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FINAL REPORT

Medaka Multigeneration Test of

Vinclozolin

Study Sponsor:

Battelle Pacific Northwest Division

Marine Sciences Laboratory

1529 West Sequim Bay Road

Sequim, Washington

United States  98382‐8415

Study Test Facility:

Maxxam Analytics  

4606 Canada Way

Burnaby, BC

V5G 1K5 Canada

Tel: 604 734 7276

Fax: 604 731 2386

Project No.:  2‐11‐1057

Study No.: TOX1105

Test Item ID:  110708A‐01

Study Completion Date: April 17, 2013
Final Report

Medaka Multigeneration Test of Vinclozolin

Study No.: TOX1105

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EXECUTIVE SUMMARY

The potential chronic effects of Vinclozolin on fish populations were assessed using a

multigeneration test with medaka (Oryzias latipes). The procedures performed in this

study were based on the draft guideline for the Medaka Multigeneration Test, dated

April 19, 2010. This test was conducted to assess the chronic effects of Vinclozolin on

medaka over a 170‐d period by observing survival, gross development, growth, and

reproduction. The potential for endocrine disruption was also evaluated by

determination of genotypic sex and Vitellogenin (VTG) levels. The test was performed

under controlled conditions using a flow‐through system to ensure constant renewal of

the test chemical, due to anticipated hydrolysis. The test organisms were exposed to

nominal concentrations of 8, 19, 48, 120, and 300 μg test item/L. In addition, a negative

control containing dilution water only was tested.  


Final Report

Medaka Multigeneration Test of Vinclozolin

Study No.: TOX1105

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DECLARATION STATEMENT

Differences in calculated means, SD and sample size between the statistical report and

the study report reflect differences in the number of fish excluded from analysis due to

lack of genotypic or histological sex determination.   At time of draft report preparation,

the complete histological sex information was not available and so more fish may have

been excluded relative to the statistical report.  

In the statistical report, the weeks‐post‐fertilization (wpf) was rounded to the nearest

week for the F1 day 119 and F2 day 168 terminations.
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TABLE OF CONTENTS

1 STUDY PURPOSE...............................................................................................................12

2 SPONSOR AND TEST FACILITY INFORMATION ...................................................................13

3 STUDY DATES (AMENDMENT #1)......................................................................................15

4 TEST AND REFERENCE SUBSTANCE INFORMATION ...........................................................16

4.1 Test Substance ...............................................................................................................16

4.2 Reference Substance......................................................................................................16

5 DESCRIPTION OF MATERIALS............................................................................................17

5.1 Materials and Apparatus................................................................................................17

6 TEST METHODS ................................................................................................................20

6.1 Test Conditions...............................................................................................................20

6.2 Study Design Rationale for a Difficult Substance...........................................................22

6.2.1 Stock and Test Solution Preparation.................................................................22

6.3 Water Quality Measurements .......................................................................................23

6.4 Test Observations ..........................................................................................................23

6.5 Feeding...........................................................................................................................24

6.6 Additional End Points.....................................................................................................26

6.6.1 Genotypic Sex Determination...........................................................................26

6.6.2 Vitellogenin‐1 (VTG‐1) Copy Number Determination.......................................26

6.6.3 Additional Phenotypic Endpoints......................................................................26

6.7 Statistical Analysis..........................................................................................................27

6.8 Test Validity Criteria.......................................................................................................27

6.9 Confirmatory Chemical Analysis ....................................................................................27

6.9.1 Confirmatory Chemical Analysis Sampling........................................................27

7 TEST RESULTS ...................................................................................................................28

7.1 Confirmatory Chemical Analysis ....................................................................................28

7.2 Water Quality.................................................................................................................33

7.3 F0 Fish Fecundity Metrics ..............................................................................................36

7.4 F1 Fish Survival Metrics..................................................................................................37

7.5 F1 Fish Weight and Length Metrics (Day 79‐80)............................................................40

7.6 F1 Fish Weight and Length Metrics (Day 119) ...............................................................47

7.7 F1 Anal Fin Papillae (Day 119)........................................................................................49
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Medaka Multigeneration Test of Vinclozolin

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7.8 F1 Fish Fecundity Metrics ..............................................................................................49

7.9 F2 Fish Survival Metrics..................................................................................................54

7.10 F2 Fish Weight and Length Metrics................................................................................56

7.11 F2 Anal Fin Papillae (Day 169/170) ................................................................................61

7.12 Test Validity Criteria.......................................................................................................62

8 DISCUSSION .....................................................................................................................63

8.1 Test Substance Concentration .......................................................................................63

8.2 Water Quality.................................................................................................................63

8.3 Summary Statistics.........................................................................................................64

8.3.1 F0 Fertility and Fecundity..................................................................................64

8.3.2 F1 Hatching Success and Survival......................................................................64

8.3.3 F1 Growth (Lengths and Weights) ....................................................................64

8.3.4 F1 Secondary Sex Characteristics (Anal Fin Papillae)........................................65

8.3.5 F1 Fertility and Fecundity..................................................................................65

8.3.6 F2 Hatching Success and Survival......................................................................65

8.3.7 F2 Growth (Lengths and Weights) ....................................................................66

8.3.8 F2 Secondary Sex Characteristics (Anal Fin Papillae)........................................66

8.4 Study Challenges ............................................................................................................66

9 ARCHIVING.......................................................................................................................68

10 LITERATURE CITED............................................................................................................69

APPENDICES

A GLP Certificate of Recognition

B Study Plan Including Amendments and Deviations

C Test Substance Information

D Certificate of Analysis

E Material Safety Data Sheet

F Confirmatory Analysis Report

G Test Results and Data Analysis

H Raw Data for Definitive Test

I Laboratory Notebooks

J Water Quality Data Summaries

K Dilution Water Chemistry

L Statistical Analysis Report

M Medaka Sexing and Vitellogenin Report
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LIST OF TABLES  

Table 4‐1: Test Substance Properties ............................................................................... 16

Table 5‐1: Source of Materials, Apparatus, and Reagents ............................................... 17

Table 6‐1: Summary of Test Conditions (Amendment #1) ............................................... 20

Table 6‐2: Acceptable Parameter Levels in Control/Dilution Water ................................ 21

Table 6‐3: Feeding Rates for Duration of Medaka Multigeneration Test......................... 24

Table 7‐1: Measured Concentrations of Test Substance in Stock Solutions Post Generator

Columns ........................................................................................................... 28

Table 7‐2: Measured Concentrations of Test Substance in Toxicity Test Samples........... 29

Table 7‐3: F0 Pooled Cumulative Egg Production............................................................. 36

Table 7‐4: F0 Pooled Cumulative Egg Production per Day ............................................... 36

Table 7‐5: F1 Medaka Egg Hatching Success .................................................................... 38

Table 7‐6: F1 Number of Hatchlings Placed in Each Replicate 7‐8 d Post Fertilization .... 38

Table 7‐7: F1 Number of Surviving Fish at Day 43, 21 Days Post Fertilization ................. 39

Table 7‐8: F1 Percentage of Surviving Fish at Day 43, 21 Days Post Fertilization ............ 39

Table 7‐9: F1 Number of Fish Remaining in Each Replicate on Day 43 ............................ 39

Table 7‐10: F1 Number of Surviving Fish at F1 Termination, Day 79/80 & Day 119 .......... 40

Table 7‐11: F1 Percent of Surviving Fish at F1 Termination Day 119 ................................. 40

Table 7‐12: F1 Treatment 1 Day 79 and 80 Fish Weights (g).............................................. 41

Table 7‐13: F1 Treatment 2 Day 79 and 80 Fish Weights (g).............................................. 41

Table 7‐14: F1 Treatment 3 Day 79 and 80 Fish Weights (g).............................................. 42

Table 7‐15: F1 Treatment 4 Day 79 and 80 Fish Weights (g).............................................. 42

Table 7‐16: F1 Treatment 5 Day 79 and 80 Fish Weights (g).............................................. 43

Table 7‐17: F1 Treatment 6 Day 79 and 80 Fish Weights (g).............................................. 43

Table 7‐18: F1 Treatment 1 Day 79 and 80 Fish Lengths (mm) .......................................... 44
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Medaka Multigeneration Test of Vinclozolin

Study No.: TOX1105

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Table 7‐19: F1 Treatment 2 Day 79 and 80 Fish Lengths (mm) .......................................... 44

Table 7‐20: F1 Treatment 3 Day 79 and 80 Fish Lengths (mm) .......................................... 45

Table 7‐21: F1 Treatment 4 Day 79 and 80 Fish Lengths (mm) .......................................... 45

Table 7‐22: F1 Treatment 5 Day 79 and 80 Fish Lengths (mm) .......................................... 46

Table 7‐23: F1 Treatment 6 Day 79 and 80 Fish Lengths (mm) .......................................... 46

Table 7‐24: F1 Female Medaka Mean Weights and Lengths (97 Days Post‐Fertilization) . 47

Table 7‐25: F1 Male Medaka Mean Weights and Lengths (97 Days Post‐Fertilization) ..... 48

Table 7‐26: F1 Medaka Anal Fin Papillae Counts (Study Day 119, 97 Days Post‐Fertilization)

......................................................................................................................... 49

Table 7‐27: F1 Cumulative Egg Production (Cumulative Eggs) ........................................... 50

Table 7‐28: F1 Cumulative Egg Production (Cumulative Eggs Per Day).............................. 51

Table 7‐29: F1 Weight Normalized Cumulative Egg Production (Cumulative Eggs/g/d).... 52

Table 7‐30: F2 Medaka Egg Hatching Success .................................................................... 54

Table 7‐31: F2 Number of Hatchlings Placed in Each Replicate 13 d Post Fertilization ..... 54

Table 7‐32: F2 Number of Surviving Fish at Day 134, 21 Days Post Fertilization ............... 55

Table 7‐33: F2 Percent of Surviving Fish at Day 134, 21 Days Post Fertilization ................ 55

Table 7‐34: F2 Number of Remaining in Each Replicate on Day 134.................................. 55

Table 7‐35: F2 Number of Surviving Fish at Test Termination, Day 169/170..................... 56

Table 7‐36: F2 Percent of Surviving Fish at Test Termination Day 169/170....................... 56

Table 7‐37: F2 Medaka (9 wpf) Mean Weights (g) Treatment 1 ........................................ 56

Table 7‐38: F2 Medaka (9 wpf) Mean Weights (g) Treatment 2 ........................................ 57

Table 7‐39: F2 Medaka (9 wpf) Mean Weights (g) Treatment 3 ........................................ 57

Table 7‐40: F2 Medaka (9 wpf) Mean Weights (g) Treatment 4 ........................................ 57

Table 7‐41: F2 Medaka (9 wpf) Mean Weights (g) Treatment 5 ........................................ 58

Table 7‐42: F2 Medaka (9 wpf) Mean Weights (g) Treatment 6 ........................................ 58

Table 7‐43: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 1..................................... 59

Table 7‐44: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 2..................................... 59

Table 7‐45: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 3..................................... 59
Final Report

Medaka Multigeneration Test of Vinclozolin

Study No.: TOX1105

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Table 7‐46: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 4..................................... 60

Table 7‐47: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 5..................................... 60

Table 7‐48: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 6..................................... 60

Table 7‐49: F2 Anal Fin Papillae Counts (Day 169/170)...................................................... 61

Table J‐1: Conductivity Measured in the Test Solutions (μS/cm) 

Table J‐2: Water Temperature in the Test Solutions (°C) 

Table J‐3: Dissolved Oxygen (DO) Concentrations in the Test Solutions (mg/L) 

Table J‐4: Measured pH in the Test Solutions
Final Report

Medaka Multigeneration Test of Vinclozolin

Study No.: TOX1105

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LIST OF FIGURES

Figure 7‐1: Treatment 2: Concentration of Vinclozolin (nominal 8 μg/L)  

in Flow‐through Test Solution Samples Over Time.......................................... 31

Figure 7‐2: Treatment 3: Concentration of Vinclozolin (nominal 19 μg/L)  

in Flow‐through Test Solution Samples Over Time.......................................... 31

Figure 7‐3: Treatment 4: Concentration of Vinclozolin (nominal 48 μg/L)  

in Flow‐through Test Solution Samples Over Time.......................................... 32

Figure 7‐4: Treatment 5: Concentration of Vinclozolin (nominal 120 μg/L)  

in Flow‐through Test Solution Samples Over Time.......................................... 32

Figure 7‐5: Treatment 6: Concentration of Vinclozolin (nominal 300 μg/L)  

in Flow‐through Test Solution Samples Over Time.......................................... 33

Figure 7‐6: Conductivity (μS/cm) measured in test vessels over the duration  

of the exposure phase. .................................................................................... 34

Figure 7‐7: Temperature measured in test vessels over the duration  

of the exposure phase. .................................................................................... 34

Figure 7‐8: Dissolved oxygen concentrations measured in test vessels over  

the durationof the exposure phase. ................................................................ 35

Figure 7‐9: pH measured in test vessels over the duration of the exposure phase. ......... 35

Figure 7‐10: F0 Cumulative Egg Production per day for each treatment group over time . 37

Figure 7‐11: F1 Cumulative Egg Production per Day per Gram Female Fish Weight........... 53
Final Report

Medaka Multigeneration Test of Vinclozolin

Study No.: TOX1105

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SECTION

1 STUDY PURPOSE

The potential of the test substance, Vinclozolin, to have chronic effects on the test fish

(medaka) was determined based on the draft guideline for the Medaka Multigeneration

Test, dated April 19, 2010. The Maxxam Study Plan, “Medaka Multigeneration Test of

Vinclozolin,” including 14 deviations and 10 amendments, (Appendix B) was followed.

The chronic effects of Vinclozolin on medaka were assessed by observing survival, gross

development, growth, and reproduction over a 170‐d period. The potential for

endocrine disruption was also evaluated by determination of genotypic sex and

Vitellogenin (VTG) levels.
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Study No.: TOX1105

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SECTION

2 SPONSOR AND TEST FACILITY INFORMATION

Personnel Name and Contact Information

Sponsor:  

Battelle Pacific Northwest

Division

Marine Sciences Laboratory

1529 West Sequim Bay Road

Sequim, Washington

United States  98382‐8415

Sponsor’s

Representative:

Irv Schultz, Ph.D.

Tel: 360 681 4566

E‐mail: irv.schultz@pnl.gov

Fax: 360 681 3600

Study Director:

(July 18, 2011 – March

6, 2012; July 23, 2012 –

August 18, 2012)

Jeremy Keating, B.Sc.

Tel: 604 639 8425

E‐mail: jkeating@maxxam.ca  

Study Director:

(March 6, 2012 – July

23, 2012; August 18 –

September 3, 2012)

Pamela Howes, B.Sc.

Tel: 604 639 8424

E‐mail: phowes@maxxam.ca  

Test Facility

Management:

Patricia Caines, B.Sc.

Manager, Product Testing Services

Tel: 604 639 2630

E‐mail: pcaines@maxxam.ca

Test Facility:

Maxxam Analytics

4606 Canada Way

Burnaby, BC  

Canada  V5G 1K5

Phone: 604 734 7276

Fax: 604 731 2386

QA Manager, BC

Region:

Said Zeinab

Tel: 604 639 2619

E‐mail: szeinab@maxxam.ca

Contributing Scientist:

(Confirmatory Analysis)

Jon Le Huray, Ph.D.

Tel: 604 638 5024

E‐mail: jlehuray@maxxam.ca

Test Site:

Maxxam Analytics

8577 Commerce Court

Burnaby, BC

Canada  V5A 4N5 Test Site Management: Patricia Caines, B.Sc.

Manager, Product Testing Services

Tel: 604 639 2630

E‐mail: pcaines@maxxam.ca
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Contributing Scientist

(Genetic

Sexing/Vitellogenin

Determination):

Michael Cobb

Tel: 360 681 4580

E‐mail: m.cobb@pnl.gov

Test Site:

Battelle Pacific Northwest

Division,  

Marine Sciences Laboratory

1529 West Sequim Bay Road

Sequim, Washington

United States  98382‐8415

Contributing Scientist

(Statistical Analysis):

Valerie Cullinan

Tel: 360 681 3662

E‐mail: val.cullinan@pnl.gov
Final Report

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Study No.: TOX1105

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SECTION

3 STUDY DATES (AMENDMENT #1)

Experimental Initiation Date: 5 March 2012

Test Initiation Date: 14 March 2012

Test Completion Date: 29 August 2012

Experimental Completion Date: 29 January 2013

Study Completion Date: 17 April 2013
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Study No.: TOX1105

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SECTION

4 TEST AND REFERENCE SUBSTANCE INFORMATION

4.1 Test Substance

The sample receipt information, including copies of the Maxxam sample login

information and other information received with the sample, is presented in Appendix

C. Copies of both the Certificate of Analysis and Material Safety Data Sheet for the test

item follow in Appendix D and E, respectively. Table 4‐1 lists the available information

on the test item.

Table 4‐1: Test Substance Properties

Name:   Vinclozolin

Source: Battelle

CAS No.: 50471‐44‐8

Lot No.: 481‐36B (Deviation #2, Amendment #2) a

Purity: 99.5% a

Molecular Formula: C12H9Cl2NO3

Solubility in Water: Very slightly soluble b

Storage Conditions: Dark, ambient temperature

Expiration Date: January 2015 (Amendment #2)a

a From Certificate of Analysis (For test substance received Feb. 5, 2012)

b From Material Safety Data Sheet for Vinclozolin

4.2 Reference Substance

No reference substance was employed in this study. Vinclozolin was used as an external

standard during the confirmatory chemical analysis.
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Study No.: TOX1105

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SECTION

5 DESCRIPTION OF MATERIALS

5.1 Materials and Apparatus

The source of materials and apparatus used in the study are listed in Table 5‐1.

Instrument logbooks and calibration records, where applicable, were archived in the

GLP Facility Record Section of the Maxxam archives.

Table 5‐1: Source of Materials, Apparatus, and Reagents

Type Brand/Model Equipment ID or Lot

Number

Acetone Fisher 114797, 115967, 117325,

120759, 120763, 123343

Acetonitrile Fisher 093525

Analytical Balance Mettler BBY2‐0260

Analytical Balance, 4‐5 decimal

places

Mettler BBY2‐0016

Analytical Balance, 4‐5 decimal

places

Mettler BBY2‐0025

Artemia Cysts (Brine shrimp) Brine Shrimp Direct 111229, 120315, 120628

Balance Weights 0.0057 g

1g

10g

100g, 200g

003694

003708

003710

003709

CitriSolv Fisherbrand 360123

Conductivity Meter   WTW BBY2‐0021

Conductivity Probe TetraCon 07120115

Conductivity Standard (996 μS/cm) Fisher CC9947, CC10651

Controlled Environment Room Bio Room 103 N/A

DO & Temperature Meter   YSI BBY2‐0015  

DO & Temperature Probe YSI 09F10039

Dry Ice Praxair Rec’d Mar 12, 2012

Ethanol Fisher H128581, H134471, 9969,

10892
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Type Brand/Model Equipment ID or Lot

Number

Formalin (Buffered) Protocol 205986, 188927

223197

Freezer TOX GLP E Danby BBY2‐0029

Freezer ‐70°C Thermo BBY2‐0176

Fume Hood Supreme Air LV BBY2‐0146

Fume Hood Supreme Air LV BBY2‐0147

Glass Wool Pyrex 19711999

GLP Refrigerated Storage N/A TOX GLP C

GLP Refrigerator #1 Woods BBY2‐0104

GLP Room Temperature Storage N/A TOX GLP D

Hartmann’s Fixative Polysciences 623648, 637373

Hydrochloric Acid Fisher 211271, 212019, 212079

Microscope camera Moticam 2 BBY2‐0267

Microscope, Dissecting Meiji BBY2‐0268

Microscope, Dissecting Meiji   BBY2‐0154

Microscope, Dissecting Meiji   BBY2‐0034

MS222 Anaesthetic Finquel TR29.03C

pH Buffers pH 4 Fisher

pH 7 Fisher

pH 10 Fisher

116550, 120488

116537, 120143

114767, 116346, 120213,

122491

pH Meter Fisher Scientific Accumet Basic   BBY2‐0118  

pH Probe Fisher Scientific Accumet UP0117261

Phosphoric Acid (10%) Fisher 85% 106508

Pipette, 1‐10 mL Eppendorf BBY2‐0122

Pipette Eppendorf Repeat Pipettor BBY2‐0002, BBY2‐0172,

BBY2‐0270, BBY2‐0275

Pipette, 20 ‐ 200 mL Eppendorf BBY2‐0135

Pipette, 1‐5 mL Gilson BBY2‐0132

Pipette, 20 μL Gilson BBY2‐0105

Pipette, 10 μL VWR BBY2‐0134

Pipette, 1‐10 mL VWR BBY2‐0128, BBY2‐0273CV

Pipette, 1‐5 mL VWR BBY2‐0120
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Study No.: TOX1105

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Type Brand/Model Equipment ID or Lot

Number

Pipette, 100 mL VWR BBY2‐0274CV

Rose Bengal Aldrich 07497PJ

RNA Later Sigma‐Aldrich 061M0795, SLBB2455

Stirplate VWR/1KA S41 S/N 03.365419

Stirplate VWR/1KA S41 S/N 03.363891

Stirplate VWR/1KA S41 S/N 03.363898

Stirplate VWR/1KA S41 S/N 03.363880

Stirplate VWR/1KA S41 S/N 03.363890

Stirplate VWR/1KA S41 S/N 03.363881

Thermometer, Digital  Min/max   Fisher BBY‐0136

Timer, Digital Fisher Scientific Mini Alarm BBY2‐0010

Top Loading Balance   Mettler   BBY2‐0024

Tubing Pump Masterflex L/S BBY2‐0142

Tubing Pump Masterflex L/S BBY2‐0143

Tubing Pump Masterflex L/S BBY2‐0144

Tubing Pump Masterflex L/S BBY2‐0171

Tubing Pump Stenner 10‐1475

UV Steriliser/ Transformer Laguna Model PT‐1650/ PT‐1594 BBY2‐0269
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Medaka Multigeneration Test of Vinclozolin

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SECTION

6 TEST METHODS

6.1 Test Conditions

The specific test conditions during the test are summarised in Table 6‐1.

Table 6‐1: Summary of Test Conditions (Amendment #1)

Parameter Test Condition

Test type Flow‐through (average flow of ~20 mL/min for each replicate)

Duration 24 weeks

Test organism species Medaka (Oryzias latipes)

Source of test animals In‐house culture; original culture was obtained from Trent

University (Peterborough, ON) in May 2010

Age at test initiation 16 ‐ 17 weeks post fertilisation (wpf) (age‐synchronised to

within 1 week) (Amendment #1)

Test solution temperature 26 ± 1°C (Deviations #6, #7)

Photo‐period 16‐h light : 8‐h dark with a 30 minute transition period

Light intensity 100 – 500 lux during testing, up to 1000 lux during sampling

and observations

Control/dilution water

Hardened, dechlorinated city water (target hardness of 60 ±

10 mg/L as CaCO3, acceptable hardness range of 40‐300 mg/L

as CaCO3); aerated before use in the test

Feeding

Brine shrimp (Artemia) nauplii, pre‐determined amounts

based on dry weight of Artemia and percentage of fish body

weight

Test vessels ~4 L glass tank (9L tanks divided in half with glass)

Test volume 1.8 L

Replicates 6

Aeration None (except egg incubators)

Controls Laboratory (dilution water) control

Saturated Columns

One column prepared weekly with  exception of test initiation

(downstream column placed in upstream position, upstream

column removed, new column put in downstream position)

Target loading rates (±30%) 8, 19, 48, 120, and 300 μg/L
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Hardened, dechlorinated city water (hardness tolerance 40‐300 mg CaCO3/L) was used

for culturing the fish and as the control/dilution water throughout the test. The

hardness was typically between 50 and 70 mg CaCO3/L through the test. This water was

aerated before use. Hardness, alkalinity, pH, dissolved oxygen concentration,

conductivity, and temperature were measured at test initiation and weekly in the

control/dilution water to ensure constant and consistent water quality. Alkalinity was

analysed according to Standard Operating Procedure for pH, Specific Conductance,

Salinity and Alkalinity (Tot. and Phenolphthalein) (BBY6SOP‐00026). Hardness was

analysed as per the Analysis of Total Hardness (Titrimetric EDTA) in Water and

Wastewater (BBY2SOP‐00005). Samples of the water were also taken prior to test

initiation for heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (e.g. Ca,

Mg, Na, K, Cl, SO4, ammonia), pesticides, TOC, and suspended solids. Alkalinity,

hardness, metals (BBY7SOP‐00002), pesticides (CAM SOP‐00301 and CAM SOP‐00307),

TOC (BBY6SOP‐00003), suspended solids (BBY6SOP‐00034), and anion and cation

(BBY6SOP‐00046, BBY7SOP‐00002, BBY6SOP‐00009, BBY6SOP‐00017) analysis were

performed non‐GLP. Table 6‐2 includes some acceptable levels for the control/dilution

water. The analytical reports are presented in Appendix K. Unionised ammonia was

calculated based on a measurement of 0.50 mg/L total ammonia, with unionised

ammonia representing <40% of total ammonia at test temperature and pH.

Table 6‐2: Acceptable Parameter Levels in Control/Dilution Water

Parameter Acceptable Level Measured Level

Particulate matter (as total suspended solids) <20 mg/L <1.0 mg/L

Total organic carbon <2 mg/L 1.36 mg/L

Unionised ammonia <0.3 mg/L <0.3 mg/L

Residual chlorine <0.3 mg/L <0.1 mg/L

Total organophosphorus pesticides <1 μg/L <RDL

Total organochlorine pesticides, plus PCBs <0.1 μg/L <RDL

Light intensity was 100‐500 lux and was measured according to the Work instruction for

the “Operation and Maintenance of Lights Meter” (BBY2 WI‐00016).

The test was initiated using fish that were between 16 to 17 wpf old (Amendment #1).

Ten fish, five of each sex, were taken for weight measurement (females were ≥250 mg,

males were ≥225 mg each, Amendment #1) to ensure that the fish being used in the

study were of appropriate size and health. Forty‐eight male and forty‐eight female fish

were anesthetised, caudal fin clipped, and segregated by pairs (one male and one

female) in a labeled retention basket (Amendment #1). The fin samples were placed in

coded/labeled 96‐well plate, allowed to air dry for approximately four hours, then

sealed and placed on dry ice for overnight shipment to Battelle for PCR analysis for the

DMY gene.  
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Once genetic sex was determined (within 48 hours), a female‐male pair was randomly

assigned to each of the 42 test chambers. Each test chamber (tank) was considered a

replicate, with 5 concentrations of test substance and one laboratory control consisting

of dilution water alone.  The tanks for the laboratory control and the 5 concentrations of

the test substance were connected to the diluter system to ensure constant refreshing

of the test substance concentration.

Prior to use, all containers and apparatus used were thoroughly cleaned according to

the Work Instruction for Cleaning of Laboratory Ware (BBY WI‐00001). While in use, the

test vessels were covered with Plexiglas to prevent evaporation, except for replicates A

and B when incubators were in place. The test organisms were fed during the exposure

period as per Section 5.6 (Deviation #1).

6.2 Study Design Rationale for a Difficult Substance  

In order to prepare the test loading rates and attain measurable concentrations of the

test substance as close as possible to the target loading rates, glass columns (packed

with glass wool) were coated with the test substance.

The resulting stock solution was pumped into the diluter system using a peristaltic pump

to a nominal concentration of 300 μg/L ± 30% (Amendment #1). The highest test

concentration was then sequentially diluted by a factor of approximately 2.5 to produce

the loading rates outlined in Table 6‐1.

6.2.1 Stock and Test Solution Preparation

Two glass columns, packed with glass wool and coated with the test substance, were

used in series. During the test, dilution water was continuously passed through the

columns at a rate which provided the test substance concentration or “stock solution”

(e.g. ~2.8 mg/L) necessary to maintain the required test loading rates (±30%). Columns

were prepared as per the procedure in the Vinclozolin Column Preparation SOP (EPA

2011); however, the test substance was weighed out in two 2.5 g aliquots, each of which

was independently added to ~37.5 mL acetone. Each of these volumes was then used to

load one side of a column. All glassware used to prepare the solutions and add them to

the columns was rinsed with acetone to ensure that all test substance was added to the

columns and there was no residual test substance on the glassware (Amendment #1).

Each column was used for 14 days. Columns were connected serially such that dilution

water flowed through the upstream column and then through the downstream column

and finally into the dilution chamber for use in the experimental setup. Columns were

used for a maximum of 7 days in each of the upstream and downstream positions; after

7 days in the upstream position, the column was removed and replaced in that position

by the column formerly occupying the downstream position. The downstream column

was then replaced with a new, pre‐flushed unused Vinclozolin‐loaded column. One
Final Report

Medaka Multigeneration Test of Vinclozolin

Study No.: TOX1105

Maxxam Analytics 23 of 70

exception was the first upstream column to be used in the test, which was only used for

a total of 7 days before being removed (Amendment #1).  

The test substance was handled according to the Work Instruction for the Management

of Test and Reference Items (BBY1 WI‐00008).

All chemicals were weighed using analytical balances, which will be operated according

to the procedures outlined in the Work Instruction “Corporate Procedure for Balance

Calibrations and Verifications” (COR WI‐00015).

6.3 Water Quality Measurements

The conductivity, pH, temperature and dissolved oxygen (DO) concentrations were

measured in one replicate of each test treatment at test initiation and weekly during the

study. Measurements were taken from replicates on a rotating schedule (e.g.: Replicate

A at test initiation, Replicate B on Day 7, and so on). Water quality was also conducted

weekly on the control/ dilution water and also included measurements of hardness and

alkalinity. Dissolved oxygen and temperature were measured according to the WI for

the Operation and Maintenance of YSI Dissolved Oxygen Meter (BBY2 WI‐00014). A

Fisher Scientific Accumet Basic pH meter was used to measure pH according to the WI

“Operation and Maintenance of Accumet Basic pH Meter” (BBY2 WI‐00015).

Conductivity was measured weekly according to the WI for the Operation and

Maintenance of WTW Conductivity Meter (BBY2 WI‐00012).  

6.4 Test Observations  

Flow to each tank was observed at least twice daily to ensure all tanks were receiving

flow of test solution. Flow rates through the generator column and to the dilution

mixing vessels were measured on weekdays. A visual inspection of the test chamber

water was made once weekly for precipitates indicative of insolubility of the test

substance.

Fish observations were performed daily to look for mortality or abnormality in the

adults, larvae, and eggs. Survival was recorded at specific time points for each

generation of fish. Lengths (mm) and weights (g) were measured from fish removed

from the experiment at specific time points.

Phenotypic sex was determined using anal fin counts at various points for the F1 and F2

generations.

The fertility (total number of eggs produced) and fecundity (total number of viable eggs

produced) were measured for both the F0 and F1 generations prior to starting the next

generation of fish.
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6.5 Feeding

Fish were fed live brine shrimp (Artemia) nauplii twice per day throughout the test

(morning and afternoon), at a feeding rate based on the size and number of fish. Table

6‐3 outlines the feeding rate per fish per day; therefore, each feeding consisted of half

of the value listed in Table 6‐3. Prior to test initiation, the dry weight to Artemia number

ratio was determined.

Artemia was prepared in separatory funnel by adding cysts to seawater and aerating

vigorously in a water bath close to a light source (Amendment #2). The cysts were left

for approximately 18 hours to hatch. To collect the newly hatched nauplii, the aeration

was stopped, and the unhatched cysts were allowed to settle to the bottom of the

funnel. The stopcock at the bottom of the funnel was opened momentarily to drain off

the unhatched cysts. The hatched nauplii were then allowed to settle for a maximum of

10 minutes. The nauplii were drained from the separatory funnel into a clean beaker.

The Artemia were poured into a sieve, rinsed with clean culture water, and resuspended

in a beaker to approximately 500 mL. A repeating pipettor was used to take 5 small (25

μL) aliquots, which were dispensed onto small filter papers. These filter papers were

inspected under a compound microscope to count the number of Artemia nauplii per 25

μL. Using the ratio of dry weight to nauplii number determined prior to test initiation,

the appropriate volume of the resuspended Artemia was dispensed into each test

replicate to match the required feeding rate per fish, as outlined in Table 6‐3.

Table 6‐3: Feeding Rates for Duration of Medaka Multigeneration Test

Test Week Test Day(s) Feeding Rate  

(dry weight)/fish/day

1 1‐7 22.5 mg

2 8‐14 22.5 mg

3 15‐21 22.5 mg

4 22‐23 22.5 mg

24‐28 No fish

5 29 No fish

30‐32 0.5 mg

33 0.6 mg

34 0.7 mg

35 0.8 mg

6 36 0.9 mg

37 1.0 mg

38 1.2 mg
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Test Week Test Day(s) Feeding Rate  

(dry weight)/fish/day

39 1.3 mg

40 1.5 mg

41 1.7 mg

42 1.8 mg

7 43 2.1 mg

44 2.3 mg

45 2.5 mg

46‐49 4.5 mg

8 50‐56 7.7 mg

9 57‐63 9.0 mg

10 64‐70 11.0 mg

11 71‐77 13.5 mg

12 78‐84 22.5 mg

13 85‐91 22.5 mg

14 92‐98 22.5 mg

15 99‐105 22.5 mg

16 106‐112 22.5 mg

17 113‐118 22.5 mg

119 No fish

18 120 No fish

18 121‐123 0.5 mg

124 0.6 mg

125 0.7 mg

126 0.8 mg

19 127 0.9 mg

128 1.0 mg

129 1.2 mg

130 1.3 mg

131 1.5 mg

132 1.7 mg

133 1.8 mg

20 134 2.1 mg

135 2.3 mg
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Test Week Test Day(s) Feeding Rate  

(dry weight)/fish/day

136 2.5 mg

137‐140 4.5 mg

21 141‐147 7.7 mg

22 148‐154 9.0 mg

23 155‐161 11.0 mg

24 162‐168 13.5 mg

6.6 Additional End Points

6.6.1 Genotypic Sex Determination

Fish were anesthetised using MS222, and a small piece of the caudal fin was removed

using dissection scissors. The caudal fins were clipped in one of two easily

distinguishable patterns: either a small piece was removed from the dorsal tip of the fin

or the ventral tip. The pieces of fin were then placed into 96 well plates, allowed to dry

for approximately 4 hours, then transferred to Battelle Sequim on dry ice for genotypic

sex determination (Amendment #1) Additional genotyping was performed by EPA

(Amendment #7).

6.6.2 Vitellogenin‐1 (VTG‐1) Copy Number Determination

The livers of sacrificed fish were removed and immediately placed into RNAlater at

room temperature. When all samples were collected, they were placed at 1 ‐ 6°C prior

to shipment. On the morning of shipment, the samples were moved to  ‐70 ± 10°C

before being transferred to Battelle for analysis (Deviation #4). The samples were

analysed by the University of Idaho (Deviation #14).

6.6.3 Additional Phenotypic Endpoints

To preserve anal fins for counting at a later date, the fish were bisected just posterior to

the body cavity, keeping the anal fin intact and connected to the posterior half of the

fish. The tissue was then laid flat and bathed in Hartmann’s fixative (Amendment #3) for

~30 seconds to prevent curling. The fins were immersed and stored in Hartmann's

fixative prior to counting. The body cavity of the remaining carcass was opened using a

scalpel or dissection scissors to allow perfusion of Hartmann’s fixative, and the entire

carcass was immersed for 72 ‐ 96 hours (Deviation #5). After this time, each carcass was

rinsed thoroughly in 70% ethanol, then placed in a transport medium (10% neutral

buffered formalin) and held at 1 – 6°C for transfer to Experimental Pathology
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Laboratories. Histopathological evaluation of medaka tissue samples collected was not

performed as a component of this study.

6.7 Statistical Analysis

Means and standard deviations of test measurements including fish weights and

lengths, anal fin papillae counts, and water quality measurements were calculated using

Microsoft Excel 2010. Additional statistical analyses were performed by Battelle, and are

included in Appendix L.

6.8 Test Validity Criteria

For a test to be valid the following conditions must be fulfilled:

 The dissolved oxygen concentration must be between  ≥  60% of air saturation

throughout the test

 The water temperature must not differ by more than ± 1.0°C between test

chambers or between successive days at any time during the test

 pH should not differ between replicates by more than ± 0.2

 There should be ≥ 80% survival of control animals after hatching through 21 days

post fertilisation (dpf) in each of the F1 and F2 generations and > 90% survival

from 21 dpf through termination for the F1 and F2 generations

 ≥ 80% hatching in control eggs in each of the F1 and F2 generations

6.9 Confirmatory Chemical Analysis

6.9.1 Confirmatory Chemical Analysis Sampling

Two sets of samples of the test solutions and from both columns from were collected

for confirmatory analysis at test initiation, day 6, day 13, day 20, day 27, and continued

weekly until day 167. Samples were also taken at test completion (day 170) for

confirmatory analysis. Samples were collected in 15 mL amber glass vials. Samples were

taken using the same replicate schedule as for water quality measurement (Section 6.3).

One set of samples were stored cold (1 to 6°C) until analysis while the second set was

stored at ‐70°C, within four hours of sampling, for possible future analysis.

Test solutions were analysed by HPLC‐UV. The machine was set with a flow rate of 0.4

mL/minute at 25°C with a wavelength of 212 nm. Aliquots of 50 μL were injected, with a

mobile phase of 50:50 acetonitrile:50 mM pH 3.3 phosphate buffer. The detailed

confirmatory analysis report is in Appendix F.
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Study No.: TOX1105

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SECTION

7 TEST RESULTS

Test results are tabulated in Appendix G and the raw data is presented in Appendix H.

Copies of the laboratory notebooks are included in Appendix I.

7.1 Confirmatory Chemical Analysis

The concentration of the Test Substance, Vinclozolin in the stock solutions post

generator columns is presented in Table 7‐1.

Table 7‐1: Measured Concentrations of Test Substance in Stock Solutions Post Generator Columns

    Concentration of Vinclozolin (μg/L)

Date Day Column 1 Column 2

27‐Feb‐12  ‐16 3726 3611

01‐Mar‐12  ‐13 3821 4033

05‐Mar‐12  ‐8 4012 4515

07‐Mar‐12  ‐6 3876 4323

14‐Mar‐12 1 3677 4344

19‐Mar‐12 6 4015 4344

26‐Mar‐12 13 3479 3911

02‐Apr‐12 20 3976 4960

09‐Apr‐12 27 3611 3976

16‐Apr‐12 34 3986 4451

23‐Apr‐12 41 3287 3356

30‐Apr‐12 48 3590 3852

07‐May‐12 55 3598 4279

14‐May‐12 62 3497 3784

22‐May‐12 70 2876 3111

28‐May‐12 76 3265 3551

04‐Jun‐12 83 3085 2993

11‐Jun‐12 90 3197 3470

18‐Jun‐12 97 3475 3769

25‐Jun‐12 104 3229 3552

03‐Jul‐12 112 3412 3738

09‐Jul‐12 118 3176 3490

16‐Jul‐12 125 3496 3608

23‐Jul‐12 132 3243 3419
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    Concentration of Vinclozolin (μg/L)

Date Day Column 1 Column 2

30‐Jul‐12 139 3416 4265

06‐Aug‐12 147 3280 3905

13‐Aug‐12 153 3311 3995

20‐Aug‐12 160 3214 3581

27‐Aug‐12 167 3257 3421

30‐Aug‐12 170 2999 3260

Average:    3469 3829

SD:    311.2 457.6

The observed concentrations of the Test Substance, Vinclozolin, in test solutions (Table

7‐2) were less than the method quantitation limit (QL) of 5 μg/L in the lab control at all

time points. The confirmatory analysis report is presented in Appendix F.

Table 7‐2: Measured Concentrations of Test Substance in Toxicity Test Samples

    Nominal Concentration (μg/L)

Date Time Point (d) 0 8 19 48 120 300

2012Mar1  ‐11 <5.0 8.3 23.4 52.1 136.8 334.5

2012Mar7  ‐7 <5.0 9.6 23.8 55.7 151.6 365.6

2012Mar14 0 <5.0 10.8 23.8 57.8 143.9 368.9

2012Mar26 13 <5.0 7.7 19.2 51.6 127.8 316.9

2012Apr2 20 <5.0 10.3 28.2 69.3 165.4 407.0

2012Apr9 27 <5.0 10.5 22.3 60.2 147.9 369.4

2012Apr16 34 <5.0 8.9 22.2 55.8 135.7 353.1

2012Apr23 41 <5.0 8.3 22.1 53.6 135.2 334.5

2012Apr30 48 <5.0 10.3 23.6 56.1 144.8 351.6

2012May7 55 <5.0 7.9 23.4 55.1 144.5 351.2

2012May14 62 <5.0 9.0 21.8 56.6 137.3 349.9

2012May22 70 <5.0 9.0 18.2 45.8 111.5 287.1

2012May28 76 <5.0 9.5 22.1 51.6 134.0 323.8

2012Jun4 83 <5.0 8.1 20.4 48.6 118.1 298.4

2012Jun11 90 <5.0 9.8 20.7 42.3 123.1 336.9

2012Jun18* 97 <5.0 8.0 21.6 51.1  ‐  326.1

2012Jun21* 100         152.5   

2012Jun25 104 <5.0 8.0 21.4 51.6 124.3 316.1

2012Jul3 112 <5.0 9.4 19.0 46.2 132.3 313.6

2012Jul9 118 <5.0 6.3 19.7 47.6 119.6 292.9

2012Jul16 125 <5.0 7.3 22.9 45.1 110.7 294.4

2012Jul23 132 <5.0 8.7 18.1 47.7 116.7 313.4

2012Jul30 139 <5.0 9.2 22.5 49.2 141.8 355.0
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Study No.: TOX1105

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    Nominal Concentration (μg/L)

Date Time Point (d) 0 8 19 48 120 300

2012Aug7 147 <5.0 12.5 22.2 47.6 128.2 321.7

2012Aug13 153 <5.0 9.5 22.9 55.3 146.7 354.9

2012Aug20 160 <5.0 8.8 20.6 52.3 125.3 326.6

2012Aug27 167 <5.0 7.0 19.6 59.3 119.3 311.4

2012Aug30 170 <5.0 8.3 19.1 47.6 121.0 303.3

Mean    <5.0 8.9 21.7 52.3 133.2 332.5

SD    ‐  1.3 2.2 5.7 13.8 28.3

n    27 27 27 27 27 27

Min (%)    <5.0

6.3

(78%)

18.1

(95%)

42.3

(88%)

110.7

(92%)

287.1

(96%)

Max (%)    <5.0

12.5

(157%)

28.2

(149%)

69.3

(144%)

165.4

(138%)

407.0

(136%)

Quantitation Limit: 5 μg Vinclozolin/L

* The Day 97 sample of 48 μg/L nominal concentration appeared to have been sampled twice, with one

sample being labeled as the 120 μg/L nominal concentration. The error was noted and the 120 μg/L

concentration re‐sampled on Day 100.

The concentration of the test substance in the control was below the quantitation limit

throughout the study.

The concentration of Vinclozolin in the flow‐through test solutions was generally within

20 % of the nominal concentration for all of the treatments. Figures 7‐1 – 7‐5 show that

the bias of the concentration was typically over the target nominal concentration the

majority of the time.   
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Figure 7‐1: Treatment 2: Concentration of Vinclozolin (nominal 8 μg/L) in Flow‐through Test Solution

Samples Over Time

Figure 7‐2: Treatment 3: Concentration of Vinclozolin (nominal 19 μg/L) in Flow‐through Test Solution

Samples Over Time
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Study No.: TOX1105

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Figure 7‐3: Treatment 4: Concentration of Vinclozolin (nominal 48 μg/L) in Flow‐through Test Solution

Samples Over Time

Figure 7‐4: Treatment 5: Concentration of Vinclozolin (nominal 120 μg/L) in Flow‐through Test Solution

Samples Over Time
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Figure 7‐5: Treatment 6: Concentration of Vinclozolin (nominal 300 μg/L) in Flow‐through Test Solution

Samples Over Time

7.2 Water Quality

The measured values, means, and standard deviation for conductivity, temperature,

dissolved oxygen concentration, and pH in the test solutions are listed in Tables J‐1 to J‐

4, respectively in Appendix J.  

Figures 7‐6 – 7‐9 shows the conductivity, temperature, dissolved oxygen and pH

measured in the test solutions for all treatments over the duration of the experiment.  
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Figure 7‐6: Conductivity (μS/cm) measured in test vessels over the duration of the exposure phase.

   

Figure 7‐7: Temperature measured in test vessels over the duration of the exposure phase.

Treatment Day

Temperature C

0 14 28 42 56 70 84 98 112 126 140 154 168

25.0

25.5

26.0

26.5

27.0

27.5

0

8

19

48

120

300

Treatment Day

Conductivity (

S/cm)

0 14 28 42 56 70 84 98 112 126 140 154 168

120

130

140

150

160

170

0

8

19

48

120

300
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Study No.: TOX1105

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Figure 7‐8: Dissolved oxygen concentrations measured in test vessels over the duration of the exposure

phase.

Figure 7‐9: pH measured in test vessels over the duration of the exposure phase.

Treatment Day

Dissolved Oxygen (mg O2/L)

0 14 28 42 56 70 84 98 112 126 140 154 168

6

7

8

8

19

48

120

300

0

Treatment Day

pH

0 14 28 42 56 70 84 98 112 126 140 154 168

7.0

7.2

7.4

7.6

7.8

8.0

0

8

19

48

120

300
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7.3 F0 Fish Fecundity Metrics  

The fecundity of the F0 medaka used in the experiment is summarized in Tables 7‐3 –

7.4. The F0 medaka cumulative egg production is presented in Table 7‐3.  

Table 7‐3: F0 Pooled Cumulative Egg Production

Treatment

1 2 3 4 5 6 Test Day Pooled Pooled Pooled Pooled Pooled Pooled

15 202 282 284 232 220 143

16 357 429 396 365 326 236

17 488 603 511 527 490 407

18 634 745 640 704 582 516

19 805 893 798 828 764 643

20 908 1009 884 948 868 762

21 1021 1087 1023 1107 996 828

22 1195 1229 1148 1252 1118 935

23 1299 1352 1254 1356 1247 1031

Mean/Day 144.3 150.2 139.3 150.7 138.6 114.6

SD 34.5 56.1 58.0 38.2 41.9 30.5

n 9 9 9 9 9 9

The F0 cumulative egg production per day is summarized in Table 7‐4.

Table 7‐4: F0 Pooled Cumulative Egg Production per Day  

Treatment

Test Day 1

Pooled

2

Pooled

3

Pooled

4

Pooled

5

Pooled

6

Pooled

15 202 282 284 232 220 143

16 179 215 198 183 163 118

17 163 201 170 176 163 136

18 159 186 160 176 146 129

19 161 179 160 166 153 129

20 151 168 147 158 145 127

21 146 155 146 158 142 118

22 149 154 144 157 140 117

23 144 150 139 151 139 115

Mean 162 188 172 173 157 126

SD 18.5 41.6 45.7 24.6 25.5 9.6

n 9 9 9 9 9 9
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The cumulative F0 egg production per day for all treatment groups is displayed in  

Figure 7‐10.

Figure 7‐10: F0 Cumulative Egg Production per day for each treatment group over time

7.4 F1 Fish Survival Metrics

The F1 medaka egg hatching data is presented in table 7‐5.  
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Table 7‐5: F1 Medaka Egg Hatching Success

   Test Substance Concentration (μg/L)

F1 Eggs Removed 0 8 19 48 120 300

Tank A 23 21 28 7 32 30

Tank B 16 14 29 37 33 40

Total 39 35 57 44 65 70

F1 Eggs Hatched        

Tank A 78 75 68 90 56 43

Tank B 72 72 59 50 47 36

Total 150 147 127 140 103 79

F1 Total Eggs        

Tank A 101 96 96 97 88 73

Tank B 88 86 88 87 80 76

Total 189 182 184 184 168 149

% Hatching Success 79.4% 81% 69% 76% 61% 53%

The F1 medaka survival data (with mean and standard deviation for final percent

survival) are presented in Tables 7‐6 – 7‐11 for each Vinclozolin treatment group. It

should be noted that in a few circumstances, more fish appear to have survived than

were originally placed in a tank or hatching vessel. This can likely be attributed to the

difficulties in counting small, fast‐moving fish in an enclosed vessel in the early stages of

the generation.

Table 7‐6: F1 Number of Hatchlings Placed in Each Replicate 7‐8 d Post Fertilization

Treatment (μg Vinclozolin/L)

Replicate 0 8 19 48 120 300

A 22 22 18 22 17 12

B 22 22 17 22 16 11

C 22 22 17 22 16 11

D 22 22 18 21 16 11

E 21 22 17 20 16 11

F 21 23 19 20 15 11
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Table 7‐7: F1 Number of Surviving Fish at Day 43, 21 Days Post Fertilization

Treatment (μg Vinclozolin/L)

Replicate 0 8 19 48 120 300

A 24 19 15 17 8 10

B 20 21 14 13 12 11

C 18 22 11 12 11 7

D 17 17 15 16 9 9

E 28 19 16 17 14 5

F 22 14 15 17 8 8

Table 7‐8: F1 Percentage of Surviving Fish at Day 43, 21 Days Post Fertilization

Treatment (μg Vinclozolin/L)

Replicate 0 8 19 48 120 300

A 109% 86% 83% 77% 47% 83%

B 91% 95% 82% 59% 75% 100%

C 82% 100% 65% 55% 69% 64%

D 77% 77% 83% 76% 56% 82%

E 133% 86% 94% 85% 88% 45%

F 105% 61% 79% 85% 53% 73%

Mean 100% 84% 81% 73% 65% 74%

SD 21% 14% 10% 13% 15% 19%

Table 7‐9: F1 Number of Fish Remaining in Each Replicate on Day 43

Treatment (μg Vinclozolin/L)

Replicate 0 8 19 48 120 300

A 10 10 10 10 8 10

B 10 10 10 10 10 10

C 10 10 10 10 10 7

D 10 10 10 10 9 9

E 10 10 10 10 10 5

F 10 10 10 10 8 8
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Table 7‐10: F1 Number of Surviving Fish at F1 Termination, Day 79/80 & Day 119

Treatment (μg Vinclozolin/L)

Replicate 0 8 19 48 120 300

A 10 8 9 10 6 9

B 7 9 7 9 10 8

C 9 10 10 10 10 8

D 10 8 10 9 9 9

E 9 10 9 8 10 6

F 10 10 9 9 9 8

Table 7‐11: F1 Percent of Surviving Fish at F1 Termination Day 119

Treatment (μg Vinclozolin/L)

Replicate 0 8 19 48 120 300

A 100% 80% 90% 100% 75% 90%

B 70% 90% 70% 90% 100% 80%

C 90% 100% 100% 100% 100% 114%

D 100% 80% 100% 90% 100% 100%

E 90% 100% 90% 80% 100% 120%

F 100% 100% 90% 90% 113% 100%

Mean 92% 92% 90% 92% 98% 101%

SD 12% 10% 11% 8% 12% 15%

7.5 F1 Fish Weight and Length Metrics (Day 79‐80)

The mean weights of the F1 fish removed from the experiment at day 79 and day 80 are

presented in Tables 7‐12  ‐  7‐17. The fish were grouped into different sexes based on

genotype before the mean and standard deviation was calculated. For grouping the fish,

genotype was considered more important than phenotype. The overall mean and

standard deviation for each treatment replicate is also presented. Some fish could not

be positively identified by genotypic sex analysis; these fish are presented as “X.”
Final Report

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Study No.: TOX1105

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Table 7‐12: F1 Treatment 1 Day 79 and 80 Fish Weights (g)GenotypicSex X M F Over

Replicate Mean SD n Mean SD n Mean SD n Mean SD 

A 0.2130*  ‐  1 0.1905 0.0205 4 0.1863 0.0215 3 0.1917 0.01

B 0.18801 0.0463 3 0.1968 0.0354 3 0.1924 0.03

C 0.17702  ‐  1 0.2110 0.0223 5 0.2553  ‐  1 0.2124 0.02

D 0.2047 0.0301 4 0.1921 0.0316 4 0.1984 0.02

E 0.2109 0.0416 5 0.2138  ‐  2 0.2117 0.03

F 0.1864 0.0352 5 0.1987 0.0305 4 0.1919 0.03

Notes: *Phenotype inconclusive, 1A1V, 1One male, IBID, had no weight measurement, 2Phenotype inconclusive, 1C4D, 3A ventrally‐clipped fish from 

generate the F replicate mating pair; therefore 9 fish were available to cull.

Table 7‐13: F1 Treatment 2 Day 79 and 80 Fish Weights (g)

GenotypicSex X M F Overa

Replicate Mean SD n Mean SD n Mean SD n Mean SD 

A 0.2032* 0.0978 3 0.1994† 0.0213 3 0.2013 0.063

B 0.1929 0.0311 4 0.2086 0.0496 4 0.2007 0.039

C 0.2037 0.0369 3 0.1916 0.0263 5 0.1961 0.028

D 0.1964  ‐  2 0.1909‡ 0.0115 4 0.1927 0.013

E 0.1835 0.0309 6 0.2165  ‐  2 0.1918 0.030

F 0.2241 0.0455 4 0.2198 0.0328 4 0.2219 0.036

Notes: *One male, 2A4V, had no weight measurement, †One female, 2A5V, had no weight measurement, ‡Two females, 2D2V & 2D3V, had no weig
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Table 7‐14: F1 Treatment 3 Day 79 and 80 Fish Weights (g)

GenotypicSex X M F Overal

Replicate Mean SD n Mean SD n Mean SD n Mean SD 

A 0.2143 0.0261 4 0.1910 0.0692 3 0.2043 0.0457 

B 0.1682 0.0297 3 0.2148* ‐  2 0.1868 0.0332 

C 0.2079  ‐  2 0.2029 0.0305 6 0.2041 0.0266 

D 0.1728  ‐  2 0.1993 0.0254 6 0.1927 0.0248 

E 0.1787 0.0263 5 0.1798  ‐  2 0.1790 0.0260 

F 0.1694 0.0049 3 0.1884 0.0339 5 0.1813 0.0276 

*One female, 3B2V, had no weight measurementTable 7‐15: F1 Treatment 4 Day 79 and 80 Fish Weights (g)

GenotypicSex X M F OveraReplicate Mean SD n Mean SD n Mean SD n Mean SD 

A 0.2024*  ‐  1 0.2018 0.0118 5 0.1883  ‐  2 0.1985 0.010

B 0.1591 0.0290 4 0.22371 0.0535 3 0.1868 0.050

C 0.1919 0.0363 5 0.2193 0.0306 3 0.2022 0.035

D 0.17052 0.0217 4 0.2038 0.0107 3 0.1847 0.024

E 0.2034 0.0090 4 0.20133  ‐  2 0.2027 0.009

F 0.21764  ‐  1 0.1613  ‐  1 0.2124 0.0273 5 0.2059 0.029

*Phenotype inconclusive, 4A4V, 1One female, 4B3V, had no weight measurement, 2One male, 4D1V, had no weight measurement, 3One fe

measurement, 4Phenotype inconclusive, 4F2V
Final Report

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Study No.: TOX1105

Maxxam Analytics 

Table 7‐16: F1 Treatment 5 Day 79 and 80 Fish Weights (g)

GenotypicSex X M F OveralReplicate Mean SD n Mean SD n Mean SD n Mean SD 

A 0.2099  ‐  2 0.2183  ‐  2 0.2141 0.0419 

B 0.2238 0.0388 4 0.2204 0.0192 4 0.2221 0.0284 

C 0.1945 0.0186 5 0.2225 0.0157 3 0.2050 0.0218 

D 0.1974 0.0311 6 0.1360  ‐  1 0.1886 0.0367 

E 0.1862 0.0249 7 0.1638  ‐  1 0.1834 0.0243 

F 0.2011 0.0305 3 0.1890* 0.0310 4 0.1942 0.0289 

*One female, 5F1V, had no weight measurementTable 7‐17: F1 Treatment 6 Day 79 and 80 Fish Weights (g)

GenotypicSex X M F OveraReplicate Mean SD n Mean SD n Mean SD n Mean SD 

A 0.1819‡ 0.0393 5 0.2487  ‐  2 0.2009 0.046

B 0.2136  ‐  2 0.2094 0.0794 4 0.2108 0.061

C 0.1682  ‐  1 0.2152 0.0170 5 0.2073 0.024

D 0.1976 0.0096 3 0.2098 0.0137 4 0.2045 0.012

E 0.2033  ‐  1 0.1931 0.0222 3 0.1957 0.018

F 0.1861†  ‐  1 0.2306  ‐  1 0.1920* 0.0371 4 0.1974 0.033

*One female, 6F4V, had no weight measurement, †Phenotype inconclusive 6F5V, ‡One male, 6A4V, had no weight measurement
Final Report

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Study No.: TOX1105

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The mean lengths of the F1 fish removed from the experiment at day 79 and day 80 are presented in Tables 

The fish were grouped into different sexes based on genotype before the mean and standard deviation was c

grouping the fish, genotype was considered more important than phenotype. The overall mean and standard 

each treatment replicate is also presented.

Table 7‐18: F1 Treatment 1 Day 79 and 80 Fish Lengths (mm)

Genotypic Sex X M F Overa

Replicate Mean SD n Mean SD n Mean SD n Mean SD 

A 27*  ‐  1 26 1.7 4 25 2.0 3 26 1.7 

B 24† 1.5 3 27 1.0 3 26 1.9 

C 26‡  ‐  1 26 0.8 5 28  ‐  1 26 1.0 

D 26 1.0 4 26 1.5 4 26 1.2 

E 27 1.8 5 27  ‐  2 27 1.5 

F 26 1.7 5 27 1.3 4 26 1.4 

*Phenotype inconclusive, 1A1V, †One male, IBID, had no length measurement, ‡Phenotype inconclusive, 1C4DTable 7‐19: F1 Treatment 2 Day 79 and 80 Fish Lengths (mm)

Genotypic Sex X M F Overa

Replicate Mean SD n Mean SD n Mean SD n Mean SD 

A 26* 4.9 3 26† 1.2 3 26 3.2 

B 25 1.5 4 26 2.2 4 26 1.8 

C 27 1.5 3 26 0.7 5 27 1.2 

D 27  ‐  2 26‡ 1.3 4 26 1.2 

E 26 1.5 6 27  ‐  2 26 1.4 

F 26 2.2 4 28 0.5 4 27 1.7 

*One male, 2A4V, had no length measurement, †One female, 2A5V, had no length measurement, ‡Two females, 2D2V & 2D3V, had no length meas
Final Report

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Study No.: TOX1105

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Table 7‐20: F1 Treatment 3 Day 79 and 80 Fish Lengths (mm)

Genotypic Sex X M F Over

Replicate Mean SD n Mean SD n Mean SD n Mean SD 

A 27 1.4 4 24 2.6 3 26 2.4 

B 26 0.6 3 27*  ‐  2 26 0.7 

C 26  ‐  2 26 0.8 6 26 0.7 

D 26  ‐  2 26 1.8 6 26 1.5 

E 26 1.2 5 26  ‐  2 26 1.5 

F 26 0.6 3 25 1.8 5 26 1.4 

*One female, 3B2V, had no length measurementTable 7‐21: F1 Treatment 4 Day 79 and 80 Fish Lengths (mm)

Genotypic Sex X M F Ove

Replicate Mean SD n Mean SD n Mean SD n Mean SD 

A 25ɣ  ‐  1 26 1.1 5 24  ‐  2 26 1.4 

B 25 0.8 5 26 2.6 3 25 1.7 

C 27 1.7 5 27 0.6 3 27 1.3 

D 26† 1.9 4 26 0.6 3 26 1.4 

E 28 1.0 4 26‡  ‐  2 27 1.3 

F 26ɣ  ‐  1 26  ‐  1 26 0.8 5 26 0.7 

*One female, 4B3V, had no length measurement, †One male, 4D1V, had no length measurement, ‡One female, 4E4V, had no length measurement

ɣPhenotype inconclusive, 4A4V, 4F2V
Final Report

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Study No.: TOX1105

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Table 7‐22: F1 Treatment 5 Day 79 and 80 Fish Lengths (mm)

Genotypic Sex X M F Ove

Replicate Mean SD n Mean SD n Mean SD n Mean SD 

A 26  ‐  2 26  ‐  2 26 1.2 

B 27 1.7 4 27* 0.6 3 27 1.3 

C 26 1.3 5 26 1.5 3 26 1.3 

D 27 1.9 6 24  ‐  1 27 2.

E 26 1.7 7 25†  ‐  1 26 1.6 

F 27 0.6 3 26 1.9 4 26 1.5 

*One female, 5B4V, had no length measurement; † One female, 5F1V, had no length measurementTable 7‐23: F1 Treatment 6 Day 79 and 80 Fish Lengths (mm)

Genotypic Sex X M F Ove

Replicate Mean SD n Mean SD n Mean SD n Mean SD 

A 26ɣ  2.1 5 27  ‐  2 26 1.8 

B 27  ‐  2 26 3.2 4 26 2.6 

C 25  ‐  1 26 1.4 5 26 1.3 

D 27 0.6 3 27 1.3 4 27 1.

E 25  ‐  1 25 0.6 3 25 0.5 

F 25†  ‐  1 28  ‐  1 26* 0.8 4 26 1.2 

*One female, 6F4V, had no length measurement, ɣOne male, 6A4V, had no length measurement, †Phenotype inconclusive 6F5V
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7.6 F1 Fish Weight and Length Metrics (Day 119)

The mean weight and lengths of the F1 medaka at Study Day 119 are presented in Tables 7‐24 and 7‐25.

Table 7‐24: F1 Female Medaka Mean Weights and Lengths (97 Days Post‐Fertilization)  

Treatment Replicate Weight

(g)

Mean

Weight (g) SD n Length(mm)

Mean

Length

(mm)

SD n

1 A 0.3481 0.4187 0.0644 5 31 32.4 1.34 5

B N/A       N/A      

C 0.4604      33      

D 0.4660      34      

E 0.4705      33      

F 0.3485      31      

2 A 0.3829 0.4519 0.0663 5 32 33.6 1.14 5

B N/A       N/A      

C 0.4979      35      

D 0.4110      34      

E 0.4248      33      

F 0.5430      34      

3 A 0.3854 0.3895 0.0216 6 33 33.5 1.05 6

B 0.3659      34      

C 0.4082      34      

D 0.3670      33      

E 0.4194      35      

F 0.3908      32      

4 A 0.4605 0.4699 0.0585 6 35 33.5 1.76 6

B 0.5470      34      

C 0.5132      34      

D 0.4470      34      

E 0.4747      34      

F 0.3770      30      

5 A 0.3605 0.4354 0.0600 6 32 33.3 1.21 6

B 0.4420      34      

C 0.4606      35      

D 0.3864      33      

E 0.4311      32      

F 0.5318      34      

6 A 0.4733 0.4999 0.0702 6 35 35.0 1.55 6

B 0.4784      34      

C 0.4593      34      

D 0.5592      35      

E 0.6091      38      

F 0.4200      34      
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* Two fish, 1BF and 2BF, were lost, and therefore had no weight or length measurements

Table 7‐25: F1 Male Medaka Mean Weights and Lengths (97 Days Post‐Fertilization)  

Treatment Replicate Weight

(g)

Mean

Weight (g) SD n Length(mm)

Mean

Length

(mm)

SD n

1 A 0.3351 0.3888 0.0598 6 33 32.3 1.03 6

B 0.4233       31      

C 0.2980       31      

D 0.4470       33      

E 0.3940       33      

F 0.4351       33      

2 A 0.4750 0.3445 0.0655 6 36 32.5 1.87 6

B 0.2964       32      

C 0.3394       31      

D 0.3157       32      

E 0.3138       33      

F 0.3265       31      

3 A 0.3151 0.3261 0.0689 6 33 33.0 1.55 6

B 0.3581       34      

C 0.3122       34      

D 0.2963       33      

E 0.2343       30      

F 0.4406       34      

4 A 0.4457 0.3661 0.0928 6 34 32.5 2.35 6

B 0.3321       30      

C 0.4170       33      

D 0.4705       36      

E 0.2965       32      

F 0.2347       30      

5 A 0.3855 0.4213 0.0641 6 34 34.5 1.97 6

B 0.3740       34      

C 0.4800       38      

D 0.4691       35      

E 0.3354       32      

F 0.4840       34      

6 A 0.3636 0.3677 0.0419 6 34 32.8 0.98 6

B 0.3475       33      

C 0.3926       32      

D 0.3680       32      

E 0.3050       32      

F 0.4294       34      
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7.7 F1 Anal Fin Papillae (Day 119)

The anal fin papillae were counted on fish sacrificed on day 119. The results of the

anal fin counts are presented in Table 7‐26.

Table 7‐26: F1 Medaka Anal Fin Papillae Counts (Study Day 119, 97 Days Post‐Fertilization)

Treatment Replicate Male Female Treatment Replicate Male Female

1 A 83 0 4 A 36 0

B 72 N/A†    B 61 0

C 70 0    C >55* 0

D 50 0    D 89 0

E 72 0    E 63 0

F 81 0    F 63 0

Mean    71.3 0 Mean    61.2 0

SD    11.7 0 SD    17.0 0

n    6 5 n    6 6

2 A 80 0 5 A 68 0

B 60 N/A†    B 69 0

C 83 0    C 86 0

D 78 0    D 82 0

E 68 0    E 77 0

F 69 0    F 0 0

Mean    73.0 0 Mean    63.7 0

SD    8.8 0 SD    32.0 0

n    6 5 n    6 6

3 A 73 0 6 A 91 0

B >45* 0    B 80 0

C 75 0    C 63 0

D 71 0    D >52* 0

E 69 0    E 78 0

F 62 0    F 70 0

Mean    65.8 0 Mean    72.3 0

SD    11.1 0 SD    13.8 0

n    6 6 n    6 6

* Values with > were used as absolute value for statistical analysis; † No fish

7.8 F1 Fish Fecundity Metrics

The fecundity metrics for the F1 fish are presented in Tables 7‐27 – 7‐29. The mean

and standard deviation of the total F1 cumulative egg production is presented in

Table 7‐27.  
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Table 7‐27: F1 Cumulative Egg Production (Cumulative Eggs)

Treatment Conc. (μg/L) Replicate Pair ID Total Mean SD n

1 0 A 1A 681 708 102 5

    B 1B N/A†      

    C 1C 731      

    D 1D 825      

    E 1E 754      

    F 1F 551      

2 8 A 2A 527 610 242 5

    B 2B N/A†      

    C 2C 219      

    D 2D 753      

    E 2E 775      

    F 2F 776      

3 19 A 3A 711 717 85 6

    B 3B 692      

    C 3C 783      

    D 3D 815      

    E 3E 727      

    F 3F 572      

4 48 A 4A 378 696 198 6

    B 4B 714      

    C 4C 575      

    D 4D 742      

    E 4E 939      

    F 4F 830      

5 120 A 5A 537 593 277 6

    B 5B 856      

    C 5C 832      

    D 5D 651      

    E 5E 588      

    F 5F 91      

6 300 A 6A 679 768 173 6

    B 6B 918      

    C 6C 903      

    D 6D 860      

    E 6E 783      

    F 6F 465      

† No fish
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The cumulative egg production for the F1 fish is presented in Table 7‐28.  

Table 7‐28: F1 Cumulative Egg Production (Cumulative Eggs Per Day)

Treatment Conc.

(μg/L) Replicate Pair ID Mean SD n

1 0 A 1A 33.3 0.9 21

0 B 1B N/A N/A 21

0 C 1C 34.5 2.2 21

0 D 1D 41.0 2.0 21

0 E 1E 37.0 1.6 21

0 F 1F 26.1 1.3 21

2 8 A 2A 25.7 0.8 21

8 B 2B N/A N/A 21

8 C 2C 8.6 3.1 21

8 D 2D 41.7 3.6 21

8 E 2E 36.2 3.6 21

8 F 2F 40.6 2.4 21

3 19 A 3A 33.3 1.8 21

19 B 3B 35.1 1.7 21

19 C 3C 40.0 1.7 21

19 D 3D 39.8 3.3 21

19 E 3E 36.9 2.2 21

19 F 3F 29.0 2.0 21

4 48 A 4A 20.2 2.1 21

48 B 4B 37.0 2.6 21

48 C 4C 30.5 1.9 21

48 D 4D 35.0 1.0 21

48 E 4E 47.6 2.2 21

48 F 4F 39.7 2.1 21

5 120 A 5A 25.4 2.6 21

120 B 5B 42.2 2.2 21

120 C 5C 42.5 3.9 21

120 D 5D 30.6 1.5 21

120 E 5E 27.8 4.4 21

120 F 5F 3.5 1.3 21

6 300 A 6A 36.5 5.4 21

300 B 6B 46.0 2.6 21

300 C 6C 42.9 2.2 21

300 D 6D 39.2 4.3 21

300 E 6E 45.0 3.6 21

300 F 6F 27.1 3.6 21
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The female weight normalized cumulative egg production per day for the F1 fish

is presented in Table 7‐29.  

Table 7‐29: F1 Weight Normalized Cumulative Egg Production (Cumulative Eggs/g/d)

Treatment Conc.

(μg/L) Replicate Mean SD n

1 0 A 95.6 2.6 21

0 B N/A N/A 21

0 C 74.9 4.7 21

0 D 88.0 4.3 21

0 E 78.7 3.4 21

   0 F 75.0 3.7 21

2 8 A 67.0 2.0 21

8 B N/A N/A 21

8 C 17.2 6.3 21

8 D 101.4 8.9 21

8 E 85.2 8.4 21

   8 F 74.8 4.4 21

3 19 A 86.5 4.6 21

19 B 95.9 4.6 21

19 C 98.0 4.1 21

19 D 108.3 8.9 21

19 E 88.1 5.3 21

   19 F 74.2 5.1 21

4 48 A 43.8 4.5 21

48 B 67.6 4.8 21

48 C 55.8 3.4 21

48 D 78.2 2.2 21

48 E 100.4 4.5 21

   48 F 105.4 5.6 21

5 120 A 70.4 7.1 21

120 B 95.4 4.9 21

120 C 92.3 8.5 21

120 D 79.1 3.9 21

120 E 64.5 10.1 21

   120 F 6.6 2.4 21

6 300 A 77.1 11.4 21

300 B 96.2 5.4 21

300 C 93.5 4.7 21

300 D 70.2 7.8 21

300 E 73.9 5.8 21

   300 F 64.6 8.5 21
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The cumulative F1 egg production per day per gram of female fish weight for all

treatment groups is displayed in Figure 7‐11.

Figure 7‐11: F1 Cumulative Egg Production per Day per Gram Female Fish Weight
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7.9 F2 Fish Survival Metrics

The F2 medaka egg hatching data is presented in Table 7‐30.

Table 7‐30: F2 Medaka Egg Hatching Success

   Test Substance Concentration (μg/L)

F2 Total Eggs 0 8 19 48 120 300

Tank A 100 89 85 92 89 86

Tank B 83 82 91 106 86 81

Total 183 171 176 198 175 167

% Hatching Success 84% 79% 37% 77% 88% 92%

F2 Eggs Hatched 0 8 19 48 120 300

Tank A 80 61 30 78 77 83

Tank B 74 74 35 74 77 70

Total 154 135 65 152 154 153

F2 Eggs Unhatched 0 8 19 48 120 300

Tank A 20 28 55 14 12 3

Tank B 9 8 56 32 9 11

Total 29 36 111 46 21 14

* Unhatched eggs were either dead, infected with fungus and removed, or did not hatch within

the hatching period.

The F2 medaka survival data (with mean and standard deviation for percent

survival) are presented in Tables 7‐31 – 7‐36 for each Vinclozolin treatment

group.  

Table 7‐31: F2 Number of Hatchlings Placed in Each Replicate 13 d Post Fertilization

Treatment (μg Vinclozolin/L)

Replicate 0 8 19 48 120 300

A 21 20 9 22 20 20

B 19 17 8 21 19 21

C 22 18 9 21 20 20

D 20 19 8 19 21 18

E 21 20 8 18 19 19

F 19 18 7 18 19 20
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Table 7‐32: F2 Number of Surviving Fish at Day 134, 21 Days Post Fertilization

Treatment (μg Vinclozolin/L)

Rep 0 8 19 48 120 300

A 16 11 7 19 18 16

B 15 15 6 17 17 15

C 18 14 8 15 20 19

D 18 18 5 15 22 12

E 19 19 7 14 18 12

F 17 15 6 13 17 10

Table 7‐33: F2 Percent of Surviving Fish at Day 134, 21 Days Post Fertilization

Treatment (μg Vinclozolin/L)

Rep 0 8 19 48 120 300

A 76% 55% 78% 86% 90% 80%

B 79% 88% 75% 81% 89% 71%

C 82% 78% 89% 71% 100% 95%

D 90% 95% 63% 79% 105% 67%

E 90% 95% 88% 78% 95% 63%

F 89% 83% 86% 72% 89% 50%

Mean 84% 82% 80% 78% 95% 71%

SD 6% 15% 10% 6% 6% 15%

Table 7‐34: F2 Number of Remaining in Each Replicate on Day 134

Treatment (μg Vinclozolin/L)

Rep 0 8 19 48 120 300

A 10 10 7 10 10 10

B 10 10 6 10 10 10

C 10 10 8 10 10 10

D 10 10 5 10 10 10

E 10 10 7 10 10 10

F 10 10 6 10 10 10
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Table 7‐35: F2 Number of Surviving Fish at Test Termination, Day 169/170

Treatment (μg Vinclozolin/L)

Rep 0 8 19 48 120 300

A 10 10 6 10 10 10

B 10 10 6 10 10 10

C 10 10 8 10 10 10

D 10 10 5 10 10 10

E 10 10 7 10 10 9

F 10 10 5 10 10 10

Table 7‐36: F2 Percent of Surviving Fish at Test Termination Day 169/170

Treatment (μg Vinclozolin/L)

Rep 0 8 19 48 120 300

A 100% 100% 86% 100% 100% 100%

B 100% 100% 100% 100% 100% 100%

C 100% 100% 100% 100% 100% 100%

D 100% 100% 100% 100% 100% 100%

E 100% 100% 100% 100% 100% 90%

F 100% 100% 83% 100% 100% 100%

Mean 100% 100% 95% 100% 100% 98%

SD 0% 0% 8% 0% 0% 4%

7.10 F2 Fish Weight and Length Metrics

The F2 medaka mean weights with standard deviation at test termination are

presented in Tables 7‐37 – 7‐42 for each Vinclozolin treatment group.

Table 7‐37: F2 Medaka (9 wpf) Mean Weights (g) Treatment 1

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 0.20112 0.01308 6 0.21588 0.04600 4 0.20702 0.02930 10

B 0.22827 0.01952 5 0.22125 0.01831 5 0.22476 0.01822 10

C 0.22684 0.02735 3 0.23175 0.03522 7 0.23028 0.03161 10

D 0.22705 0.02397 5 0.24558 0.04470 5 0.23631 0.03520 10

E 0.22352 0.04039 5 0.21431 0.02259 5 0.21891 0.03123 10

F 0.22376 0.02135 3 0.22889 0.03165 7 0.22735 0.02784 10
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Table 7‐38: F2 Medaka (9 wpf) Mean Weights (g) Treatment 2

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 0.22030 0.05083 7 0.22909 0.03165 3 0.22294 0.04430 10

B 0.21624 0.04950 5 0.20901 0.03041 5 0.21263 0.03892 10

C 0.18581 0.03101 3 0.21483 0.03206 7 0.20612 0.03310 10

D 0.24451 0.05035 5 0.22065 0.03543 5 0.23258 0.04293 10

E 0.18812 0.01656 3 0.22656 0.03700 7 0.21502 0.03631 10

F 0.22805 0.01464 6 0.22697 0.01822 4 0.22762 0.01517 10

Table 7‐39: F2 Medaka (9 wpf) Mean Weights (g) Treatment 3

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 0.17203 0.06370 5 0.23139  ‐  1 0.18192 0.06192 6

B 0.20693 0.10897 3 0.17307 0.13163 3 0.19000 0.10966 6

C 0.16709 0.03018 3 0.22933 0.00582 5 0.20599 0.03629 8

D 0.20241  ‐  2 0.22413 0.09148 3 0.21544 0.06581 5

E 0.20509 0.02226 4 0.21156 0.01370 3 0.20786 0.01795 7

F 0.17847 0.01320 3 0.21781 0.00176 2 0.19420 0.02349 5

Table 7‐40: F2 Medaka (9 wpf) Mean Weights (g) Treatment 4

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 0.20072 0.01753 7 0.22023 0.03074 3 0.20657 0.02244 10

B 0.21307 0.02916 7 0.20181 0.02068 3 0.20969 0.02630 10

C 0.20161 0.02424 4 0.22484 0.01290 6 0.21555 0.02079 10

D 0.23389 0.05116 5 0.23168 0.05833 5 0.23279 0.05174 10

E 0.22201 0.02945 3 0.22531 0.03404 7 0.22432 0.03111 10

F 0.20727 0.01064 4 0.21975* 0.00988 6 0.21476 0.01155 10

* One fish (4F5) could not have its genotype determined. It was counted as female based on

phenotype.
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Table 7‐41: F2 Medaka (9 wpf) Mean Weights (g) Treatment 5

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 0.20552 0.02157 4 0.22660 0.01127 6 0.21817 0.01855 10

B 0.21775 0.02097 3 0.22769 0.03599 7 0.22471 0.03138 10

C 0.20165 0.02508 5 0.23506 0.02279 5 0.21836 0.02865 10

D 0.21009 0.00273 4 0.22765* 0.02546 6 0.22062 0.02109 10

E 0.20593 0.03811 7 0.24460 0.02016 3 0.21753 0.03751 10

F 0.20562 0.02676 6 0.22108 0.03660 4 0.21180 0.03013 10

* One fish (5D1) could not have its genotype determined. It was counted as female based on

phenotype.

Table 7‐42: F2 Medaka (9 wpf) Mean Weights (g) Treatment 6

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 0.18178 0.04557 4 0.22151 0.03941 6 0.20562 0.04445 10

B 0.20831 0.05024 3 0.20229 0.03479 7 0.20409 0.03710 10

C 0.22975 0.03786 4 0.20316* 0.01594 6 0.21380 0.02842 10

D 0.20116 0.03325 5 0.22975 0.05423 5 0.21546 0.04501 10

E 0.19087 0.03421 5 0.22687 0.02403 4 0.20687 0.03409 9

F 0.21873 0.00901 4 0.20900† 0.05203 6 0.21289 0.03945 10

*† Two fish (6C10 and 6F2) could not have their genotype determined. They were counted as female

based on phenotype.
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The F2 medaka mean lengths with standard deviation are presented in Tables 7‐

43 – 7‐48 for each Vinclozolin treatment group.

Table 7‐43: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 1

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 27.3 0.8 6 26.8 1.3 4 27.1 1.0 10

B 28.8 0.8 5 27.8 0.4 5 28.3 0.8 10

C 28.0 1.0 3 27.4 1.9 7 27.6 1.6 10

D 28.6 0.9 5 27.8 1.3 5 28.2 1.1 10

E 27.8 1.3 5 26.8 1.5 5 27.3 1.4 10

F 28.0 1.0 3 27.4 0.8 7 27.6 0.8 10

Table 7‐44: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 2

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 27.7 1.4 7 27.7 1.5 3 27.7 1.3 10

B 27.8 2.4 5 25.8 2.9 5 26.8 2.7 10

C 26.3 1.2 3 27.4 1.5 7 27.1 1.4 10

D 28.6 1.9 5 28.2 1.6 5 28.4 1.7 10

E 27.3 0.6 3 28.0 1.2 7 27.8 1.0 10

F 28.7 1.4 6 28.0 0.8 4 28.4 1.2 10

Table 7‐45: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 3

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 26.4 1.8 5 28.0  ‐  1 26.7 1.8 6

B 27.0 4.0 3 24.0 7.0 3 25.5 5.4 6

C 25.0 2.0 3 28.4 0.9 5 27.1 2.2 8

D 27.5  ‐  2 26.0 3.6 3 26.6 2.7 5

E 28.5 1.0 4 28.0 1.0 3 28.3 1.0 7

F 26.3 2.1 3 27.5 0.7 2 26.8 1.6 5
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Table 7‐46: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 4

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 26.6 1.4 7 26.0 4.4 3 26.4 2.4 10

B 27.7 1.6 7 27.3 0.6 3 27.6 1.3 10

C 27.5 1.0 4 28.2 0.8 6 27.9 0.9 10

D 28.0 1.6 5 27.2 3.6 5 27.6 2.6 10

E 28.0 1.7 3 28.3 1.3 7 28.2 1.3 10

F 27.3 0.5 4 26.8* 0.8 6 27.0 0.7 10

* One fish (4F5) could not have its genotype determined. It was counted as female based on

phenotype.

Table 7‐47: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 5

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 26.5 1.3 4 27.2 0.8 6 26.9 1.0 10

B 27.0 1.0 3 27.3 1.1 7 27.2 1.0 10

C 27.0 1.2 5 28.0 2.0 5 27.5 1.6 10

D 27.0 0.8 4 27.5* 1.0 6 27.3 0.9 10

E 27.4 1.3 7 28.3 0.6 3 27.7 1.2 10

F 27.8 1.9 6 28.0 1.4 4 27.9 1.7 10

* One fish (5D1) could not have its genotype determined. It was counted as female based on

phenotype.

Table 7‐48: F2 Medaka (9 wpf) Mean Lengths (mm) Treatment 6

Genotypic Sex M     F     Overall   

Replicate Mean SD n Mean SD n Mean SD n

A 26.3 2.5 4 27.0 2.0 6 26.7 2.1 10

B 27.7 2.1 3 26.9 1.5 7 27.1 1.6 10

C 28.5 1.3 4 26.8* 1.2 6 27.5 1.4 10

D 27.2 2.9 5 27.2 3.1 5 27.2 2.9 10

E 27.2 1.9 5 28.0 0.8 4 27.6 1.5 9

F 28.3 1.0 4 26.7† 3.0 6 27.3 2.5 10

*† Two fish (6C10 and 6F2) could not have their genotype determined. They were counted as female

based on phenotype.
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7.11 F2 Anal Fin Papillae (Day 169/170)  

The F2 anal fin papillae counts are presented in Table 7‐49.

Table 7‐49: F2 Anal Fin Papillae Counts (Day 169/170)

Genotype: M F

Treatment Replicate Mean SD n NMWOP Mean SD n NFWP

1 A 55 14 6 0 0 0 4 0

B 67 8.8 5 0 0 0 5 0

C 56 9.9 3 0 0 0 7 0

D 60 7.1 5 0 0 0 5 0

E 55 11 5 0 0 0 5 0

F 61 4.9 4 0 0 0 6 0

2 A 32 31 7 3 0 0 3 0

B 53 10 5 0 0 0 5 0

C 24 42 3 2 0 0 7 0

D 43 26 5 1 0 0 5 0

E 56 6.4 3 0 0 0 7 0

F 30 26 6 2 0 0 4 0

3 A 55 33 5 1 0 0 1 0

B 15 26 3 2 0 0 3 0

C 25 23 3 0 0 0 5 0

D 52  ‐  2 0 0 0 3 0

E 52 14 4 0 0 0 3 0

F 43 17 3 0 0  ‐  2 0

4 A 46 7.6 7 0 0 0 3 0

B 47 6.1 7 0 0 0 3 0

C 36 21 4 0 0 0 6 0

D 34 32 5 2 0 0 5 0

E 26 26 3 1 0 0 7 0

F 37 20 4 0 0 0 6 0

5 A 20 24 4 2 6.5 16 6 1

B 0 0 3 3 0 0 7 0

C 29 18 5 1 0 0 5 0

D 45 6.2 4 0 5.8 14 6 1

E 44 15 7 0 0 0 3 0

F 39 21 6 1 0 0 3 0

6 A 9.3 11 4 2 0 0 7 0

B 19 14 3 0 0 0 7 0

C 24 17 4 0 0 0 6 0

D 25 16 5 1 0 0 5 0

E 32 23 5 1 0 0 4 0

F 18 13 4 1 0 0 6 0

NMWOP: Number of Males Without Papillae, NFWP: Number of Females With Papillae
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7.12 Test Validity Criteria

For a test to be valid the following conditions must be fulfilled:

 The dissolved oxygen concentration must be between ≥ 60% of air saturation

throughout the test

 At the lowest measured temperature during the test, 25.6°C, 60% air

saturation would be approximately 4.9 mg O2/L. As the lowest measured

dissolved oxygen concentration was 5.8 mg/L, this test validity criterion

was passed.

 The water temperature must not differ by more than ± 1.0°C between test

chambers or between successive days at any time during the test

 At one point during the study (Day 147), the difference in water

temperature between two measured test vessels was 1.3°C. At three

points, the difference in temperature between weekly temperature

readings exceeded 1.0°C (1.1 – 1.3°C). However, these deviations are

minor, and did not occur frequently. Therefore, it is not expected that the

results of the study are significantly affected (Deviation #11).

 pH should not differ between replicates by more than ± 0.2

 At two points during the study (Days 41 and 62), the change in pH from

the previous week’s readings in all nominal concentrations (Day 41) and

all nominal concentrations except for the 300 μg/L concentration (Day

62) was greater than 0.2 units. However, these deviations are minor, and

did not occur frequently. Therefore, it is not expected that the results of

the study are significantly affected (Deviation #12).

 There should be ≥ 80% survival of control animals after hatching through 21

days post fertilisation (dpf) in each of the F1 and F2 generations and > 90%

survival from 21 dpf through termination for the F1 and F2 generations

 In the F1 generation, there was 100% survival at 21 dpf, and 92% survival

through termination. In the F2 generation, there was 84% survival at 21

dpf, and 100% survival through termination. Therefore this test validity

criterion was passed.

 ≥ 80% hatching in control eggs in each of the F1 and F2 generations

 There was 79% and 84% hatching of control eggs in the F1 and F2

generations, respectively. Therefore, this test validity criterion was not

met. However, the percent hatching was only slightly below the criterion,

and the remainder of the test showed no effects from this difference.

Therefore, it is not expected that the results of the study are significantly

affected (Deviation #10).
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SECTION

8 DISCUSSION

A sample of test substance, Vinclozolin, was tested to assess its potential to

cause chronic effects and endocrine disruption to the freshwater fish, Oryzias

latipes, using procedures based on the draft guideline for the Medaka

Multigeneration Test, dated April 19, 2010. The test was conducted using a flow‐

through system at a range of nominal concentrations from 8 to 300 μg/L over a

170 day period. During that time, survival, growth, reproduction, and gross

development were assessed. The potential for endocrine disruption was also

evaluated by determination of genotypic sex, secondary sex characteristics (such

as anal fin papillae), and Vitellogenin levels.

8.1 Test Substance Concentration

To ensure constant exposure of the test organisms to the test substance, the

concentration of the test substance was measured weekly throughout the test in

each of the 5 test concentrations and the control. Measured concentrations

ranged from 78% to 157% of nominal (Table 7‐2, Figures 7‐1 to 7‐5). The majority

of the measured concentrations were above nominal during the study, despite

initial concerns regarding the water solubility of the test item. It is possible that

the water hardness at Maxxam (typically 50 – 70 mg CaCO3/L) allowed a higher

saturation level (as compared to the expected 2.8 mg/L) for Vinclozolin to be

dissolved in the water after passing through the two generator columns (Table 7‐

1).

8.2 Water Quality

Water quality parameters (dissolved oxygen, temperature, pH, conductivity, and

hardness) were also performed weekly. The temperature ranged from 25.6 to

27.0°C over the course of the study. As noted in Section 7.12, there were four

points during the study where the difference between tanks on the same day, or

the difference between successive days, was greater than 1.0°C. Additionally,

there were two time points where the pH of a concentration changed by greater

than 0.2 between measurements. These differences are minor, and were rare

during the course of the test, so it is not expected that they would significantly

affect the outcome of the study.
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8.3 Summary Statistics

Summary statistical analyses (mean and standard deviation, where appropriate)

were performed on various data points from different time points during the

test. However, no comparative analysis has been performed, and therefore

statistical comparisons of results between concentrations are not possible at this

time.

8.3.1 F0 Fertility and Fecundity

Fecundity and fertility of the F0 generation were measured over a 9 day period,

and are presented in Section 7.3. A general trend of diminishing egg production

per day was observed in all concentrations (See Figure 7.10). Graphically, the 300

μg/L concentration appears to be lower than the other concentrations, but it is

not certain if this difference is statistically significant.

8.3.2 F1 Hatching Success and Survival

Generally, hatching success was varied across the concentrations for the F1

generation (Section 7.4). Some eggs and fish were lost by being caught in the

stainless steel mesh that encased the hatching vessel, while others died or were

lost for unknown reasons. In addition, during the culturing phase leading up to

the study, many eggs were infected with a fungus, which may have played a role

in diminished hatching success during the study. The controls showed 79.4%

hatching success, just slightly below the test validity criterion (Deviation #10).

The number of hatchlings surviving from hatching to 21 days post fertilisation

(dpf) appeared to be higher in the controls and at lower concentrations. From 21

dpf to sacrifice, the survival of the fish was >90% for all treatments, with no

obvious dose‐related effect.

It should be noted that in a few circumstances, more fish appear to have

survived than were originally placed in a tank or hatching vessel. This can likely

be attributed to the difficulties in counting small, fast‐moving fish in an enclosed

vessel in the early stages of the generation. These anomalies can be observed in

Table 7‐6 to 7‐11 and the raw data presented in Appendix H.

8.3.3 F1 Growth (Lengths and Weights)

The growth of the F1 generation was determined twice, at Day 79/80, and at Day

119, by measuring the length and weight of each fish sacrificed (Sections 7.5 and

7.6). The Day 79/80 data was separated based on fish genotype in addition to

concentration and replicate, while the Day 119 data was based on concentration

and replicate only. Note that there were several fish in the Day 79/80 dataset

that did not have a clear genotypic sex determined; these fish are presented

with a genotype of “X”.

No clear trends were evident at either time point for either measurement.
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8.3.4 F1 Secondary Sex Characteristics (Anal Fin Papillae)

Anal fin papillae of the F1 generation were counted twice, at Day 79/80, and at

Day 119 (Section 7.7).  

Counts performed on the Day 79/80 fish proved to be exceptionally challenging.

Many of the fins were curled and degraded, with papillae not visible in most

specimens. Those fins that did have papillae present were difficult to count, so

the data generated from these fins should be considered qualitative rather than

quantitative. It is suspected that one of the ingredients of the Hartmann’s

fixative that was used in the study, acetic acid, may have contributed to the

difficulties in counting by degrading calciferous tissues, such as the papillae.

When fin counting was initially attempted, the tissue was found to be very

cloudy. A series of clearing steps, involving overnight dehydration steps with

increasing concentrations of ethanol (50%, 70%, 100%), followed by a clearing

solution (CitriSolv) increased the clarity of the fins enough to allow for some

counts to be performed. However, many fins were still too degraded to provide

reliable counts. In addition, during this clearing process, the Treatment 1 and

Treatment 2 fins were found to have dried out due to evaporation of the 100%

ethanol in the storage plates. Therefore, these counts will be presented as raw

data, and will not be subjected to statistical analysis.

By contrast, the Day 119 fins were clear enough to allow quantitative counts.

This may be due to the greater tissue mass present. There was no clear trend in

papillae counts in the Day 119 fins, though one fish, which was genotypically

male and able to successfully fertilise embryos, did not have any papillae.

Furthermore, its fin looked phenotypically female in shape and structure.

8.3.5 F1 Fertility and Fecundity

Fecundity and fertility of the F1 generation were measured over a 21 day period,

and are presented in Section 7.8. There does not appear to be any clear trends in

either the fertility or fecundity at this test point. In contrast to the F0 data, the

egg production per day per gram of female fish for the F1 generation seemed to

be relatively constant over the course of their reproductive phase (Figure 7‐7).

There was no observable concentration/response observed for the F1 egg

production.   

8.3.6 F2 Hatching Success and Survival

Generally, hatching success was varied across the concentrations for the F2

generation (Section 7.9). Most notable was the 19 μg/L concentration, which had

very poor hatching success (37%) when compared to other concentrations. No

reason could be determined for this effect. The controls showed 85% hatching

success, above the test validity criterion.
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The number of hatchlings surviving from hatching to 21 days post fertilisation

(dpf) did not appear to correlate with concentration. From 21 dpf to sacrifice,

survival of the fish was above 95% throughout all concentrations, with no

obvious dose‐related effect.

8.3.7 F2 Growth (Lengths and Weights)

The growth of the F2 generation was determined at the end of the study (Day

169/170), by measuring the length and weight of each fish sacrificed (Section

7.10). The data were separated based on fish genotype in addition to

concentration and replicate.

No clear trends were evident for either measurement.

8.3.8 F2 Secondary Sex Characteristics (Anal Fin Papillae)

Anal fin papillae of the F2 generation were counted at the end of the test on Day

169/170 (Section 7.11).

Counts performed on the fresh fish were significantly easier than those

performed on preserved tissues, and provided good quantitative results. It is

worth noting that 26 genotypically identified males did not exhibit anal fin

papillae, and 2 genotypically identified females possessed papillae. There also

appeared to be a trend of fewer papillae on average as concentration increased.

8.4 Study Challenges

A number of challenges arose over the course of the study, many of which are

discussed in the Summary Statistics section above.

Another issue that bears mention is the clipping for genotypic sex identification

of the F1 fish on Day 79. During the clipping process, the dorsally‐clipped fish in

treatments 1, 2, and replicates A through D of treatment 3 were pooled after

clipping. Therefore, the fin clips for these fish could not be positively cross‐

referenced to the fish. In order to proceed with choosing a male/female pair for

each replicate, pairs were created from these replicates using ventrally‐clipped

fish wherever possible. When not possible, pairs were created by selecting a

phenotypically distinct fish. The presence of viable eggs in the segregation tubes

was also used as an indicator of the sex of the fish.

Due to the length of the study, algal growth started to become quite prevalent.

The tanks were siphoned regularly to remove debris, and cleaned periodically

using a foam brush, but many tanks could not be completely cleaned. It is

uncertain if the presence of this algal growth would have any adverse effects on

the study; while test substance concentrations did not appear to have been

affected, perhaps contamination by a particular algae in the 19 μg/L

concentration could have affected the health of the fish in those tanks.
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Delays during the shipment of the F2 fish livers to Battelle resulted in the

complete loss of dry ice inside the shipping container. This allowed the sample

racks to move about freely in the cooler, breaking open and/or crushing a

number of the liver sample vials in the process. In total, 24 liver samples were

lost.

Three of the test validity criteria for this study were not met. The validity

criterion for water temperature was quite stringent. Water temperature should

have not deviated by more than 1.0°C, There were four times during the study

where this criterion was not met (Deviation #11).  

The second validity criteria stated that pH should not differ between replicates

by more than 0.2. This criterion was not met on two occasions for several test

concentrations (Deviation #12).  

The third criterion, that hatching success in the controls was to be  ≥80%, was

slightly missed by the F1 generation controls (79.4%) (Deviation #10).  

While each of these validity criteria was not met, the first two test validity

criterion exceedances were extremely minor in nature, and not likely to affect

the overall results of the study. The third could have had a long term affect if the

hatching success had been significantly lower than the 80% mark. However, with

the F1 hatching success being 79.4%, and the remainder of the test showing no

effects from this deviation, it is unlikely to have significantly affected the results

of the study.
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SECTION

9 ARCHIVING

9.1 Records

All original study records will be transferred to EPA. Facility records will be

maintained at Maxxam.

These records will be archived by Maxxam Analytics and may be ultimately

stored at Iron Mountain, 8825 Northbrook Court (Head Office), Burnaby, BC,

Canada V5J 5J1.

No material will be destroyed or removed from the archive without written

permission of the study sponsor. Archiving was done based on the Work

Instruction “Archiving and Storage of GxP Records” (BBY1 WI‐00010).  

9.2 Sample Retention and Disposal

Test solutions taken for confirmatory analysis will be kept, at a minimum, until

the study completion date. A sample of test substance will be archived by

Maxxam Analytics for possible future identity confirmation and will be retained

for the duration of the marketing or research permit or until the expiry date. The

test substance will be archived at Maxxam Analytics 4606 Canada Way, Burnaby,

BC, Canada V5G 1K5.  

Any unused portion of the test substance will be kept for a minimum of three

months after the completion of the study. After this period, the unused portion

of the test substance will be discarded at the Sponsor’s expense or returned to

the Sponsor.
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SECTION

10 LITERATURE CITED

Archiving and Storage of Records (BBY1 WI‐00010).

Cleaning of Laboratory Ware (BBY WI‐00001).

Corporate Procedure for Balance Calibrations and Verifications (COR WI‐00015).

Corporate Procedure for Volume Verification (COR WI‐00017)

Determination of Analysis of Total Hardness (Titrimetric EDTA) in Water and

Wastewater (BBY2SOP‐00005).

Determination of Free and Total Chlorine in Water (BBY6SOP‐00046).

Determination of Metals in Environmental Samples Using CRC ICPMS (BBY7SOP‐

00002).

Determination of Sulfate by Konelab (BBY6SOP‐00017).

Determination of TOC and DOC in Water and Wastewater (BBY6SOP‐00003).

Determination of Total Suspended Solids in Waters and Wastewaters (BBY6SOP‐

00034).

Final Study Plan Medaka Multigeneration Test of Vinclozolin (Study Number

TOX1105) July 18, 2011.

Health and Safety Policies (MEHS WI‐00013).

Management of Test and Reference Items (BBY1 WI‐00008).

Medaka Culture Receipt and Maintenance (BBY2 WI‐00011).

Medaka Multigeneration Test (Draft Guideline) April 19, 2010.

Microsoft (2012). Microsoft® Office Excel 2010. Microsoft Corporation,

Redmond, WA.

Nitrogen (Ammonia) in Waters by Konelab using Automated Phenate Method

(BBY6SOP‐00009).

Operation and Maintenance of Accumet Basic pH Meters (BBY2 WI‐00015).

Operation and Maintenance of Light Meters (BBY2 WI‐00016).

Operation and Maintenance of WTW Conductivity Meter (BBY2 WI‐00012).

Operation and Maintenance of YSI Dissolved Oxygen Meter (BBY2 WI‐00014).

Operation and Maintenance of YSI Model 3100 Conductivity Meter (BBY2 WI‐

00013).

Organochlorine Pesticides and PCBs in Solids, Water and Biological Matrix (CAM

SOP‐00307).
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pH, Specific Conductance, Salinity and Alkalinity (Tot. and Phenolphthalein)

(BBY6SOP‐00026).

Quality Assurance Project Plan for Task Order #32: Medaka Multi‐Generation

Test Interlaboratory Evaluation Version 1. Battelle Pacific Northwest Division.

July 2010.

Quality Manual (COR WI‐00001).

Reports for GXP Studies (BBY1 W1‐00007).

Semivolatile Organics Acid/Base Neutral Extractables in Water and Soil (CAM

SOP‐00301).

Study Plans, Amendments, and Deviations for GXP Studies (BBY1 WI‐00006).

United States Environmental Protection Agency (EPA) Principles of Good

Laboratory Practice (40CFR Part 160).

United States Environmental Protection Agency (EPA) Vinclozolin Column

Preparation SOP, 2011 (included in Amendment 1).