A
Fish
Two
Generation
Toxicity
Test
Detailed
Review
Paper
Endocrine
Disruptor
Methods
Validation
Subcommittee
December
2002
Leslie
Touart
Detailed
Review
Paper:

A
Fish
Two
Generation
Toxicity
Test
Detailed
Review
Paper
WORK
PERFORMED
BY:

and
SpringbornSmithers
Laboratories
LLC
On
behalf
of
the
United
States
Environmental
Protection
Agency
EPA
CONTRACT
NUMBER
68­
W­
01­
023
METHODS
USED
IN
THIS
ANALYSIS
°
On­
line
Literature
Search
 
"
Dialog"
On­
Line
search
with
database
Biosis
Previews
Aquatic
Science
and
Fisheries
Abstracts
 
Endocrine
disruptor
screening
methods
for
fathead
minnow,
zebrafish,
medaka,
sheepshead
minnow
 
Key
Words
"
estrogen*
or
testosteron*
or
endocrin*

or
antiandrogen*
or
androgen*
or
hormon*
or
thyroxin*
or
*
thyroid
*
method,
protocol
etc 

 
7453
records
were
refined
down
to
601
records
METHODS
USED
IN
THIS
ANALYSIS
Cont.

Interviews
With
The
Following
Experts
Tom
Hutchinson
Reynaldo
Patino
Taisen
Iguchi
Alf
Lundgren
Dan
G.
Cyr
Nancy
Denslow
METHODS
USED
IN
THIS
ANALYSIS
Cont.

External/
Internal
Peer
Review
°
Dr.
Dave
Hinton
 
Duke
Unv.
USA
°
Dr.
John
Sumpter
 
Brunell
Unv.
UK
°
Dr.
Gary
Thorgaard
­
Univ.
of
Wash.
USA
°
EPA
Technical
Experts
OVERVIEW
AND
SCIENTIFIC
BASIS
°
Evidence
exists
that
EDCs
affect
sexual
differentiation,

development,
and
reproduction
in
fish
°
Fish
life
cycle
test
methods
have
been
standardized
for
decades
°
Existing
methods
do
not
assess
transgenerational
effects
and
lack
relevant
biochemical,
morphological,
and
behavioral
endpoints
°
The
proposed
Fish
Two
Generation
Test
addresses
these
EDC
relevant
omissions
Test
Species
°
Fathead
minnow
(
Pimephales
promelas)

°
Medaka
(
Oryzias
latipes)

°
Zebrafish
(
Danio
rerio)

°
Sheepshead
minnow
(
Cyprinodon
variegatus)

 
small
size
at
maturity
 
ease
of
culture
 
maintenance
costs
 
asynchronous
spawners
Fathead
Minnow
Family
Cyprinidae
°
35
to
75
mm
length
°
Extensive
aquatic
toxicity
in
USA
°
generation
time
about
4
months
°
sexually
dimorphic
°
females
produce
50
to
250
embryos
per
spawn
Fathead
Minnow
Strengths
 
Large
enough
to
collect
individual
blood
plasma
samples
 
Distinct
secondary
sex
characteristics
in
both
sexes
 
Large
historical
regulatory
database
 
Many
laboratories
are
familiar
with
culture
and
testing
 
Spawn
on
a
substrate
 
High
fertilization
rate
 
Indigenous
to
North
America
Weaknesses
 
Relatively
long
life
cycle
 
Relatively
high
variability
in
fecundity
 
Relative
size
of
the
fish
requires
more
space
for
culture
and
testing
 
Intersex
condition
is
less
frequently
observed
compared
to
other
fishes.

 
Genome
poorly
characterized
Medaka
Family
Adrianichthyidae
°
indigenous
to
Japan,
Taiwan,
and
southeastern
Asia
°
Generation
interval
of
2
to
3
months
°
sexually
dimorphic
°
25
mm
to
50
mm
length
°
females
produce
10
to
30
eggs
per
spawn
°
estimated
to
be
over
500
cultivated
strains
 
Genetically
Engineered
/
Inbred
Strains
in
Toxicity
Testing
Medaka
Strengths
 
Relatively
short
life
cycle
 
Relatively
small
fish,

making
culture
and
testing
possible
in
smaller
space
 
Female
sex
determined
during
embryo
stage
vs.

male
sex
determined
after
hatch
 
Sex­
linked
color
strain
Weaknesses
 
Smaller
size
reduces
individual
blood
sample
volumes
compared
to
fathead
minnow
 
Less
distinctive
secondary
sex
characteristics
 
Regulatory
data
base
less
extensive
compared
to
fathead
minnow.

 
Limited
use
in
short­
term
tests
in
the
U.
S.
A.
Zebrafish
Family
Cyprinidae
°
Native
to
East
India
and
Burma
°
4
cm
to
5
cm
in
length
°
Extensive
aquatic
toxicity
in
Europe
°
Difficult
to
sex
zebrafish
°
Sexual
maturity
in
10
to
12
weeks
°
150
to
400
eggs
per
female
°
Development
of
transgenic
zebrafish
Zebrafish
Strengths
 
Short
life
cycle
 
Small
fish,
making
culture
and
testing
possible
in
smaller
spaces
 
Male
fish
go
through
a
hermaphroditic
phase
as
juveniles
 
Widely
used
in
other
medical
and
genetic
research
 
Frequently
used
in
Europe
for
regulatory
purposes
 
Transgenic
fish
increasingly
available
 
Anticipated
that
entire
genome
will
be
sequenced
soon.
Weaknesses
 
Small
size
makes
individual
blood
plasma
samples
not
likely
 
Minimal
secondary
sex
characteristics
 
Limited
US
regulatory
data
base
 
Limited
testing
experience
in
the
US
Sheepshead
Minnow
Family
Cyprinodonitidae
°
Native
to
Atlantic
and
Gulf
of
Mexico
estuaries
°
35
mm
to
50
mm
length
°
Tolerates
wide
ranges
in
temperature
(
0
to
40
º
C)
and
salinity
(
0.1
ppt
to
149
ppt)

°
Sexually
dimorphic
°
Sexual
maturity
in
60
days
°
Low
variability
in
fecundity
°
Large
historical
regulatory
database
Sheepshead
Minnow
Strengths
 
Very
short
life
cycle
(<
60days
to
sexual
maturity),
seawater
costs
may
be
offset
by
shorter
exposure
times
for
testing
 
Males
large
enough
for
individual
blood
plasma
samples
 
Distinct
sexual
dimorphism
 
Relatively
low
variability
in
fecundity
 
Relatively
large
historical
regulatory
database
 
Many
laboratories
are
familiar
with
culture
and
testing
 
Relatively
small
fish
making
culture
and
testing
possible
in
smaller
space
Weaknesses
 
Estuarine/
marine
species,

salinity
of
15
to
30
ppt
recommended,
however,
lower
salinity
may
be
possible
(
5
ppt)

 
Culture
requires
a
large
number
of
females
to
produce
enough
eggs
in
a
24­
hr
period
to
initiate
a
life­
cycle
test
 
Limited
information
on
reproductive
endocrinology
 
Small
size
makes
individual
blood
plasma
samples
not
likely
Routes
of
Administration
of
Chemical
Exposure
°
Aqueous
°
Dietary
exposures
°
Direct
injection
techniques
 
Intravascular
 
intraperitoneal
Measurement
Endpoints
°
Growth
and
Morphological
Alterations
 
Gonadosomatic
Index
 
Histology
Techniques
 
Sexual
Differentiation
 
Secondary
Sex
Characteristics
°
Measures
of
Reproductive
Performance
 
Fecundity
 
Gamete
Viability
 
Changes
in
Spawning
Behavior
°
Biochemical
Measures
 
Vitellogenin
Induction
 
Tissue
Steroid
Concentrations
 
Thyroid
hormones
MEASUREMENT
OF
BIOCHEMICAL
ENDPOINTS
°
Sex
Steroids
in
Tissues
Estrogens/
Androgens/
Progestins
 
Radioimmunoassay
(
RIA)

 
Enzyme­
linked
Immunosorbent
Assay
(
ELISA)

 
Liquid/
Gas
Chromatography
with
Mass
Selective
Detection
(
LC/
GC­
MS)
Measurement
of
Vitellogenin
°
Indirect
Quantification
of
Vitellogenin
Protein
 
Alkaline­
labile
Phosphate
Assay
°
Direct
Quantification
of
Vitellogenin
Protein
 
RIA
 
Enzyme­
linked
Immunosorbent
Assay
 
Antibody­
capture
 
Antigen­
capture
 
Direct
Enzyme­
linked
Immunosorbent
Assay
 
Sandwich
Enzyme­
linked
Immunosorbent
Assay
°
Quantifying
Vitellogenin
mRNA
 
Ribonuclease
Protection
Assay
(
RPA)

 
Quantitative
Reverse
Transcription­
Polymerase
Chain
Reaction
(
QRT­
PCR)

°
Mass
spectrometry
(
MS)
CANDIDATE
PROTOCOLS
1)
Partial
Life­
Cycle
Test
{
Adult
(
P)
to
Juvenile
(
F1)}

2)
Full
Life­
Cycle
Test
{
Egg
(
P)
to
Juvenile
(
F1)}

3)
Multi­
Generation
Test
{
Egg
(
P)
to
Juvenile
(
F2)}

4)
Two
Generation
Test
{
Adult
(
P)
to
Juvenile
(
F2)}
Partial
Life­
Cycle
Test
{
Adult
(
P)
to
Juvenile
(
F1)}

A
partial
life­
cycle
toxicity
test,
which
exposes
P
adult,

sexually
mature
fish
and
the
early
life
cycle
of
F1
fish,
(
can
be
used
to
estimate
the
NOEC)

A
pre­
exposure
reproductive
evaluation
is
conducted
on
the
P
fish.
Biological
endpoints
evaluated:

°
P
pre­
exposure,
secondary
sexual
characteristics
and
fecundity/
reproduction
(
e.
g.,
eggs/
female)

°
P
post­
exposure,
survival,
secondary
sexual
characteristics,

fecundity/
reproduction
(
e.
g.,
eggs/
female),
GSI,

histopathology,
and
protein
and
sex
steroid
biomarkers
(
e.
g.,

VTG)

°
F1
hatching
success,
survival,
growth
(
length
and
weight)
Full
Life­
Cycle
Test
{
Egg
(
P)
to
Juvenile
(
F1)}

A
full
life
cycle
test
developed
(
Benoit
1981­
fathead
minnows
)
and
(
Hansen
et
al.
1978
­
sheepshead
minnow).

Initiated
with
fertilized
eggs
(
P)
and
the
fish
are
continuously
exposed
through
reproductive
maturity,

followed
assessment
of
the
early
development
of
the
F1
generation.

Biological
endpoints
evaluated:

°
P
embryo
time­
to­
hatch,
hatching
success,
larval
survival
and
length,
weight
of
thinned
fish,
survival,
secondary
sexual
characteristics,
fecundity/
reproduction
(
e.
g.,

eggs/
female),
growth.

°
F1
hatching
success,
survival,
growth
(
length
and
weight)
Multi­
Generation
Test
{
Egg
(
P)
to
Juvenile
(
F2)}

Multi­
generation
toxicity
test,
exposes
all
life­
stages
of
two
generations
of
fish
Test
is
initiated
with
eggs
and
two
full
generations
of
fish
are
exposed
during
the
test
(
can
estimate
the
NOEC)

Biological
endpoints
evaluated:

C
P
and
F1
hatching
success,
survival,
growth
(
length
and
weight),
time­
to­
maturity,
sex
ratio,
secondary
sexual
characteristics,
fecundity/
reproduction
(
e.
g.,
eggs/
female),

and
protein
and
sex
steroid
biomarkers
(
e.
g.,
VTG).

°
F2
hatching
success,
survival
and
growth.
Two
Generation
Test
{
Adult
(
P)
to
Juvenile
(
F2)}

Two
generation
life­
cycle
toxicity
test,
exposes
the
adult
P,

full
F1generation,
and
measures
F2
viability
Variables
including
the
time­
line
of
the
test,
the
number
of
fish
required
in
the
test,
and
obtaining
endpoints
such
as
Vtg
plasma
levels
will
be
associated
with
the
different
species.

Biological
endpoints
evaluated:

°
P
Survival,
secondary
sex
characteristics,
reproductive
behavior,
spawning
activity,
fecundity,
fertilization
success
°
F1
hatching
success,
survival,
growth
(
length
and
weight),

time­
to­
maturity,
sex
ratio,
secondary
sexual
characteristics,
fecundity/
reproduction
(
e.
g.,
eggs/
female),

and
protein
and
sex
steroid
biomarkers
(
e.
g.,
VTG).

°
F2
hatching
success,
survival
and
growth.
Timeline
forTwo­
Generation
Protocol
with
the
Fathead
Minnow
|

 

F2
 

 
//

F1
 

 

Parental
Time
(
weeks)

­
3
0
3
18
21
25
Significant
Data
Gaps
°
Male­
specific
effects
of
estrogen
agonists
other
than
VTG
induction.

°
The
effects
of
anti­
estrogens
°
The
effects
of
androgen
agonists
and
antagonists
°
Baseline
data
for
thyroid
hormone
levels
in
test
species.

°
The
effects
of
thyroid
hormone
agonists
(
or
thyroid
stimulation)
on
reproduction.

°
Published
methods
of
sexual
differentiation
for
fathead
and
sheepshead
minnows
IMPLEMENTATION
CONSIDERATIONS
°
Pre­
validation
studies
following
the
ICCVAM
validation
process
 
Recommend
evaluating
the
increased
sensitivity
of
a
two­
generation
design
over
the
existing
fish
full
lifecycle
standard
practice
 
Recommend
determining
and
optimizing
specific
two
generation
protocol
variables
for
the
candidate
species
 
Recommend
demonstration
of
sensitivity,
reliability
and
reproducibility
for
each
species
in
the
recommended
protocol
°
Validation
of
the
study
design
through
interlaboratory
comparisons
