COMPARATIVE EVALUATION OF VITELLOGENIN METHODS FOR FATHEAD MINNOW, MEDAKA AND

ZEBRA FISH

Photos: Biosense Laboratories

Endocrine Disruptor Methods Validation Endocrine Disruptor Methods Validation SubcommitteeSubcommitteeAugust 2003August 2003

Endocrine Disruptor Methods Validation Endocrine Disruptor Methods Validation SubcommitteeSubcommitteeAugust 2003August 2003

EPA Work Assignments: 2-19 and 2-26

Presented by: Michael L. Blanton and Dr. Irv Schultz

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COMPARATIVE EVALUATION OF VITELLOGENIN COMPARATIVE EVALUATION OF VITELLOGENIN METHODS FOR FATHEAD MINNOW, MEDAKA METHODS FOR FATHEAD MINNOW, MEDAKA AND ZEBRA FISHAND ZEBRA FISH

COMPARATIVE EVALUATION OF VITELLOGENIN COMPARATIVE EVALUATION OF VITELLOGENIN METHODS FOR FATHEAD MINNOW, MEDAKA METHODS FOR FATHEAD MINNOW, MEDAKA AND ZEBRA FISHAND ZEBRA FISH

WORK PERFORMED BY

On behalf of the United States Environmental Protection AgencyEPA CONTRACT NUMBER 68-W-01-023

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IntroductionIntroductionIntroductionIntroduction

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.

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VTG BackgroundVTG BackgroundVTG BackgroundVTG 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.

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Methods for Measuring VTG Methods for Measuring VTG induction in fishesinduction in fishes

Methods for Measuring VTG Methods for Measuring VTG induction in fishesinduction in fishes

Enzyme-Linked Immunosorbant Assays (ELISA) An enzyme immunoassay utilizing an enzyme-

labeled immunoreactant (antigen/antibody) and an immunosorbent (antigen/antibody bound to a solid support – i.e, a polystyrene microliter plate)

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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 preferred methods for quantifying fish VTG mRNA have emerged,

the ribonuclease protection assay (RPA) the quantitative reverse transcription-

polymerase chain reaction (QRT-PCR)

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

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

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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 Methods for Measuring VTG induction induction in fishes- Mass Spectrometry in fishes- Mass Spectrometry

Methods for Measuring VTG Methods for Measuring VTG induction induction in fishes- Mass Spectrometry in fishes- Mass Spectrometry

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Participants in the Participants in the Fathead Minnow VTG Study Fathead Minnow VTG Study Participants in the Participants in the Fathead Minnow VTG Study Fathead Minnow VTG Study

WA 2-19 Fathead Minnow participants1. University of Florida, USA2. University of Idaho, USA3. Oregon State University, USA4. US EPA Duluth, USA5. University of Exeter, USA6. Brixham Environmental Laboratory, UK7. Battelle Richland, USA8. Battelle Sequim, USA 9. Molecular Light Technology, UK10. Biosense, Norway 11. INERIS, France12. Cemagref, France13. University of Windsor, Canada14. University of Southern Denmark, Denmark15. Finnish Environmental Institute, Finland

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

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

WA 2-26 Zebrafish/Medaka Zebrafish/Medaka Participants1. Biosense Laboratories, Norway 2. Centre for Fish and Wildlife Health, University of Bern,

Switzerland3. Department of Pathology, Vet. Medicine, Swedish University

of Agricultural Sciences, Sweden4. EnBioTec Laboratories, Ltd., Japan5. Environmental and Symbiotic Sciences, Prefectural University

of Kumamoto, Japan6. Institute of Biology, University of Southern Denmark, Denmark7. Los Angeles County Sanitation District, USA8. National Institute for Environmental Studies (NIES), Japan9. Notox Safety & Environmental Research, the Netherlands10. Phylonix Pharmaceuticals, Inc., USA11. Unité d’Evaluation des Risques Ecotoxicologiques (INERIS),

France.

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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 laboratoriesDetermine 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 Objectives Fathead Minnow VTG StudyFathead Minnow VTG Study

Objectives Objectives Fathead Minnow VTG StudyFathead Minnow VTG Study

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Objectives Objectives Zebrafish/Medaka VTG StudyZebrafish/Medaka VTG StudyObjectives Objectives Zebrafish/Medaka VTG StudyZebrafish/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

Phot

o: B

org

2003

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Methods Fathead Minnow VTG Study (contd)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 β 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

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

Sample PreparationObtained approximately 400 zebrafish and 400 medaka Exposed subset of each group to 300 ng/L 17 β 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.

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

Shipment to participating labs

• Subsampled 20-µL 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 participating labs on 2 June 2003 with documentation, information, and standardized chain-of-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 femalepositive controlPurified vitellogenin as calibration standard for the species (S)

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Methods zebrafish Medaka VTG Study (contd)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]

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

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

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C: Medaka liver D. Medaka whole body

Results Results Zebrafish/Medaka VTG StudyZebrafish/Medaka VTG StudyResults Results Zebrafish/Medaka VTG StudyZebrafish/Medaka VTG Study

Expected trend was as follows:1 uninduced male < 2 uninduced female < 3 induced male < 4 induced female >> 5 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

VT

G C

on

cen

trati

on

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

Mea

n V

TG

Co

nce

ntr

atio

n

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

Mea

n V

TG

Co

nce

ntr

atio

n 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

Mea

n V

TG

Co

nce

ntr

atio

n 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

Code 0 = Blank; Code 1 = Uninduced Male; Code 2 = Uninduced Female; Code 3 = Induced male; Code 4 = Induced Female; Code 5 = Positive Control

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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

VT

G C

on

cen

trat

ion

(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

VT

G C

on

cen

trat

ion

(ug

/mL

)

Lab 1 Lab 4 Lab 5

Lab 6 Lab 7 Lab 11

Lab 13 Max detection Limit

Results Fathead Results Fathead Minnow VTG StudyMinnow VTG Study

Results Fathead Results Fathead Minnow VTG StudyMinnow VTG Study

Expected trend was as follows:

1 uninduced male < 2 uninduced female < 3 induced male < 4 induced female >>5 positive control

Results showed good relative tracking of trend, particularly Plasma, but higher variability in absolute measured values

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mRNA average for liver samples

0.1

1

10

100

1000

10000

100000

1000000

1 2 3 4

Lab 1 (pg VTG mRNA / ug totalRNA)

Lab 2 (mRNA VTG / total RNA)

Lab 15 (fmol VTG mRNA / ugtotal 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 TaqManLaboratory 15 HPA (hybridization protection assay)

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2 1 9 P U 1 8 E x p o s e d P l a s m a :P u r i f i e d u s i n g m i c r o c o n 1 0 0 w i t h 6 0 u g B S A ( 6 u g i n j e c t e d )

- 0 . 0 0 5

0 . 0 0 5

0 . 0 1 5

0 . 0 2 5

0 . 0 3 5

0 . 0 4 5

1

57

5

11

49

17

23

22

97

28

71

34

45

40

19

45

93

51

67

57

41

63

15

68

89

74

63

80

37

86

11

91

85

97

59

T i m e ( 6 0 0 / m i n )

S e r i e s 1

B S A

V t g

R a n k P r o b a b i l i t y Z s c o r e P r o t e i n I n f o r m a t i o n a n d S e q u e n c e A n a l y s e T o o l s ( T ) % p I

+ 1 1 . 0 e + 0 0 0 2 . 0 8 g i | 4 5 7 2 5 5 2 | g b | A A D 2 3 8 7 8 . 1 | A F 1 3 0 3 5 4 _ 1 v i t e l l o g e n i n p r e c u r s o r [ P i m e p h a l e s p r o m e l a s ]

1 7 9 . 0

- - g i | 1 5 7 7 8 5 6 2 | g b | A A L 0 7 4 7 2 . 1 | A F 4 1 4 4 3 2 _ 1 v i t e l l o g e n i n [ C y p r i n u sc a r p i o ]

1 39 . 1

+ 22 . 8 e - 0 0 5 0 . 1 4 T g i | 6 0 0 6 0 1 1 | r e f | N P _ 0 0 5 4 9 2 . 1 | ( N M _ 0 0 5 5 0 1 ) i n t e g r i n a l p h a 3 , i s o f o r m b , [ H o m o s a p i e n s ]

1 4

6 . 5

+ 3 6 . 4 e - 0 0 7 - T g i | 2 1 3 6 2 2 8 7 | r e f | N P _ 6 5 3 0 9 9 . 1 | R I K E N c D N A 2 2 1 0 4 0 2 G 2 2 [ M u s m u s c u l u s ]2 5

9 . 1

4 2 . 3 e - 0 0 7 - T g i | 2 0 3 4 4 3 3 6 | r e f | X P _ 1 1 1 7 7 2 . 1 | s i m i l a r t o p u t . g a g a n d p o l g e n e ( a a 1 - 8 1 4 ) [ M u s m u s c u l u s ]1 9

9 . 6

+ 5 9 . 8 e - 0 0 8 - T g i | 1 6 5 5 0 8 8 1 | d b j | B A B 7 1 0 7 2 . 1 | ( A K 0 5 6 0 0 6 ) u n n a m e d p r o t e i n p r o d u c t [ H o m o s a p i e n s ] 2 7

9 . 8

6 1 . 4 e - 0 0 8 - T g i | 1 3 3 8 5 1 6 4 | r e f | N P _ 0 7 9 9 8 2 . 1 | R I K E N c D N A 4 4 3 2 4 0 5 K 2 2 [ M u s m u s c u l u s ]1 7

5 . 2

+ 7 1 . 1 e - 0 0 8 -T g i | 1 4 7 3 5 3 7 1 | r e f | X P _ 0 2 7 0 5 4 . 1 | ( X M _ 0 2 7 0 5 4 ) K I A A 0 6 7 4 p r o t e i n [ H o m o s a p i e n s ]

1 9

5 . 0

+ 8 7 . 6 e - 0 0 9 - T g i | 6 0 0 5 9 4 4 | r e f | N P _ 0 0 9 0 5 8 . 1 | ( N M _ 0 0 7 1 2 7 ) v i l l i n 1 ; V i l l i n - 1 [ H o m o s a p i e n s ]1 5

6 . 0

9 6 . 8 e - 0 0 9 - T g i | 2 1 3 9 1 4 7 2 | g b | A A K 5 8 4 8 0 . 1 | v i t e l l o g e n i n 1 [ D a n i o r e r i o ] 1 2

8 . 9

k D a

1 4 6 . 2 6

1 4 8 . 2 4

1 1 8 . 7 4

6 6 . 1 3

8 5 . 0 0

6 4 . 0 1

7 6 . 5 1

9 2 . 4 9

9 2 . 6 8

1 2 8 . 0 2

V t g A n i o n E x c h a n g e F r a c t i o n f r o m t r e a t e d p l a s m a 2 1 9 P T 1 8 :d a t a b a s e s e a r c h w i t h p e p t i d e m a s s e s

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

Rela

tive

abun

danc

e

Comparison of MALDI -MS spectra from Vtg Digests

vitellogenin precursor [ Pimephales promelas ]Rank: 1 Probability 1.0 e +000Z 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 +000Z 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 l)

MALDI-MS

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

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Summary Results Summary Results Fathead Minnow Fathead Minnow (contd)(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 valuesIntra-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 HomogenatesmRNA 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

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Results Fathead Minnow, Results Fathead Minnow, Zebrafish/Medaka VTG Study Zebrafish/Medaka VTG Study (contd)(contd)

Results Fathead Minnow, Results Fathead Minnow, Zebrafish/Medaka VTG Study Zebrafish/Medaka 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)

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Discussion and Recommendations Discussion and Recommendations Fathead minnow, Zebrafish/Medaka Fathead minnow, Zebrafish/Medaka

VTG StudyVTG Study

Discussion and Recommendations Discussion and Recommendations Fathead minnow, Zebrafish/Medaka Fathead minnow, Zebrafish/Medaka

VTG StudyVTG StudyVarious studies have demonstrated utility of VTG as biomarker indicating endocrine disruption in fishBased on present results, most laboratories and methods considered are capable of distinguishing changes in VTG levels in Fathead minnow, zebrafish and/or medakaIssues need to be resolved before VTG measurement could be used as reliable screening and testing toolRecommendations: Develop specific performance criteria for VTG analytical methods Use single, standardized protocol for each fish species to quantify

VTG in interlaboratory validation trials