Document ID: EPA-HQ-OPPT-2003-0027-0027
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-08-13T04:00Z

COMPARATIVE
EVALUATION
OF
VITELLOGENIN
METHODS
FOR
FATHEAD
MINNOW,
MEDAKA
AND
ZEBRA
FISH
Photos
:
Biosense
Laboratories
Endocrine
Disruptor
Methods
Validation
Endocrine
Disruptor
Methods
Validation
Subcommittee
Subcommittee
August
2003
August
2003
EPA
Work
Assignments:
2­
19
and
2­
26
Presented
by:

Michael
L.
Blanton
and
Dr.
Irv
Schultz
2
COMPARATIVE
EVALUATION
OF
VITELLOGENIN
METHODS
FOR
FATHEAD
MINNOW,
MEDAKA
AND
ZEBRA
FISH
COMPARATIVE
EVALUATION
OF
VITELLOGENIN
COMPARATIVE
EVALUATION
OF
VITELLOGENIN
METHODS
FOR
FATHEAD
MINNOW,
MEDAKA
METHODS
FOR
FATHEAD
MINNOW,
MEDAKA
AND
ZEBRA
FISH
AND
ZEBRA
FISH
WORK
PERFORMED
BY
On
behalf
of
the
United
States
Environmental
Protection
Agency
EPA
CONTRACT
NUMBER
68­
W­
01­
023
3
Introduction
Introduction
Introduction
Purpose
of
study:
Survey
of
Methods
for
measurement
of
Vitellogenin
(
VTG)

to
conduct
a
survey
of
existing
VTG
analytical
methods
for
suitability
in
a
routine
screening
program.

Study
was
not
intended
to
be
a
Method
Validation

the
comparison
was
not
intended
to
be
a
validation
of
a
given
method,
but
an
evaluation
across
methods
to
ascertain
the
qualitative
and/
or
quantitative
comparability
of
the
variety
of
methods
currently
available.
4
VTG
Background
VTG
Background
VTG
Background
VTG
is
a
phospholipoglycoprotein
precursor
to
egg
yolk
protein
that
normally
occurs
in
sexually
active
female
oviparous
fishes,
but
can
be
induced
to
occur
in
males
in
response
to
estrogenic
substances.

The
measurement
of
a
biochemical
marker,

VTG
in
oviparous
vertebrates
is
generally
agreed
to
be
a
good
indicator
for
estrogenic
and
anti­
estrogenic
effects
and
is
proposed
as
one
of
several
endpoints
in
the
fish
screening
assay.
5
Methods
for
Measuring
VTG
induction
in
fishes
Methods
for
Measuring
VTG
Methods
for
Measuring
VTG
induction
in
fishes
induction
in
fishes
Enzyme­
Linked
Immunosorbant
Assays
(
ELISA)

An
enzyme
immunoassay
utilizing
an
enzymelabeled
immunoreactant
(
antigen/
antibody)
and
an
immunosorbent
(
antigen/
antibody
bound
to
a
solid
support
 
i.
e,
a
polystyrene
microliter
plate)
6
mRNA
detection
An
alternative
to
measuring
the
VTG
protein
is
to
quantify
the
messenger
ribonucleic
acid
(
mRNA)
for
VTG
that
codes
for
the
protein.

Two
methods
for
quantifying
fish
VTG
mRNA
have
emerged,

the
ribonuclease
protection
assay
(
RPA)

the
quantitative
reverse
transcriptionpolymerase
chain
reaction
(
QRT­
PCR)

Methods
for
Measuring
VTG
induction
in
fishes
cont.

Methods
for
Measuring
VTG
Methods
for
Measuring
VTG
induction
in
fishes
cont.

induction
in
fishes
cont.
7
Mass
Spectrometry
(
MALDI­
MS)

In
general,
MS
approaches
to
protein
quantification
attempt
to
measure
the
protein
largely
in
its
intact
form
or
rely
on
digestion
procedures
(
chemical
or
enzymatic)
to
reduce
the
size
of
the
protein
into
smaller
fragments.

The
MS
technique
allows
both
the
direct
measurement
of
the
VTG
mass
and
generation
of
peptide­
fingerprinting
data
for
further
identification
(
Wunschel
and
Wahl,
2002).

Methods
for
Measuring
VTG
induction
in
fishes­
Mass
Spectrometry
Methods
for
Measuring
VTG
Methods
for
Measuring
VTG
induction
induction
in
fishes
in
fishes­
Mass
Spectrometry
Mass
Spectrometry
8
Participants
in
the
Fathead
Minnow
VTG
Study
Participants
in
the
Participants
in
the
Fathead
Minnow
VTG
Study
Fathead
Minnow
VTG
Study
WA
2­
19
Fathead
Minnow
participants
1.
University
of
Florida,
USA
2.
University
of
Idaho,
USA
3.
Oregon
State
University,
USA
4.
US
EPA
Duluth,
USA
5.
University
of
Exeter,
USA
6.
Brixham
Environmental
Laboratory,
UK
7.
Battelle
Richland,
USA
8.
Battelle
Sequin,
USA
9.
Molecular
Light
Technology,
UK
10.
Biosense,
Norway
11.
INERIS,
France
12.
Cemagref,
France
13.
University
of
Windsor,
Canada
14.
University
of
Southern
Denmark,
Denmark
15.
Finnish
Environmental
Institute,
Finland
9
Participants
Zebrafish/
Medaka
VTG
Study
(
contd)

Participants
Participants
Zebrafish/
Medaka
VTG
Study
Zebrafish/
Medaka
VTG
Study
(
contd)

(
contd)

WA
2­
26
Zebrafish/
Medaka
Zebrafish/
Medaka
Participants
1.
Biosense
Laboratories,
Norway
2.
Centre
for
Fish
and
Wildlife
Health,
University
of
Bern,

Switzerland
3.
Department
of
Pathology,
Vet.
Medicine,
Swedish
University
of
Agricultural
Sciences,
Sweden
4.
EnBioTec
Laboratories,
Ltd.,
Japan
5.
Environmental
and
Symbiotic
Sciences,
Prefectural
University
of
Kumamoto,
Japan
6.
Institute
of
Biology,
University
of
Southern
Denmark,
Denmark
7.
Los
Angeles
County
Sanitation
District,
USA
8.
National
Institute
for
Environmental
Studies
(
NIES),
Japan
9.
Notox
Safety
&
Environmental
Research,
the
Netherlands
10.
Phylonix
Pharmaceuticals,
Inc.,
USA
11.
Unité
d'Evaluation
des
Risques
Ecotoxicologiques
(
INERIS),

France.
10
Prepare
standard
series
of
plasma,
liver
and
whole
body
homogenates
of
fathead
minnow
(
Pimephales
promelas)
to
provide
a
range
of
VTG
and
mRNA
concentrations
produced
in
male
and
female
fish
(
plus
a
positive
control)
for
evaluation
by
participating
laboratories
Determine
the
comparability
of
various
methods
for
the
analysis
of
vitellogenin
in
fathead
minnows
by
means
of
statistical
analysis
of
the
results
from
eleven
laboratories

The
results
from
8
ELISA
laboratories,
3
mRNA
laboratories
and
one
Mass
spectrometric
(
MS)
method
are
presented
Objectives
Fathead
Minnow
VTG
Study
Objectives
Objectives
Fathead
Minnow
VTG
Study
Fathead
Minnow
VTG
Study
11
Objectives
Zebrafish/
Medaka
VTG
Study
Objectives
Objectives
Zebrafish/
Medaka
VTG
Study
Zebrafish/
Medaka
VTG
Study
Prepare
standard
series
of
liver
and
whole
body
homogenates
of
medaka
and
zebrafish
for
evaluation
by
participating
laboratories;
the
series
represents
a
range
of
vitellogenin
concentrations
in
male
and
female
fish,
plus
a
positive
control
Determine
the
comparability
of
ELISA
methods
for
the
analysis
of
vitellogenin
of
the
two
species
by
means
of
statistical
analysis
of
the
results
from
eleven
laboratories
Photo:

Borg
2003
12
Methods
Fathead
Minnow
VTG
Study
(
contd)

Methods
Fathead
Minnow
VTG
Study
(
contd)

Methods
Fathead
Minnow
VTG
Study
(
contd)

Sample
Preparation
(
Battelle)

Obtained
approximately
600
Fathead
minnows

Exposed
subset
of
100M
and
190F
to
300
ng/
L
17
 
estradiol
in
a
7­

day
static
renewal
treatment
to
induce
vitellogenin
production.

Remaining
fish
(
210M
and
190F
remained
unexposed).

Day
2
of
exposure
fish
were
sacrificed
and
liver
tissue
harvested
for
mRNA
standard
series
(
80
EM/
80
UM
and
160EF/
160UF)

Prepared
vitellogenin
standard
series
from
plasma
collected
from
caudal
vessels
into
heparinized
hematocrit
tubes.

whole
body
of
exposed
and
unexposed,
male
and
female
fish
by
grinding
tissue
with
ice
cold
ELISA
assay
buffer
(
1:
1
ratio,
fish:
buffer
by
wt),
centrifuging,
quick­
freezing
supernatant
on
liquid
nitrogen

Spiked
unexposed
male
tissue
of
each
species
with
known
quantity
of
purified
vitellogenin
from
corresponding
species
as
positive
control
13
Methods
Zebrafish/
Medaka
VTG
Study
(
contd)

Methods
Methods
Zebrafish/
Medaka
Zebrafish/
Medaka
VTG
Study
(
contd)

VTG
Study
(
contd)

Sample
Preparation

Obtained
approximately
400
zebrafish
and
400
medaka

Exposed
subset
of
each
group
to
300
ng/
L
17
 
estradiol
in
a
7­
day
static
renewal
treatment
to
induce
vitellogenin
production

Prepared
vitellogenin
standard
series
from
liver
and
whole
body
of
exposed
and
unexposed,
male
and
female
fish
of
each
species
by
grinding
tissue
with
ice
cold
ELISA
assay
buffer
(
1:
2
ratio,
fish:
buffer
by
wt),

centrifuging,
quick­
freezing
supernatant
on
liquid
nitrogen

Spiked
unexposed
male
tissue
of
each
species
with
known
quantity
of
purified
vitellogenin
from
corresponding
species
as
positive
control
________________________

1Blood
plasma
was
also
collected,
but
its
analysis
was
subsequently
deleted
from
study.
14
Methods
Fathead,
Zebrafish/
Medaka
VTG
Study
(
contd)

Methods
Fathead,

Methods
Fathead,
Zebrafish/
Medaka
Zebrafish/
Medaka
VTG
Study
(
contd)

VTG
Study
(
contd)


Shipment
to
participating
labs
°
Subsampled
20­
µ
L
aliquots
of
homogenates
and
assembled
triplicate
vials
of
each
sample
type
as
standard
series,
each
vial
labeled
with
unique
code
for
blind­
analysis:

°
Shipped
frozen
at
­
80
°
C
in
super­
insulated
packaging
to
elev
participating
labs
2
June
2003
with
documentation,
information,
and
standardized
chainof
custody
and
data­
reporting
forms
 
Zebra
fish
 
Zebra
fish
 
W2
 
Zebra
fish
 
L2
 
Zebra
fish
 
S2
Standard
Series:
Whole
Body,
Liver
(
W,
L)

 
uninduced
male
 
uninduced
female
 
induced
male
 
induced
female
 
positive
control
Purified
vitellogenin
as
calibration
standard
for
the
species
(
S)
15
Methods
zebrafish
Medaka
VTG
Study
(
contd)

Methods
zebrafish
Medaka
VTG
Study
(
contd)

Methods
zebrafish
Medaka
VTG
Study
(
contd)

Sample
analysis
(
participating
labs)

zebrafish

Sandwich
enzyme
immunoassay,
VTG
zebrafish
antibody
(
Biosense
2002)
(
6
labs)


Sandwich
ELISA,
VTG
zebrafish
monoclonal
antibody
(
EnBio
2002;
Nishi
2002)
(
1
lab)


Direct
noncompetitive
sandwich
ELISA,
anti­
zebrafish
lipovitellin,

polyclonal
antibody
(
Holbech
et
al.
2001)
(
1
lab)


Modified
Holbech
et
al.
2001
(
Borg
2003,
unpublished)
(
1
lab)


[
Competitive
binding
assay
(
Brion
et
al.
2002)
(
1
lab);
data
received
too
late
for
inclusion
in
statistical
analysis;
see
appendix]
16
Methods
Zebrafish/
Medaka
VTG
Study
(
contd)

Methods
Methods
Zebrafish/
Medaka
VTG
Study
Zebrafish/
Medaka
VTG
Study
(
contd)

(
contd)

Sample
analysis
(
participating
labs)

Medaka

Sandwich
enzyme
immunoassay,
VTG
medaka
antibody
(
Biosense
2003)
(
5
labs)


Sandwich
ELISA,
VTG
medaka
monoclonal
antibody
(
EnBio
2002;
Nishi
2002)
(
2
labs)


Direct
sandwich
ELISA,
VTG
medaka
monoclonal
and
biotinylated
polyclonal
antibodies
(
Transgenic
2002)
(
1
lab)


[
Competitive
binding
assay
(
Brion
et
al.
2002)
(
1
lab);
data
received
too
late
for
inclusion
in
statistical
analysis;
see
appendix]
17
Methods
Fathead
Minnow
VTG
Study
(
contd)

Methods
Methods
Fathead
Minnow
VTG
Study
Fathead
Minnow
VTG
Study
(
contd)

(
contd)

Sample
analysis
(
participating
labs)

Fathead
Minnow

ELISA
(
8
methods,
11
participating
labs)

­
Carp
based
polyclonal
and
monoclonal
antibodies,
sandwich
ELISA
(
Biosense
2002)
(
3
labs)

­
Carp
based
polyclonal
antibodies,
competitive
ELISA
(
1
lab)

­
Carp
based
polyclonal
and
monoclonal
antibodies,
sandwich
ELISA
(
1
lab)

­
Trout
based
polyclonal
antibodies
in
a
competitive
ELISA
(
2
labs)

­
Fathead
minnow
based
polyclonal
antibodies,
competitive,
antibody­
capture
(
1
lab)

­
Fathead
minnow
based,
monoclonal
antibody,
direct
ELISA
(
1
lab)

­
Zebrafish
based
polyclonal
antibodies,
competitive
ELISA
(
1
lab)

­
Zebrafish
based
anti­
lipovitellin
direct
non­
competitive
sandwich
ELISA
(
1
lab)

mRNA
(
3
methods,
3
participating
labs)

­
mRNA
­
RT­
PCR
­
mRNA
­
qRT­
PCR
TaqMan
­
mRNA
­
HPA
(
hybridization
protection
assay)

Mass
Spectrometric
(
1
method,
1
participating
lab)

­
Matrix
assisted
laser
desorption/
ionization
mass
spectrometry
(
MALDI­
MS)
(
1
lab)
18
C:
Medaka
liver
D.
Medaka
whole
body
Results
Zebrafish/
Medaka
VTG
Study
Results
Results
Zebrafish/
Medaka
VTG
Study
Zebrafish/
Medaka
VTG
Study
Expected
trend
was
as
follows:

uninduced
male<
uninduced
female<
induced
male<
induced
female>>
positive
control
1
10
100
1,000
10,000
100,000
1,000,000
10,000,000
100,000,000
0
1
2
3
4
5
6
Concentration
Code
Mean
VTG
Concentration
Lab
1
Lab
2
Lab
4
Lab
5
Lab
7
Lab
8
Lab
9
Lab
10
Lab
12
Max
Detection
Limit
1
10
100
1,000
10,000
100,000
1,000,000
10,000,000
100,000,000
0
1
2
3
4
5
6
Concentration
Code
Mean
VTG
Concentration
Lab
1
Lab
2
Lab
4
Lab
5
Lab
7
Lab
8
Lab
9
Lab
10
Lab
12
Max
Detection
Limit
A:
zebrafish
liver
B:
zebrafish
whole
body
1
10
100
1,000
10,000
100,000
1,000,000
10,000,000
0
1
2
3
4
5
6
Concentration
Code
Mean
VTG
Concentration
Lab
1
Lab
2
Lab
4
Lab
5
Lab
11
Lab
11
Lab
11
Lab
12
1
10
100
1,000
10,000
100,000
1,000,000
10,000,000
100,000,000
0
1
2
3
4
5
6
Concentration
Code
Mean
VTG
Concentration
Lab
1
Lab
2
Lab
4
Lab
5
Lab
11
Lab
11
Lab
11
Lab
12
 
Results
showed
good
relative
tracking
of
trend,

particularly
in
medaka,
but
higher
variability
in
absolute
measured
values
Best
fit:
medaka
liver
results
19
Code
0
=
Blank;
Code
1
=
Uninduced
Male;
Code
2
=
Uninduced
Female;

Code
3
=
Induced
male;
Code
4
=
Induced
Female;
Code
5
=
Positive
Control
Homogenate
0.00
0.01
0.10
1.00
10.00
100.00
1000.00
10000.00
100000.00
0
1
2
3
4
5
6
Concentration
Code
VTG
Concentr
ation
(

ug/

mL
Lab
1
Lab
4
Lab
5
Lab
6
Lab
7
Lab
8
Lab
11
Lab
13
Max
detection
Limit
Plasma
0.00
0.01
0.10
1.00
10.00
100.00
1000.00
10000.00
100000.00
1000000.00
0
1
2
3
4
5
6
Concentration
Code
VTG
Concentration
(

ug/

m
Lab
1
Lab
4
Lab
5
Lab
6
Lab
7
Lab
11
Lab
13
Max
detection
Limit
Results
Fathead
Minnow
VTG
Study
Results
Fathead
Results
Fathead
Minnow
VTG
Study
Minnow
VTG
Study
Expected
trend
was
as
follows:

uninduced
male<
uninduced
female<
induced
male<
induced
female>>
positive
control
 
Results
showed
good
relative
tracking
of
trend,
particularly
Plasma,
but
higher
variability
in
absolute
measured
values
20
mRNA
average
for
liver
samples
0.1
1
10
100
1000
10000
100000
1000000
1
2
3
4
Lab
1
(
pg
VTG
mRNA
/
ug
total
RNA)

Lab
2
(
mRNA
VTG
/
total
RNA)

Lab
15
(
fmol
VTG
mRNA
/
ug
total
RNA)

All
three
methods
distinguished
between
Uninduced
and
Induced
fish
mRNA
Liver
Results
Code
1
=
Uninduced
Male;
Code
2
=
Uninduced
Female;

Code
3
=
Induced
male;
Code
4
=
Induced
Female;

Laboratory
1
RT­
PCR
Laboratory
2
qRT­
PCR
TaqMan
Laboratory
15
HPA
(
hybridization
protection
assay)
21
219PU18
Exposed
Plasma:

Purified
using
microcon
100
with
60
ug
BSA
(
6
ug
injected)

­
0
.005
0.005
0.015
0.025
0.035
0.045
1
5
75
1
14
9
172
3
229
7
2
8
71
3
44
5
4
01
9
459
3
516
7
5
7
41
6
3
15
6
88
9
7
46
3
803
7
8
6
11
9
1
85
9
75
9
Time
(
600/
min)
Series1
BSA
Vtg
Rank
Probability
Z
score
Protein
Information
and
Sequence
Analyse
Tools
(
T)
%
pI
+
1
1.0e+
000
2.08
gi|
4572552|
gb|
AAD23878.1|
AF130354_
1
vitellogenin
precursor
[
Pimephales
promelas]
17
9.0
­
­
gi|
15778562|
gb|
AAL07472.1|
AF414432_
1
vitellogenin
[
Cyprinus
carpio]
13
9.1
+
2
2.8e­
005
0.14
T
gi|
6006011|
ref|
NP_
005492.1|
(
NM_
005501)
integrin
alpha
3,
isoform
b,
[
Homo
sapiens]
14
6.5
+
3
6.4e­
007
­
T
gi|
21362287|
ref|
NP_
653099.1|
RIKEN
cDNA
2210402G22
[
Mus
musculus]
25
9.1
4
2.3e­
007
­
T
gi|
20344336|
ref|
XP_
111772.1|
similar
to
put.
gag
and
pol
gene
(
aa
1­
814)
[
Mus
musculus]
19
9.6
+
5
9.8e­
008
­
T
gi|
16550881|
dbj|
BAB71072.1|
(
AK056006)
unnamed
protein
product
[
Homo
sapiens]
27
9.8
6
1.4e­
008
­
T
gi|
13385164|
ref|
NP_
079982.1|
RIKEN
cDNA
4432405K22
[
Mus
musculus]
17
5.2
+
7
1.1e­
008
­
T
gi|
14735371|
ref|
XP_
027054.1|
(
XM_
027054)
KIAA0674
protein
[
Homo
sapiens]
19
5.0
+
8
7.6e­
009
­
T
gi|
6005944|
ref|
NP_
009058.1|
(
NM_
007127)
villin
1;
Villin­
1
[
Homo
sapiens]
15
6.0
9
6.8e­
009
­
T
gi|
21391472|
gb|
AAK58480.1|
vitellogenin
1
[
Danio
rerio]
12
8.9
kDa
146.26
148.24
118.74
66.13
85.00
64.01
76.51
92.49
92.68
128.02
Vtg
Anion
Exchange
Fraction
from
treated
plasma
219PT18:

database
search
with
peptide
masses
1000
1500
2000
2500
3000
219PU18
VTG
AX
fraction
Digest
1000
1500
2000
2500
3000
Mass
(
m/
z)
219PS18
VTG
AX
fraction
Digest
1000
1500
2000
2500
3000
Standard
curve
20ug
Each
Vtg
&
BSA:

VTG
AX
fraction
digest
Relative
abundance
Comparison
of
MALDI­
MS
spectra
from
Vtg
Digests
vitellogenin
precursor
[
Pimephales
promelas]

Rank:
1
Probability
1.0
e
+
000
Z
score:
1.98
%
coverage:
17
vitellogenin
precursor
[
Pimephales
promelas]

Rank:
1
Probability
1.0
e
+
000
Z
score:
1.67
%
coverage:
25
vitellogenin
precursor
[
Pimephales
promelas]

Rank:
1
Probability
1.0
e
+
000
Z
score:
1.51
%
coverage:
15
Directly
analyze
VTG
from
fathead
minnow
plasma
using
a
combination
of
liquid
chromatography
and
mass
spectrometry.

A
membrane
filtration
pre­
purification
step
was
coupled
to
an
analytical
scale
anion
exchange
separation.
This
approach
to
MALDI­
MS
analysis
small
plasma
sample
(<
10
µ
l)
MALDI­
MS
22
Summary
Results
Zebrafish/
Medaka
VTG
Study
(
contd)

Summary
Results
Summary
Results
Zebrafish/
Medaka
VTG
Zebrafish/
Medaka
VTG
Study
Study
(
contd)

(
contd)

Quantification
of
VTG
was
not
absolute,
but
depended
on
method
used
and
on
the
laboratory
conducting
the
analysis
(
one
method
can
yield
results
thousands
of
times
higher
than
those
of
another
method,

but
with
similar
relative
trends)

Within­
run
variability
was
typically
low
(
CVs<
10%
for
zebrafish,
<
14%

for
medaka)
in
75%
of
results,
with
some
higher
outliers
that
accounted
for
a
broader
overall
range
of
values
Intra­
assay
variability
also
covered
broad
range
over
all,
but
75%
of
results
were
within
a
fairly
tight
distribution
(
CVs<
30%
zebrafish,
<
41%

medaka);
objective
of
detection
of
the
standard
series
was
generally
met
Little
variation
in
values
when
two
different
standards
were
used
for
calibration
for
medaka
for
all
three
methods;
similarly
low
for
zebrafish
VTG
values
for
two
methods,
but
high
for
one
method
23
Summary
Results
Fathead
Minnow
(
contd)

Summary
Results
Summary
Results
Fathead
Minnow
Fathead
Minnow
(
contd)

(
contd)

Quantification
of
VTG
was
not
absolute,
but
depended
on
method
used
and
on
the
laboratory
conducting
the
analysis
(
one
method
can
yield
results
thousands
of
times
higher
than
those
of
another
method,
but
with
similar
relative
trends)

Within­
run
variability
was
relatively
low
for
both
Plasma
and
Homogenate
CVs
range
0­
173%
mean
32%,
and
0
­
173%
mean
34%
respectively
with
some
higher
outliers
that
accounted
for
a
broader
overall
range
of
values
Intra­
assay
variability
also
covered
broad
range
for
both
sample
types
75%
of
the
CVs
were
less
than
51%.
This
high
level
of
intra­
assay
variability
indicates
that
the
methods
provide
a
high
degree
of
variability
when
replicate
samples
are
analyzed.

Detection
of
the
standard
series
trend
was
generally
met
for
Plasma
and
Homogenates
mRNA
levels
were
variable,
with
the
labs
reporting
generally
higher,

similar,
or
lower
levels
in
the
unexposed/
exposed
males
vs.
the
levels
found
in
unexposed/
exposed
females.

MALDI­
MS
allowed
detection
of
VTG
confidently
identifying
VTG
by
matching
experimental
data
with
sequences
in
protein
databases.

However,
problems
with
sample
degradation
after
thawing
prevented
quantitative
estimates
of
VTG
in
plasma
samples
24
Results
Fathead
Minnow,

Zebrafish/
Medaka
VTG
Study
(
contd)

Results
Fathead
Minnow,

Results
Fathead
Minnow,

Zebrafish/
Medaka
Zebrafish/
Medaka
VTG
Study
VTG
Study
(
contd)

(
contd)

Tukey's
Honestly
Significant
Difference
multiple
comparison
test
of
ranked
average
VTG
concentrations:
did
neighboring
means
significantly
differ?

Most
methods
could
detect
difference
between
uninduced
males
and
other
treatments;

differences
were
less
clear
cut
among
other
groups.

There
are
biological
reasons
as
well
as
differences
in
the
chemical
processes
of
the
various
methods
that
explain
the
results.

Positive
controls
were
spiked
at
a
specific,
concentration;
%
recovery
varied
widely
(
orders
of
magnitude)

Within­
lab
variability
low,
but
among
laboratories
and
methods
moderate
(
medaka)
to
high
(
zebrafish)
For
Fathead
minnow
both
within
and
among
laboratories
the
variability
was
relatively
high.
Results
are
typical
of
those
seen
in
other
studies
(
e.
g.,
Brion
et
al.

2002;
Holbech
et
al.
2001)
for
variability
of
positive
control
spike
recovery
Sources
of
variability
could
not
be
precisely
defined
within
the
confines
of
this
study.
Variability
due
to
the
following
factors
not
considered
in
this
statistical
analysis:

Shipping,
storage,
and
handling
of
samples
and
other
materials

Time
and
resource
restrictions

Technical
differences
among
protocols,
such
as
method
detection
limits

Biological
differences
among
fish
(
e.
g.,
reproductive
status
of
fish
during
treatment)
25
Discussion
and
Recommendations
Fathead
minnow,
Zebrafish/
Medaka
VTG
Study
Discussion
and
Recommendations
Discussion
and
Recommendations
Fathead
minnow,

Fathead
minnow,
Zebrafish/
Medaka
Zebrafish/
Medaka
VTG
Study
VTG
Study
Various
studies
have
demonstrated
utility
of
VTG
as
biomarker
indicating
endocrine
disruption
in
fish
Based
on
present
results,
most
laboratories
and
methods
considered
are
capable
of
distinguishing
changes
in
VTG
levels
in
Fathead
minnow,
zebrafish
and/
or
medaka
Issues
need
to
be
resolved
before
VTG
measurement
could
be
used
as
reliable
screening
and
testing
tool
Recommendations:

Develop
specific
performance
criteria
for
VTG
analytical
methods

Use
single,
standardized
protocol
for
each
fish
species
to
quantify
VTG
in
interlaboratory
validation
trials