ovary
on
one
side
and
an
testis
on
the
other,
ovarian
size
ranged
form
well­
developed
mature
female
to
extremely
small
with
a
few
oocytes
present.

Of
the
five
sites
where
intersex
was
found,
four
had
detectable
atrazine.
Of
the
four
sites
where
no
intersex
was
observed,
only
one
contained
detectable
levels
of
atrazine.
According
to
the
authors
the
relationship
between
detection
of
atrazine
and
prevalence
of
intersex
"
approached
significance"
(
P
=
0.07).
At
one
site
treated
with
copper
sulfate
in
1994,
1
frog
of
33
collected
had
an
ovotestis.
In
1995,
no
relationship
between
the
detection
of
atrazine
and
the
prevalence
of
intersex.
No
intersex
was
identified
in
frogs
collected
from
a
pond
treated
with
endothall
in
1995.
Lead
residues
measured
in
1994
and
1995
were
not
associated
with
the
prevalence
of
intersex.

Of
the
frogs
collected
from
PCB
and
control
sites,
only
1
frog
with
an
ovotestis
was
identified
from
the
control.
Sex
ratios
differed
significantly
(
probability
not
given)
between
contaminated
and
control
sites.
In
13
juveniles
from
control
and
13
from
contaminated
sites,
gonadal
tissue
was
immature
and
could
not
be
identified
for
histological
preparation.
According
to
the
authors,
the
association
between
sex
ratios
of
PCB/
PCDF
contaminated
and
control
groups
revealed
a
significant
difference
(
p
=
0.0007).

While
a
wide
range
of
chemical
residue
analyses
were
conducted,
no
data
are
provided
on
the
results
of
these
analyses.
It
is
not
clear
why
the
study
focuses
primarily
on
atrazine;
however,
the
authors
suggest
that
there
may
be
a
trend
between
atrazine
residues
and
the
proportion
of
animals
exhibiting
intersex.
The
only
statistically
significant
relationship
though
was
between
sex
ratios
in
PCB/
PCDF
contaminated
sites
relative
to
controls;
however,
the
sample
size
for
making
this
determination
was
low
(
n=
4).

In
this
paper,
Reeder
et
al.
(
1998)
discussed
the
range
of
chemical
residues
in
the
field
collection
sites
and
how
these
chemicals,
combined
with
environmental
conditions,
could
impact
gonadal
development.
These
factors
contributed
to
the
limited
utility
of
this
study
because
the
investigation
did
not
demonstrate
a
significant
effect
of
chemical
residues
on
the
prevalence
of
intersex
in
cricket
frogs.
This
study
underscored
the
need
to
have
focused
study
designs
with
sufficient
power
in
terms
of
sample
size
to
discriminate
effects
if
they
exist.
Also,
the
report
acknowledged
that
little
is
known
about
natural
intersex
rates
in
cricket
frogs.
Without
a
better
Data
Evaluation
Report
on
Forms
and
Prevalence
of
Intersexuality
and
Effects
of
Environmental
Contaminants
on
Sexuality
in
Cricket
Frogs
(
Acris
crepitans)
EPA
MRID
Number:
None
Page
3
of
10
understanding
of
the
biology
of
the
cricket
frog
and
the
toxicological
phenomenon
being
examined,
it
is
difficult
to
interpret
the
significance
of
the
reported
observations.

I.
MATERIALS
AND
METHODS
GUIDELINE
FOLLOWED:
Nonguideline
Study
COMPLIANCE:
Not
conducted
under
full
Good
Laboratory
Practices
A.
MATERIALS:

1.
Test
Material
Atrazine
Description:

Lot
No./
Batch
No.
:
Not
reported
Purity:
Stability
of
Compound
Under
Test
Conditions:
Not
reported
Storage
conditions
of
test
chemicals:
Not
reported
2.
Test
organism:

Species:
Cricket
frogs
(
Acris
crepitans)
.

Age
at
test
initiation:
Weight
at
study
initiation:
(
mean
and
range)
not
reported
Length
at
study
initiation:
(
mean
and
range)
not
reported
Source:
Field­
collected
along
pond
banks
in
sites
distributed
across
Illinois
B.
STUDY
DESIGN:

Objective:
to
assess
the
prevalence
of
gonadal
intersexuality
in
adult
and
juvenile
cricket
frogs
(
Acris
crepitans)
and
to
determine
whether
sexuality
is
altered
in
response
to
exposure
to
environmental
contaminants
Data
Evaluation
Report
on
Forms
and
Prevalence
of
Intersexuality
and
Effects
of
Environmental
Contaminants
on
Sexuality
in
Cricket
Frogs
(
Acris
crepitans)
EPA
MRID
Number:
None
Page
4
of
10
1.
Experimental
Conditions
a)
Range­
finding
Study:
In
1993,
a
pilot
study
focused
on
pond
site
identification
and
preliminary
assessment
of
histologic
lesions
in
Illinois
cricket
frogs,
20
sites
selected.

b)
Definitive
Study
In
1994
and
1995
frogs
were
collected
from
specific
sites
based
on
suitable
habitat
and
presence
of
cricket
frogs;
ponds
were
of
comparable
size
and
were
distributed
across
the
state
of
Illinois.
Table
1
.
Experimental
Parameters
Parameter
Details
Acclimation:
period:
conditions:
(
same
as
test
or
not)
Feeding:
Health:
(
any
mortality
observed)
NA
Duration
of
the
test
NA
Test
condition
static/
flow
through
Type
of
dilution
system­
for
flow
through
method.

Renewal
rate
for
static
renewal
NA
Aeration,
if
any
NA
Test
vessel
Material:
(
glass/
stainless
steel)
Size:

Fill
volume:
NA
Source
of
dilution
water
Quality:
NA
Data
Evaluation
Report
on
Forms
and
Prevalence
of
Intersexuality
and
Effects
of
Environmental
Contaminants
on
Sexuality
in
Cricket
Frogs
(
Acris
crepitans)
EPA
MRID
Number:
None
Parameter
Details
Page
5
of
10
Water
parameters:
Hardness
pH
Dissolved
oxygen
Total
Organic
carbon
Particulate
Matter
Ammonia
Nitrite
Metals
Pesticides
Chlorine
Temperature
Salinity
Intervals
of
water
quality
measurement
NA
Number
of
replicates/
groups:
negative
control:
0.004%
ethanol
treated
ones:
NA
Number
of
organisms
per
replicate
/
groups:
In
1993,
gonads
of
50
intact
cricket
frogs
from
18
sites.
In
1994,
gonads
of
242
frogs
examined
from
8
sites
(
24
­
39
frogs
from
the
various
sites).
In
1995,
gonads
of
40
frogs
examined
from
7
sites
(
4
­
7
frogs
per
site).

Biomass
loading
rate
NA
Test
concentrations:
nominal:
NA
Solvent
(
type,
percentage,
if
used)
NA
Lighting
NA
Feeding
NA
Data
Evaluation
Report
on
Forms
and
Prevalence
of
Intersexuality
and
Effects
of
Environmental
Contaminants
on
Sexuality
in
Cricket
Frogs
(
Acris
crepitans)
EPA
MRID
Number:
None
Parameter
Details
Page
6
of
10
Recovery
of
chemical
Level
of
Quantitation
Level
of
Detection
NA
Positive
control
{
if
used,
indicate
the
chemical
and
concentrations}
NA
Other
parameters,
if
any
NA
2.
Observations:
Table
2:
Observations
Criteria
Details
Parameters
measured
including
the
sublethal
effects/
toxicity
symptoms
prevalence
of
gonadal
abnormalities
in
terms
of
testicular
oocytes.

Observation
intervals
NA
Were
raw
data
included?
No
Other
observations,
if
any
Study
A
In
1994,
eight
sites
with
similar
size
studied
(
5
in
south,
2
in
central
and
1
in
northeast
Illinois).
In
1995,
seven
of
the
ponds
studied
in
1994
and
one
pond
in
the
agricultural
area
of
east­
central
Illinois,
studied.

In
1993,
gonads
of
50
intact
cricket
frogs
from
18
sites
in
east­
central
and
southern
Illinois
examined
for
gross
morphology.
In
1994,
gonads
of
242
frogs
examined
from
8
sites
(
24
­
39
frogs
from
the
various
sites).
In
1995,
gonads
of
40
frogs
examined
from
7
sites
(
4
­
7
frogs
per
site).

Animals
from
1993
subject
to
whole
body
histologic
sections
while
samples
from
1994
and
1995
were
subject
to
gonad
histology.

Composite
water
and
sediment
samples
collected
in
acetone­
rinsed
containers
from
multiple
locations
in
each
pond
studied
in
1994
and
1995.
Samples
collected
at
the
end
of
May
and
again
in
late
Data
Evaluation
Report
on
Forms
and
Prevalence
of
Intersexuality
and
Effects
of
Environmental
Contaminants
on
Sexuality
in
Cricket
Frogs
(
Acris
crepitans)
EPA
MRID
Number:
None
Page
7
of
10
July/
early
August
in
1994
and
at
the
end
of
May
in
1995.
Additional
water
samples
were
collected
at
sites
11
and
12
in
June
1998
following
an
aquatic
plant
kill.
Water
analyzed
(
State
of
Illinois
Animal
Disease
Laboratory,
Centralia,
IL)
for
herbicides,
fungicides,
insecticides,
PCBs
and
metals
(
Table
1;
source:
Reeder
et
al.
1998).

Study
B
In
mid­
July
1995,
16
juvenile
cricket
frogs
collected
from
three
sites
in
Crab
Orchard
National
Wildlife
Refuge
in
southern
Illinois
where
point
contamination
with
PCBs,
PCDFs,
and
PCDDs
had
previously
occurred.
On
the
same
sampling
days,
16
juvenile
frogs
also
collected
from
each
of
three
control
sites
located
outside
the
area
of
PCB
and
PCDF
contamination
but
within
a
16­
km
radius
of
the
Crab
Orchard
site.

Fisher's
Exact
Test
used
to
compare
prevalence
of
intersex
gonads
and
detection
of
atrazine
at
those
sites.
Fisher's
Exact
test
also
used
to
compare
sex
ratios
among
control
and
PCB/
PCDF
sites
using
SigmaStat
software.

II.
RESULTS
and
DISCUSSION:

Study
A
Although
cricket
frog
testicular
tissue
is
typically
heavily
pigmented,
about
20%
of
males
had
testes
with
reduced
or
absent
pigmentation
particularly
on
the
right
side;
however,
normal
spermatogenesis
was
observed
in
both
pigmented
and
nonpigmented
testes.
Mature
females
contained
large
egg
mass
with
bipolar
eggs
(
tan/
black
coloring).

Of
the
55
adult
and
juvenile
male
and
female
frogs
collected
in
1993,
2
(
3.6%)
had
both
an
ovary
and
testis.
In
the
testis
of
one,
spermatogenesis
was
normal;
in
the
other,
an
immature
ovary
was
present
as
well
as
a
testis
with
no
active
spermatogenesis.
Of
the
243
frogs
examined
in
1994,
6
(
2.5%)
contained
both
ovary
and
testis;
five
of
the
affected
animals
had
areas
of
normal
spermatogenesis
in
the
testis
interspersed
with
oocytes.
One
animal
had
a
mature
ovary
and
mature
Data
Evaluation
Report
on
Forms
and
Prevalence
of
Intersexuality
and
Effects
of
Environmental
Contaminants
on
Sexuality
in
Cricket
Frogs
(
Acris
crepitans)
EPA
MRID
Number:
None
Page
8
of
10
testis
with
normal
spermatogenesis.
Of
the
43
frogs
examined
in
1995,
only
one
(
2.3%)
had
an
ovotestis.
Across
all
three
sampling
years,
the
prevalence
of
intersex
was
2.8%
(
report
claims
2.6%).
In
specimens
with
an
ovary
on
one
side
and
an
testis
on
the
other,
ovarian
size
ranged
form
welldeveloped
mature
female
to
extremely
small
with
a
few
oocytes
present.

Of
the
five
sites
where
intersex
was
found,
four
had
detectable
atrazine.
Of
the
four
sites
where
no
intersex
was
observed,
only
one
contained
detectable
levels
of
atrazine.
According
to
the
authors
the
relationship
between
detection
of
atrazine
and
prevalence
of
intersex
"
approached
significance"
(
P
=
0.07).
At
one
site
treated
with
copper
sulfate
in
1994,
1
frog
of
33
collected
had
an
ovotestis.
In
1995,
no
relationship
between
the
detection
of
atrazine
and
the
prevalence
of
intersex.
No
intersex
was
identified
in
frogs
collected
from
a
pond
treated
with
endothall
in
1995.
Concentrations
of
lead
from
both
years
not
associated
with
intersex
either.

Study
B
Of
the
frogs
collected
from
PCB
and
control
sites,
only
1
frog
with
an
ovotestis
was
identified
from
the
control.
Sex
ratios
differed
significantly
(
probability
not
given)
between
contaminated
and
control
sites.
In
13
juveniles
from
control
and
13
from
contaminated
sites,
gonadal
tissue
was
immature
and
could
not
be
identified
for
histological
preparation.
According
to
the
authors,
the
association
between
sex
ratios
of
PCB/
PCDF
contaminated
and
control
groups
revealed
a
significant
difference
(
p
=
0.0007).
.

F.
REVIEWER'S
COMMENTS:

It
is
not
clear
what
was
driving
the
sampling
design
used
in
this
study.
Sample
size
varied
considerably
between
years:
1993
(
55
frogs),
1994
(
243
frogs)
and
1995
(
43
frogs).

There
are
several
discrepancies
between
the
tables
and
the
text.
In
discussing
the
prevalence
of
intersex
relative
to
atrazine,
the
authors
refer
to
5
sites
with
residues
and
4
sites
without
residues
in
the
1994
sampling;
however,
according
to
the
methods
section
only
8
sites
(
not
9)
were
sampled
in
1994.

In
study
A,
a
wide
range
of
chemicals
were
analyzed
in
both
water
and
sediment
at
each
of
the
collection
sites
in
1994
and
1995;
however,
the
results
section
does
not
discuss
the
outcome
of
these
analyses
but
rather
focuses
primarily
on
atrazine
thus
implying
that
it
was
either
the
only
chemical
were
residues
were
detected
or
that
it
was
the
only
chemical
residues
where
the
researchers
could
Data
Evaluation
Report
on
Forms
and
Prevalence
of
Intersexuality
and
Effects
of
Environmental
Contaminants
on
Sexuality
in
Cricket
Frogs
(
Acris
crepitans)
EPA
MRID
Number:
None
Page
9
of
10
identify
a
significant
relationship.
Since
no
data
are
provided
on
residue
levels,
it
isn't
possible
for
the
Agency
to
verify
whether
a
correlation
existed
between
atrazine
residues
and
the
prevalence
of
intersex;
however,
the
results
section
suggests
that
if
a
relationship
did
exist
in
1994,
it
was
transient
since
no
relationship
existed
in
1995.
Further,
it
isn't
clear
whether
the
lack
of
a
"
relationship"
between
atrazine
residues
in
1995
and
atrazine
residues
was
a
result
of
only
one
intersex
animal
being
found
in
spite
of
atrazine
residues
or
whether
there
were
no
detectable
atrazine
residues.
The
article
implies
that
the
authors
attempted
to
correlate
atrazine
residues
only
to
cases
where
intersex
was
determined.
If
this
is
the
case
then
the
analysis
is
somewhat
biased
since
zero
prevalence
would
not
be
captured.
Atrazine
residues
at
each
sites
should
have
been
compared
to
the
rate
of
intersex
at
each
site;
however,
it
doesn't
appear
that
the
study
was
designed
to
anticipate
such
an
analysis.
The
utility
of
calculating
an
overall
prevalence
of
abnormalities
seems
unsound,
as
it
unnecessarily
combines
data
from
sites
identified
as
contaminated
or
control.

In
study
B,
the
methods
section
suggests
that
they
collected
16
frogs
from
control
and
from
contaminated
sites.
The
results
section
states
that
the
gonads
of
13
frogs
from
each
location
were
too
immature
to
determine
sex.
This
implies
that
over
80%
of
the
animals
were
too
immature
to
determine
sex.
Therefore,
the
effective
sample
size
for
determining
sex
ratio
differences
was
low
with
three
animals
in
each
group.

Presentation
of
the
analytical
chemistry
data
is
not
clear...
are
blank
entries
indicative
of
no
analyses
or
no
measurable
quantities?
The
authors
note
the
variety
of
chemical
residues
measured
in
the
field
studies
and
how
varying
levels
of
response
to
the
wide
range
of
chemicals
might
influence
the
frog's
sensitivity
to
the
chemicals.
Classifying
a
site
categorically
as
atrazine
contaminated
based
on
the
sampling
plan
is
misleading.
The
real
question
is:
what
were
the
atrazine
concentrations
during
the
developmental
stage
of
interest?
Given
this
lack
of
information,
it
is
difficult
to
accept
the
correlational
association
of
gonadal
abnormalities
and
atrazine
presence,
as
it
has
no
basis
in
the
biology
nor
toxicology.
Additionally
the
authors
note
how
environmental
factors
such
as
temperature
may
impact
gonadal
development.
Given
the
number
of
potentially
confounding
effects,
it
is
difficult
to
place
much
weight
on
the
statistical
relationships
developed
in
what
amounts
to
a
reconnaissance
survey.
These
factors
contribute
to
the
limited
utility
of
this
study
since
the
study
does
not
demonstrate
a
significant
affect
of
chemical
residues
on
the
prevalence
of
intersex
in
cricket
frogs.
The
study
underscores
to
need
to
have
focused
study
designs
with
sufficient
power
in
terms
of
sample
size
to
discriminate
effects
should
they
exist.
Data
Evaluation
Report
on
Forms
and
Prevalence
of
Intersexuality
and
Effects
of
Environmental
Contaminants
on
Sexuality
in
Cricket
Frogs
(
Acris
crepitans)
EPA
MRID
Number:
None
Page
10
of
10
The
report
admits
in
the
introduction
that
virtually
nothing
is
known
about
intersex
conditions
in
cricket
frogs.
Perhaps
cricket
frogs
exhibit
ovotestes
normally
at
some
low
level
and
the
presence
and
absence
of
this
condition
at
any
one
site
is
a
function
of
small
sample
sizes.
In
general,
this
study
is
another
one
that
puts
the
"
cart
ahead
of
the
horse."
That
is,
they
conducted
a
field
study,
replete
with
complications
and
uncertainties
without
having
a
firm
understanding
of
either
the
biology
of
the
organism
that
they
were
studying
or
the
toxicological
phenomenon
that
they
attempted
to
study.
It
clearly
does
not
contribute
to
the
understanding
of
whether
or
not
atrazine
poses
any
reproductive
risk
to
anurans.
