Document ID: EPA-HQ-OPP-2003-0024-0033
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-06-03T04:00Z

Data
Evaluation
Report
on
Hermaphroditic,
Demasculinized
Frogs
After
Exposure
to
the
Herbicide
Atrazine
at
Low
Ecologically
Relevant
Doses
EPA
MRID
Number:
None
Data
Requirement:
:
EPA
DP
Barcode
None
EPA
MRID
Not
Assigned
EPA
Guideline
Open
Literature
Test
material:
Purity:
not
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
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/
30/
2003
CITATION:
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.
Data
Evaluation
Report
on
Hermaphroditic,
Demasculinized
Frogs
After
Exposure
to
the
Herbicide
Atrazine
at
Low
Ecologically
Relevant
Doses
EPA
MRID
Number:
None
Page
2
of
9
EXECUTIVE
SUMMARY:

The
objective
of
this
study
was
to
test
the
hypothesis
that
atrazine,
at
ecologically
relevant
low
doses,
may
interfere
with
metamorphosis
and
sex
differentiation
via
endocrine­
disrupting
mechanisms.
African
clawed
frogs
(
Xenopus
laevis)
were
exposed
to
atrazine
concentrations
ranging
from
0.01
to
200

g/
L
from
96­
hr
post­
hatch
through
complete
tail
resorption
(
Nieuwkoop­
Faber
Stage
66).
At
the
end
of
the
exposure
period,
animal
growth
(
length
and
weight),
time
to
metamorphosis,
gonadal
abnormalities
and
size
(
cross­
sectional
diameter)
of
the
larynx
dilator
muscle
were
recorded.
Exposure
to
atrazine
concentrations

0.1

g/
L
resulted
in
gonadal
abnormalities
in
16
­
20%
of
the
animals
at
all
doses
tested
except
for
0.1
ug/
L.
Abnormalities
included
multiple
gonads
or
hermaphrodites
(
multiple
testes
and
ovaries
in
the
same
animal).
These
abnormalities
were
not
observed
in
the
controls.
In
general,
males
typically
exhibited
larger
diameter
laryngeal
muscle
diameters
than
females.
In
this
study,
though,
atrazine
at
concentrations
as
low
as
1

g/
L
(
1
ppb),
significantly
decrease
the
proportion
of
males
that
were
at
or
above
the
mean
laryngeal
size
in
the
control
males.
According
to
the
authors,
these
results
suggested
a
threshold
effect
at

1

g/
L
in
which
80%
of
the
exposed
males
exhibited
larynx
sizes
below
the
average.
The
authors
hypothesized
that
the
co­
occurrence
of
oocytes
and
testicular
tissue
(
hermaphroditism)
and
the
decreased
male
larynx
muscle
size
(
demasculinizing)
were
consistent
with
increased
endogenous
estrogen
concentrations
and
that
one
possible
mechanism
would
be
through
increased
aromatase
activity.
In
support
of
their
hypothesis,
the
researchers
demonstrated
that
adult
male
Xenopus
exposed
to
atrazine
at
25

g/
L
showed
significantly
reduced
plasma
testosterone.

This
study
states
that
the
results
have
been
repeatedly
verified,
but
additional
data
have
not
yet
been
provided
in
the
open
literature
or
submitted
to
EPA.
It
would
be
helpful
if
supporting
studies
were
available
to
verify
the
results
of
this
specific
study.
In
addition,
more
information
on
the
measurement
endpoints,
effects
in
the
field,
percentage
of
males
and
females
in
the
study,
numbers
of
frogs
exhibiting
abnormalities,
and
sensitivity
of
Xenopus
laevis
compared
to
North
American
frogs
would
be
useful.
Also,
the
lack
of
a
dose­
response
relative
to
the
phenomenon
of
hermaphroditism
makes
it
difficult
to
interpret
cause­
effect
relationships.
Although
there
appears
to
be
a
dose­
dependent
reduction
in
laryngeal
muscle
area
relative
to
atrazine
concentrations,
the
reliance
on
the
proportion
of
animals
falling
below
the
average
is
an
indirect
measure
of
the
effect.
A
direct
comparison
of
measured
laryngeal
muscle
area
between
controls
and
exposed
animals
would
further
clarify
the
magnitude
and
extent
of
any
developmental
effects.
Information
on
how
diminished
dilator
muscle
area
or
the
gonadal
deformities
might
relate
to
the
reproductive
success,
growth
or
survival
of
the
affected
species
in
the
environment
would
also
provide
further
insights
on
the
ecological
relevancy
of
effects.
This
laboratory
study
was
not
conducted
for
the
purposes
of
reregistration
and
many
of
the
details
typically
monitored
in
a
study
conducted
under
Good
Laboratory
Practice
standards
were
not
recorded.
Data
Evaluation
Report
on
Hermaphroditic,
Demasculinized
Frogs
After
Exposure
to
the
Herbicide
Atrazine
at
Low
Ecologically
Relevant
Doses
EPA
MRID
Number:
None
Page
3
of
9
I.
MATERIALS
AND
METHODS
GUIDELINE
FOLLOWED:
Nonguideline
study
COMPLIANCE:
Not
conducted
under
full
Good
Laboratory
Practices
A.
MATERIALS:

1.
Test
Material
Atrazine
Description:
Not
reported
Lot
No./
Batch
No.
:
Not
reported
Purity:
Not
reported
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
Experiment
1
and
2:
96­
hr
larvae;
Experiment
3:
adults
(
age
not
reported)
Weight
at
study
initiation:
(
mean
and
range)
not
reported
Length
at
study
initiation:
(
mean
and
range)
not
reported
Source:
Eggs
and
sperm
obtained
from
three
adults
(
3
males
and
3
females)
from
long­
term
captive
colony
maintained
at
the
University
of
California,
Berkeley,
for
Experiment
1
and
from
adults
obtained
from
Nasco
(
Fort
Atkinson,
WI)
for
Experiment
2.
Adults
from
long­
term
captive
colony
maintained
at
UC
Berkeley
for
Experiment
3.

B.
STUDY
DESIGN:

Objective:
To
test
the
hypothesis
that
atrazine
may
interfere
with
metamorphosis
and
sex
differentiation
at
ecologically
relevant
low
doses
via
endocrine­
disrupting
mechanisms.
1.
Experimental
Conditions
a)
Range­
finding
Study:
Not
reported
b)
Definitive
Study
Data
Evaluation
Report
on
Hermaphroditic,
Demasculinized
Frogs
After
Exposure
to
the
Herbicide
Atrazine
at
Low
Ecologically
Relevant
Doses
EPA
MRID
Number:
None
Page
4
of
9
Table
1
.
Experimental
Parameters
Parameter
Details
Acclimation:
period:
Conditions:
(
same
as
test
or
not)
Feeding:
Health:
(
any
mortality
observed)
Experiments
1
and
2:
Embryos
hatched
in
0.3x
modified
mammalian
Ringer's
solution;
at
96­
hrs
post­
hatch,
larvae
transferred
to
aerated
10%
Holtfreter's
solution.
Fed
solution
of
ground
Purina
rabbit
chow
daily.
No
mortality
reported.
Experiment
3:
Adults
acclimated
in
10%
Holtfreter's
solution
for
5
days,
unaerated;
animals
fed
Purina
trout
chow
daily;
water
renewed
every
72
hours.

Duration
of
the
test
Experiment
1
and
2:
Hatching
(
NF
Stage
48)
until
complete
tail
resorption
(
NF
Sate
66)
Experiment
3:
Adults
(
age
not
reported)
exposed
for
46
days.

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
Experiments
1
and
2
with
larvae
employed
aeration;
Experiment
3
with
adults
did
not
employ
aeration
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
Hermaphroditic,
Demasculinized
Frogs
After
Exposure
to
the
Herbicide
Atrazine
at
Low
Ecologically
Relevant
Doses
EPA
MRID
Number:
None
Parameter
Details
Page
5
of
9
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:
Experiment
1
and
2:
3
replicates
Number
of
organisms
per
replicate
/
group:
Experiment
1
and
2:
30
larvae
per
replicate
Biomass
loading
rate
Experiment
1
and
2:
30
larvae/
4
L
Test
concentrations:
nominal:
Experiment
1:
0.01,
0.1,
1.0,
10.0,
and
25

g/
L
Experiment
2:
0.1,
0.4,
0.8,
1.0,
25,
and
200

g/
L
Experiment
3:
25

g/
L
Solvent
(
type,
percentage,
if
used)
Experiments
1
and
2:
0.004%
ethanol
in
10%
Holtfreter's
solution.
Experiment
3:
10%
Holtfreter's
solution
Holtfreter's
medium:

Lighting
12
hrs
light,
12
hrs
dark
Data
Evaluation
Report
on
Hermaphroditic,
Demasculinized
Frogs
After
Exposure
to
the
Herbicide
Atrazine
at
Low
Ecologically
Relevant
Doses
EPA
MRID
Number:
None
Parameter
Details
Page
6
of
9
Feeding
Experiments
1
and
2:
Purina
rabbit
chow
ad
libitum
(
personal
communication,
T.
Hayes,
2002)

Experiment
3:
Purina
trout
chow
daily
Recovery
of
chemical
Level
of
Quantitation
Level
of
Detection
Concentrations
confirmed
by
PTRL
West,
Richmond,
Ca
and
Iowa
Hygenic
Laboratory,
Univ.
of
Iowa,
Iowa
City,
IO).
Not
reported.
Not
reported.

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
Experiment
1
and
2:
mortality,
time
to
metamorphosis,
weight
and
length
at
metamorphosis,
sex
based
on
gross
morphology
(
all
animals)
and
histology
(
10
animals/
tank),
laryngeal
size
based
on
largest
crosssectional
area
of
M.
dilator
larngis
(
transverse
histology
from
10
males
and
10
females
from
each
replicate).

Observation
intervals
Not
reported
Were
raw
data
included?
No
Other
observations,
if
any
Experiment
3:
plasma
testosterone
in
blood
collected
by
decapitation
using
radioimmuno­
assay.
Testosterone
antisera
obtained
from
Endocrine
Sciences
(
Calabasas,
CA).

II.
RESULTS
and
DISCUSSION:

Statistical
analysis
was
conducted
using
SYSTAT
®
(
SPSS,
Chicago);
sex
ratios
and
mortality
were
determined
using
a
G
test
with
Wilkin's
g­
adjustment
;
and
time
to
metamorphosis
and
growth
(
length
and
weight
at
metamorphosis)
were
analyzed
using
ANOVA.
Animals
were
scored
based
on
the
size
of
their
larynx
in
relationship
to
the
mean
laryngeal
size
for
controls.
After
scoring,
a
G
test
was
conducted
to
determine
the
Data
Evaluation
Report
on
Hermaphroditic,
Demasculinized
Frogs
After
Exposure
to
the
Herbicide
Atrazine
at
Low
Ecologically
Relevant
Doses
EPA
MRID
Number:
None
Page
7
of
9
affect
of
atrazine
treatment
followed
by
Kendall's
ranked
coefficient
to
determine
whether
the
percentage
of
below­
average
animals
varied
with
the
dose
of
atrazine.

Experiments
1
and
2:

At
the
doses
tested,
atrazine
exposure
had
no
significant
(
P
>
0.05)
effect
on
mortality,
time
to
metamorphosis,
length,
or
weight
at
metamorphosis.
Males
and
females
were
sexually
differentiated
at
metamorphosis
based
on
gonadal
morphology
and
histology.
All
atrazine
doses
tested
except
0.01

g/
L
produced
gonadal
abnormalities
in
16
­
20%
of
the
animals.
Abnormalities
included
multiple
gonads
or
hermaphrodites
(
multiple
testes
and
ovaries.
These
abnormalities
were
not
observed
in
the
controls.
The
paper
contains
histological
sections
through
the
gonads
of
an
animal
exposed
to
1

g/
L
atrazine
that
was
classified
as
a
hermaphrodite.
The
sections
in
the
histological
slides
showed
a
transition
from
bilateral
testicular
tissue
to
ovarian
tissue,
moving
caudally
along
the
gonads.

Control
males
had
larger
larynges
than
females
at
metamorphosis,
but
males
exposed
to

1

g/
L
had
reduced
larynges
in
both
Experiments
1
and
2.
In
the
caption
of
Figure
3,
the
authors
noted
that
atrazine

1

g/
L
reduced
laryngeal
size
in
males
but
did
not
affect
females.
Larynges
of
animals
in
Experiment
2
were
larger
than
in
Experiment
1,
suggesting
a
population
difference
in
absolute
size
of
the
larynges.
Similar
to
Experiment
1
though,
exposure
to
atrazine
at

1

g/
L
significantly
reduced
laryngeal
size
in
males.
Logtransformed
percent
of
animals
above
control
mean
laryngeal
size
indicated
that
atrazine
exposure

1

g/
L
significantly
decreased
the
proportion
of
males
that
were
at
or
above
the
mean
control
males
(
G
test,
p
<
0.05).
It
also
suggested
a
threshold
effect
at

1

g/
L
in
which
80%
of
the
exposed
males
were
below
average.
The
percentage
of
below­
average
animals
suggested
a
dose
effect
with
increasing
atrazine
doses
(
Kendal
rank
coefficient;
p
<
0.01).

Experiment
3:

An
extrapolatation
of
Figure
4
of
the
paper
indicated
that
plasma
testosterone
levels
in
control
males
averaged
4
ng/
mL.
Plasma
testosterone
in
atrazine­
treated
males
and
control
females
averaged
roughly
0.1
and
0.2
ng/
mL,
respectively,
and
were
significantly
lower
(
ANOVA,
P
<
0.05)
than
control
males.

The
authors
hypothesized
that
the
16
­
20%
incidence
of
multiple
gonads/
hermaphroditism
and
smaller
than
average
larynxes
in
males
were
caused
by
atrazine­
disrupting
steroidogenesis.
They
also
concluded
that
sexually
mature
males
suffered
a
10­
fold
decrease
in
plasma
testosterone.
According
to
the
authors,
the
current
findings
suggested
that
atrazine
inhibits
testosterone
and
induces
estrogen
synthesis
by
an
induction
of
aromatase
and
consequent
transformation
of
androgens
(
testosterone)
to
estrogen.
They
hypothesized
that
the
loss
of
masculine
features,
i.
e.,
decreased
laryngeal
muscle
size,
may
be
due
to
decreased
androgens,
and
the
induction
of
ovaries
may
result
from
increased
estrogen
synthesis
and
secretion.
According
to
the
authors,
the
current
data
raised
concerns
regarding
the
effects
of
atrazine
on
amphibians:
exposure
to
0.1

g/
L
produced
hermaphrodites
and
exposure
to
1

g/
L
resulted
in
a
reduction
in
laryngeal
size.

F.
REVIEWER'S
COMMENTS:

This
nonguideline,
published
study
was
useful
in
identifying
a
potential
hazard
to
amphibians
and
presented
information
on
measurement
endpoints,
such
as
gonadal
deformities
and
laryngeal
muscle
diameter.
The
study,
however,
did
not
show
a
clear
dose
response
that
demonstrates
a
causal
relationship
between
atrazine
exposure
Data
Evaluation
Report
on
Hermaphroditic,
Demasculinized
Frogs
After
Exposure
to
the
Herbicide
Atrazine
at
Low
Ecologically
Relevant
Doses
EPA
MRID
Number:
None
Page
8
of
9
and
developmental
effects.
While
the
paper
states
that
the
study
results
have
been
repeated
in
51
trials,
the
Agency
did
not
receive
these
replicated
data.
Results
of
these
repeated
trials
would
help
the
Agency
verify
the
results
found
in
this
study.
Because
of
the
variability
and
nature
of
the
measurement
endpoints,
the
statistical
analyses
in
this
study
relied
heavily
on
nonparametric
methods.
Access
to
the
raw
data
supporting
this
study
would
help
the
Agency
to
verify
the
study's
conclusions
regarding
mortality,
time
to
metamorphosis,
growth,
gonadal
development,
laryngeal
diameter
(
size)
or
plasma
testosterone
levels.
Although
the
range
of
gonadal
abnormalities
was
provided,
the
prevalence
of
gonadal
effects
is
difficult
to
interpret.
EPA
needs
more
specific
data
on
the
range
of
gonadal
abnormalities,
the
incidence
for
each
dose
tested,
the
relative
percentages
of
males
and
females,
the
numbers
of
animals
involved
in
the
16­
20%
abnormality
incidence,
and
the
impact
of
gonadal
abnormalities
on
reproductive
success
in
the
environment.
Without
this
information,
it
is
difficult
to
interpret
the
error
bars
in
Figure
4
of
the
paper
and
to
establish
the
ecological
relevancy
of
the
data.
EPA
also
needs
clarifying
information
concerning
the
sample
sizes
involved
in
the
analyses,
the
number
of
doses
samples
and
the
method
of
blood
collection
(
i.
e.,
decapitation
could
contaminate
blood
samples).

In
its
evaluation
of
this
study,
the
Agency
had
questions
concerning
the
use
of
ethanol
as
a
co­
solvent.
Since
atrazine
concentrations
used
in
these
studies
were
less
than
the
30
mg/
L
water
solubility
limit,
a
co­
solvent
should
not
be
necessary.
Previous
studies
conducted
by
the
authors
(
personal
communication:
Tyrone
Hayes
2002)
used
dihydrotestosterone
and
17­
 
estradiol
as
positive
controls
and
required
ethanol
as
a
co­
solvent.
The
current
study,
though,
did
not
utilize
these
steroids
and
a
co­
solvent
should
not
have
been
necessary.
The
Agency
also
has
questions
concerning
the
conditions
of
the
study.
The
study
states
that
30
organisms
with
an
estimated
maximal
weight
of
1.5
g
were
held
in
4
liters
of
test
solution.
These
conditions
would
result
in
a
loading
rate
of
11.3
g/
L,
which
is
27
times
the
recommended
loading
rate
for
static
renewal
toxicity
tests.

While
the
paper
discusses
that
the
study
results
have
been
repeated
in
51
trials,
none
of
the
data
are
presented;
therefore,
it
is
not
possible
to
gauge
the
researchers'
success
at
replicating
the
current
results.

Although
only
the
range
of
gonadal
abnormalities
(
16
­
20%)
is
provided,
no
information
is
presented
on
what
the
incidence
was
for
each
dose
tested.
The
data
suggest
that
there
was
a
threshold
effect
level
at
0.1

g/
L
but
that
the
effect
did
not
increase
with
increasing
dose.

While
the
authors
suggest
that
up­
regulation
of
aromatase
activity
is
a
plausible
rationale
for
the
observed
effects
and
that
reduced
testosterone
levels
in
atrazine
treated
adult
male
Xenopus
further
support
this
hypothesis,
no
direct
measure
of
aromatase
activity.
Further
testing
with
additional
dose
concentrations
is
a
needed
to
establish
a
causal
relationship
between
atrazine
exposure
and
gonadal
effects
in
amphibians.
is
provided
nor
can
a
dose
response
be
established.
Additionally,
if
aromatase
activity
were
increased
and
there
was
subsequent
decline
in
testosterone,
estrogen
levels
would
likely
increase;
however,
there
are
no
data
available
on
the
estrogen
concentrations.
Since
a
single
concentration
(
25

g/
L)
of
atrazine
was
used
to
test
the
hypothesis
concerning
aromatase,
it
isn't
possible
to
establish
a
dose
response.

The
ecological
relevancy
of
this
data
is
not
clear.
No
information
is
available
on
whether
similar
gonadal
abnormalities
impact
reproductive
success
in
the
environment.

The
lab
study
utilizes
relatively
sub­
standard
conditions
in
that
30
organisms
with
an
estimated
maximal
weight
of
1.5
g
were
held
in
4
liters
of
test
solution.
This
results
in
a
loading
rate
of
11.3
g/
L,
which
is
27
times
the
recommended
loading
rate
for
static
renewal
toxicity
tests.
Data
Evaluation
Report
on
Hermaphroditic,
Demasculinized
Frogs
After
Exposure
to
the
Herbicide
Atrazine
at
Low
Ecologically
Relevant
Doses
EPA
MRID
Number:
None
Page
9
of
9
As
reported,
the
prevalence
of
gonadal
effects
is
difficult
to
interpret.
The
results
section
apparently
combines
the
results
of
both
larval
studies
and
fails
to
provide
dose
response
information
and
any
indication
of
variance.
Since
the
presentation
of
dose
response
data
is
a
fundamental
standard
for
toxicological
studies,
this
omission
is
curious,
and
suggests
that
the
report
is
incomplete
for
some
reason.
Also,
what
is
the
relative
contribution
of
the
two
major
abnormality
types
to
the
overall
prevalence
of
gonadal
abnormalities?
There
are
no
data
presented
on
the
relative
percentages
of
males
and
females
in
the
study,
nor
any
other
data
indicating
numbers
on
which
the
apparent
16­
20%
abnormality
incidence
was
calculated.

Results
from
the
adult
atrazine
exposure
do
not
indicate
the
sample
sizes
involved
in
the
analysis.
In
fact,
it
does
not
indicate
the
basic
information
on
how
many
males
were
exposed
or
held
as
controls.
Since
this
information
is
not
available,
one
cannot
make
sense
of
the
error
bars
(
what
are
they
representing?)
in
Figure
4,
nor
whether
or
not
this
is
a
credible
analysis.
In
the
RIA
section
of
the
Methods
&
Materials,
the
report
states
that
plasma
was
sampled
at
3
doses.
However,
the
Adult
Treatments
section
indicates
that
only
one
dose
was
utilized.
The
method
of
blood
collection,
decapitation,
seems
rather
crude
and
likely
to
contaminate
blood
with
other
bodily
fluids,
which
could
confound
the
analysis.
A
more
acceptable
approach,
for
example,
would
be
a
cardiac
puncture.

H.
REFERENCES:

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