Data
Evaluation
Report
on
Response
of
the
Amphibian
Tadpole
(
Xenopus
laevis)
to
Atrazine
During
Sexual
Differentiation
of
the
Testes
EPA
MRID
Number:
None
Data
Requirement:
:
EPA
DP
Barcode
None
EPA
MRID
Not
Assigned
EPA
Guideline
Open
Literature
Test
material:
Purity:
99%
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
30,
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:
Tavera­
Mendoza,
L.,
S.
Ruby,
P.
Brousseau,
M.
Fournier,
D.
Cyr
and
D.
Marcogliese.
2001.
Response
of
the
amphibian
tadpole
(
Xenopus
laevis)
to
atrazine
during
sexual
differentiation
of
the
testis.
Environmental
Toxicology
and
Chemistry
21:
527
­
531.
Data
Evaluation
Report
on
Response
of
the
Amphibian
Tadpole
(
Xenopus
laevis)
to
Atrazine
During
Sexual
Differentiation
of
the
Testes
EPA
MRID
Number:
None
Page
2
of
8
EXECUTIVE
SUMMARY:

In
an
effort
to
examine
the
effects
of
atrazine
on
gonadal
differentiation
and
reproductive
impairments,
male
African
clawed
frog
larvae
(
Nieuwkoop­
Faber
Stage
56)
were
exposed
to
mean­
measured
concentrations
of
atrazine
at
18

g/
L
(
nominal:
21

g/
L)
under
static
conditions
for
48
hours.
Animals
were
fasted
during
the
48
hours
test.
Total
testicular
volume
decreased
significantly
(
p
=
0.004)
from
0.026
±
0.003
mm3
in
controls
to
0.01
±
0.001
m3
in
atrazine­
treated
tadpoles
representing
a
57%
decrease
after
48
hours
of
exposure.
The
number
of
spermatogonial
cell
nests
decreased
significantly
(
p
<
0.001)
from
an
mean
of
242.4
±
35.7
in
controls
to
72.9
±
21.8
in
atrazine­
exposed
tadpoles
representing
a
70%
reduction.
The
number
of
nursing
cells
declined
significantly
(
p
<
0.001)
from
a
mean
of
9.62
±
0.17
in
controls
to
2.35
±
0.36
in
atrazine
treated
tadpoles.
Testicular
resorption
was
observed
in
70%
of
the
male
tadpoles
exposed
to
atrazine
relative
to
controls;
failure
of
full
development
of
the
testis
(
aplasia)
was
observed
in
10%
of
the
testes
examined.
Histological
examination
of
the
pituitary
suggested
that
tissues
were
actively
secreting
hormones
based
on
the
absence
of
chromophores.

This
study
provides
useful
information
on
hazard
identification
and
measurement
endpoints,
such
as
gonadal
abnormalities.
However,
the
study
does
not
provide
sufficient
information
to
establish
a
dose­
response
relationship
because
only
one
concentration
of
atrazine
was
tested.
Other
information
which
is
needed
to
more
fully
interpret
the
significance
of
this
study
include:
documentation
of
the
measurement
endpoints,
elaboration
on
the
ecological
relevancy
of
the
measurement
endpoints,
and
clarification
of
the
number
of
exposed
animals.
Data
Evaluation
Report
on
Response
of
the
Amphibian
Tadpole
(
Xenopus
laevis)
to
Atrazine
During
Sexual
Differentiation
of
the
Testes
EPA
MRID
Number:
None
Page
3
of
8
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:
99%
(
Sigma
Chemical
Co.,
St.
Louis,
MO)
Stability
of
Compound
Under
Test
Conditions:
Not
reported
Storage
conditions
of
test
chemicals:
Not
reported
2.
Test
organism:

Species:
African
clawed
frog
(
Xenopus
laevis)
.
Age
at
test
initiation:
Niewkoop­
Faber
stage
54
tadpoles
just
prior
to
gonadal
differentiation.
Weight
at
study
initiation:
(
mean
and
range)
not
reported
Length
at
study
initiation:
(
mean
and
range)
not
reported
Source:
Xenopus
1
(
Dexter,
MI,
USA)
B.
STUDY
DESIGN:

Objective:
To
examine
the
effects
of
atrazine
on
gonadal
differentiation
and
reproductive
impairments.

1.
Experimental
Conditions
a)
Range­
finding
Study:
Exposure
concentration
based
on
atrazine
residues
recently
reported
in
St.
Lawrence
Rive
Valley
region
of
Quebec,
CA
b)
Definitive
Study
Data
Evaluation
Report
on
Response
of
the
Amphibian
Tadpole
(
Xenopus
laevis)
to
Atrazine
During
Sexual
Differentiation
of
the
Testes
EPA
MRID
Number:
None
Page
4
of
8
Table
1
.
Experimental
Parameters
Parameter
Details
Acclimation:
period:
conditions:
(
same
as
test
or
not)
Feeding:
Health:
(
any
mortality
observed)
1
week
100­
L
glass
aquaria
containing
15­
L
dechlorinated
Montreal
tap
water
at
21oC
on
a
12:
12
(
light:
dark)
cycle
Fed
specially
prepared
tadpole
diet
(
Boreal,
St.
Catherines,
Ontario,
CA)
twice
per
week
during
acclimation
Duration
of
the
test
48
hours
starting
at
NF
stage
56
Test
condition
static/
flow
through
Type
of
dilution
system­
for
flow
through
method.

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

Fill
volume:
glass
100­
L
15­
L
fill
volume
Source
of
dilution
water
Quality:
City
of
Montreal
(
Canada)
tap
water
Data
Evaluation
Report
on
Response
of
the
Amphibian
Tadpole
(
Xenopus
laevis)
to
Atrazine
During
Sexual
Differentiation
of
the
Testes
EPA
MRID
Number:
None
Parameter
Details
Page
5
of
8
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
pH
=
7.6
not
reported
not
reported
not
reported
not
reported
not
reported
not
reported
not
reported
not
reported
21oC
not
reported
not
reported
Number
of
replicates/
groups:
negative
control:
treated
ones:
2
replicates
per
treatment
Number
of
organisms
per
replicate
/
groups:
16
tadpoles/
replicate
Biomass
loading
rate
16
tadpoles/
15
L
Test
concentrations:
nominal:
0
and
21

g/
L
Solvent
(
type,
percentage,
if
used)
none
Lighting
12
hours
dark,
12
hours
light
Feeding
no
feeding
during
48­
hr
exposure
Recovery
of
chemical
Level
of
Quantitation
Level
of
Detection
Water
samples
collected
at
0
and
48
hours
for
quantitation
by
HPLC/
MS
Positive
control
{
if
used,
indicate
the
chemical
and
concentrations}
Data
Evaluation
Report
on
Response
of
the
Amphibian
Tadpole
(
Xenopus
laevis)
to
Atrazine
During
Sexual
Differentiation
of
the
Testes
EPA
MRID
Number:
None
Parameter
Details
Page
6
of
8
Other
parameters,
if
any
2.
Observations:
Table
2:
Observations
Criteria
Details
Parameters
measured
including
the
sublethal
effects/
toxicity
symptoms
Observation
intervals
Were
raw
data
included?

Other
observations,
if
any
Gonad­
kidney
prepared
for
histological
analysis.
Brain
along
with
attached
pituitary
stained
to
determine
if
cells
in
the
pituitary
were
actively
secreting
hormones.
Index
of
total
testicular
volume
,
estimated
number
of
spermatogonial
cell
nests
and
nurse
cell
integrity
measured
in
each
testis.

For
the
three
testicular
parameters,
data
were
ranked
to
satisfy
the
assumption
of
a
normal
distribution
and
then
subjected
to
one­
way
ANOVA
followed
by
Tukey's
test
(
SPSS10
program;
SPSS,
Chicago,
IL
II.
RESULTS
and
DISCUSSION:

Exposure
measured
at
0
and
48
hours.
Control
values
contained
atrazine
at
<
0.05

g/
L
while
the
nominal
21

g/
L
tank
contained
atrazine
at
18

g/
L.
Therefore,
mean­
measured
atrazine
concentrations
were
roughly
87%
of
nominal.

Total
testicular
volume
decreased
significantly
(
p
=
0.004)
from
0.026
±
0.003
mm3
in
controls
to
0.01
±
0.001
m3
in
atrazine­
treated
tadpoles
representing
a
57%
decrease
after
48
hours
of
exposure.
The
number
of
spermatogonial
cell
nests
decreased
significantly
(
p
<
0.001)
from
an
mean
of
242.4
±
35.7
in
controls
to
72.9
±
21.8
in
atrazine­
exposed
tadpoles
representing
a
70%
reduction.
The
number
of
nursing
cells
declined
significantly
(
p
<
0.001)
from
a
mean
of
9.62
±
0.17
in
controls
to
2.35
±
0.36
in
atrazine
treated
tadpoles.
Testicular
resorption
was
observed
in
70%
of
the
male
tadpoles
exposed
to
atrazine
relative
to
controls;
failure
of
full
development
of
the
testis
(
aplasia)
was
observed
in
10%
of
the
testes
examined.
Data
Evaluation
Report
on
Response
of
the
Amphibian
Tadpole
(
Xenopus
laevis)
to
Atrazine
During
Sexual
Differentiation
of
the
Testes
EPA
MRID
Number:
None
Page
7
of
8
Histological
sections
of
the
pituitary
revealed
only
undifferentiated
chromophobes
in
both
control
and
treated
tadpoles.
No
evidence
was
found
of
chromophobes,
indicating
that
the
pituitary
was
actively
secreting
hormones.

The
study
authors
conclude
that
their
findings
suggest
that
the
developing
germ
cells
in
tadpole
testes
during
testicular
differentiation
are
highly
sensitive
to
atrazine
and
that
because
these
are
primary
sources
of
germ
cells
for
the
life
of
the
organism,
the
effects
would
not
be
transient
and
recovery
could
not
occur.
The
authors
suggest
that
atrazine
may
disrupt
sexual
differentiation
by
altering
testosterone
metabolism
through
upregulation
of
CYP19
and
consequent
increased
activity
of
aromatase.
Increased
aromatase
activity
could
increase
transformation
of
testosterone
to
estrogen.
Alternatively
they
suggest
that
atrazine
could
directly
block
testosterone
and/
or
dihydrotestosterone
at
the
receptor
level.
The
authors
claim
that
the
potential
for
extrapolating
their
results
to
other
vertebrates
is
high
given
how
highly
conserved
testosterone
and
estrogen
receptor
 
ligand
binding
sites
are
as
well
as
the
mRNA
nucleotide
sequence
for
both
5 ­
reductase
and
aromatase.

This
is
a
nonguideline
study
and
does
not
provide
the
level
of
detail
in
terms
of
methodology
and
results
that
EPA
typically
uses
to
evaluate
studies.
The
methodology
section
contains
some
inconsistencies
in
terms
of
the
number
of
animals
used
in
the
study.
This
study
provides
data
on
the
effects
of
atrazine
on
testicular
development;
however,
with
only
a
single
concentration
of
atrazine
tested,
it
isn't
possible
to
characterize
a
dose
response.
The
measurement
endpoints
are
relatively
undocumented
in
Xenopus
laevis
and
it
is
unclear
how
the
effects
discussed
in
this
paper
may
impact
reproductive
capacity,
growth
and/
or
survival
of
affected
animals.
Taken
at
face
value,
the
effects
observed
in
the
testes
after
only
48
hours
of
exposure
are
rather
remarkable.
However,
the
scope
of
this
study
is
quite
limited
and
is
unrepeated,
even
within
this
laboratory.

F.
REVIEWER'S
COMMENTS:

According
to
Nieuwkoop­
Faber,
stage
54
corresponds
to
age
±
26
days
with
a
length
corresponding
to
58
­
65
mm..
Stage
56
corresponds
to
age
±
38
days
with
lengths
ranging
from
70
­
100
mm.

According
to
the
methods
section,
there
are
two
replicates
per
treatment
and
there
are
two
treatments
(
0
and
21

g/
L);
therefore
there
are
4
tanks
in
total
with
16
tadpoles
per
tank.
This
should
yield
64
tadpoles
in
the
study;
however
the
authors
claim
that
the
total
number
of
tadpoles
for
the
experiment
was
48.

According
to
the
study,
the
total
number
of
males
was
24
and
the
distribution
of
males
in
each
of
the
tanks
was
7
and
9
in
the
controls
and
8
and
8
in
the
treated
tanks
(
7+
9+
8+
8=
32
males)
not
24
males.

Study
used
dechlorinated
tap
water.
EPA
recommends
the
use
of
dechlorinated
water
unless
chlorine
residue
analysis
is
conducted
to
verify
that
the
water
is
indeed
dechlorinated.
Additionally,
a
limited
number
(
pH
and
water
temperature)
of
water
quality
parameters
are
reported.

Stock
solution
of
atrazine
prepared
by
dissolving
0.15
g
atrazine
in
1
L
distilled
water
and
ultrasonicating
for
6
hours
in
an
ice
bath.
This
is
relatively
harsh
treatment
for
a
compound.
Why
not
make
up
a
more
dilute
solution?
Stock
solution:
0.1485
mg
a.
i./
mL
(
21
mL)
=
0.3119
mg
a.
i./
15L
=
20.79

g
a.
i./
L
treatment
solution.
Data
Evaluation
Report
on
Response
of
the
Amphibian
Tadpole
(
Xenopus
laevis)
to
Atrazine
During
Sexual
Differentiation
of
the
Testes
EPA
MRID
Number:
None
Page
8
of
8
While
apparently
substantial
declines
in
testicular
volume
(
57%)
and
spermatogonial
cell
nests
(
70%)
occurred
following
exposure
to
atrazine
18

g
a.
i./
L
and
testicular
resorption
was
observed
in
70%
of
the
male
tadpoles
exposed
to
atrazine
relative
to
controls,
no
data
are
provided
on
the
extent
to
which
reproductive
success
would
be
impaired.
With
a
single
dose
of
atrazine
tested,
there
is
no
way
to
determine
whether
the
effect
is
doserelated
Also,
the
methodology
section
contained
some
inconsistencies
on
the
actual
number
of
frogs
used
in
the
study;
the
actual
sample
sizes
are
not
clear.
However,
in
general
while
sample
reported
sizes
were
not
high,
the
treatments
were
replicated.

Taken
at
face
value,
the
effects
observed
in
the
testes
after
only
48
hours
of
exposure
are
rather
remarkable.
However,
the
scope
of
this
study
is
quite
limited
and
is
unreapeated,
even
within
this
laboratory.
Unfortunately,
the
study
does
not
include
measurements
on
the
gonads
of
organisms
prior
to
the
exposure.

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.
