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STUDY TITLE

4-tert-Octylphenol: Larval Amphibian Growth and Development Assay Using Xenopus laevis 

AUTHOR

Simon Dinehart, Ph.D. 

STUDY INITIATION DATE

30 June 2011 

STUDY COMPLETION DATE

28 December 2012 

SPONSOR

Battelle-Pacific Northwest Division

Marine Research Operations

1529 West Sequim Bay Road 

Sequim, Washington 98382 

PERFORMING LABORATORIES

ABC Laboratories, Inc. (In-Life Phase)

7200 E. ABC Lane 

Columbia, Missouri 65202 

United States

Battelle-Pacific Northwest Division (Genetic Sexing & Statistics)

Marine Research Operations

1529 West Sequim Bay Road 

Sequim, Washington 98382 

PROJECT IDENTIFICATION

ABC Study No. 66086




ABC Study No. 66086 

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TABLE OF CONTENTS

Page

TITLE PAGE ...................................................................................................................................1 

STATEMENT OF GLP COMPLIANCE ........................................................................................2

QUALITY ASSURANCE STATEMENT ......................................................................................3

STUDY PERSONNEL ....................................................................................................................4

SIGNATURE PAGE .......................................................................................................................5

TABLE OF CONTENTS .................................................................................................................6

LIST OF TABLES ...........................................................................................................................7

LIST OF FIGURES .........................................................................................................................8

LIST OF APPENDICES ................................................................................................................10

EXECUTIVE SUMMARY ...........................................................................................................11

1.0 INTRODUCTION .............................................................................................................13

2.0 MATERIALS AND METHODS .......................................................................................13

2.1 Test Substance and Reference Substances .............................................................13

2.2 Test Species ...........................................................................................................14

2.3 Dilution Water .......................................................................................................14

2.4 Test Methods ..........................................................................................................14

2.4.1 Saturator Solution Preparations .................................................................14

2.4.2 Exposure System ........................................................................................15

2.4.3 Biological Methods ....................................................................................16

2.5 Analytical Method .................................................................................................21

2.5.1 Preparation of Analytical Standard and Matrix Spiking Solutions ............21

2.5.2 Test Solution Analysis ...............................................................................21

2.5.3 Instrument Conditions ................................................................................22

2.5.4 Calculations for 4-tert-octylphenol ............................................................22

2.6 Genetic Sexing .......................................................................................................23

2.7 Statistical Analyses ................................................................................................24

3.0 RESULTS AND DISCUSSION ........................................................................................25

3.1 Analytical Results ..................................................................................................25

3.2 Biological Results ..................................................................................................26

3.3 Genotypic/Phenotypic Sex Ratios..........................................................................38

3.4 Water Quality .........................................................................................................38

3.5 Vitellogenin Results ...............................................................................................39

3.6 Plasma Thyroxine (T4) Results .............................................................................39

3.7 Validity and Performance Criteria .........................................................................42

4.0 CONCLUSIONS................................................................................................................42
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5.0 RECORDS .........................................................................................................................43

PROTOCOL DEVIATIONS .........................................................................................................44

REFERENCES ..............................................................................................................................48

LIST OF TABLES

Page

Table 3.1.1. Summary of analytical results for the Xenopus laevis 4-tert-octylphenol inlife exposure. ..........................................................................................................26

Table 3.2.1. Mean (and standard deviation) time to NF stage 62 during the Xenopus 

laevis 4-tert-octylphenol exposure. ........................................................................27

Table 3.2.2. Mean (and standard deviation) whole body weight and snout-to-vent length 

for NF stage 62 larvae during the Xenopus laevis 4-tert-octylphenol 

exposure. ................................................................................................................28

Table 3.2.3. Mean (and standard deviation) whole body weight and snout-to-vent length 

at the NF stage 66 cull of the Xenopus laevis 4-tert-octylphenol exposure. ..........30

Table 3.2.4. Mean (and standard deviation) whole body weight and snout-to-vent length 

at the .......................................................................................................................32

Table 3.2.5. Mean (standard deviation) proportion of female and male frogs and total 

number of frogs having nuptial pads at the termination of the Xenopus laevis

4-tert-octylphenol exposure. ..................................................................................35

Table 3.2.6. Proportion of total frogs within each treatment having nuptial pads at the 

termination of the Xenopus laevis 4-tert-octylphenol exposure. ...........................36

Table 3.2.7. Mean (and standard deviation) liver weight:whole body weight for male and 

female frogs at the termination of the Xenopus laevis 4-tert-octylphenol 

exposure. ................................................................................................................37

Table 3.3.1. Frog phenotypic and genotypic sex ratios at the termination of the Xenopus 

laevis 4-tert-octylphenol exposure. ........................................................................38

Table 3.4.1. Ranges for Water Quality Measurements During the 113-Day Exposure 

with the African Clawed Frog, Xenopus laevis, Exposed to 4-tertoctylphenol .............................................................................................................39

Table 3.6.1. Mean (and standard deviation) plasma thyroxine (T4) for NF Stage 62 frogs 

during the Xenopus laevis 4-tert-octylphenol exposure.........................................40

Table 3.6.2. Mean (and standard deviation) plasma thyroxine (T4) for frogs at the 

termination of the Xenopus laevis 4-tert-octylphenol exposure. ...........................40

Table 4.0.1. Calculated No-Observable-Effect Concentration and Lowest-ObservableEffect Concentration based on mean measured 4-tert-octylphenol 

concentrations for parameter measured for Xenopus laevis exposed to 4-tertoctylphenol. ............................................................................................................43
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LIST OF FIGURES

Page

Figure 3.2.1. Boxplot of time to NF stage 62 during the Xenopus laevis 4-tert-octylphenol 

exposure based on the replicate analysis. The box represents the 

interquartile range, the circle is the mean, the asterisk is an outlier, and the 

horizontal line is the median. .................................................................................27

Figure 3.2.2. Boxplot of time to NF stage 62 during the Xenopus laevis 4-tert-octylphenol 

exposure based on the Kaplan-Meier product-limit estimator analysis. The 

box represents the interquartile range, the circle is the mean, the asterisk is 

an outlier, and the horizontal line is the median. ...................................................28

Figure 3.2.3. Boxplot of whole body weight for NF stage 62 larvae during the Xenopus 

laevis 4-tert-octylphenol exposure. The box represents the interquartile 

range, the circle is the mean, and the horizontal line is the median. .....................29

Figure 3.2.4. Boxplot of snout-to-vent length for NF stage 62 larvae during the Xenopus 

laevis 4-tert-octylphenol exposure. The box represents the interquartile 

range, the circle is the mean, the asterisk is an outlier, and the horizontal line 

is the median. .........................................................................................................29

Figure 3.2.5. Boxplot of whole body weight for frogs at the NF stage 66 cull of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the 

interquartile range, the circle is the mean, the asterisk is an outlier, and the 

horizontal line is the median. .................................................................................30

Figure 3.2.6. Boxplot of snout-to-vent length for frogs at the NF stage 66 cull of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the 

interquartile range, the circle is the mean, the asterisk is an outlier, and the 

horizontal line is the median. .................................................................................31

Figure 3.2.7. Boxplot of whole body weight for male frogs at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the 

interquartile range, the circle is the mean, and the horizontal line is the 

median. Gender is based on genotypic sex. ..........................................................32

Figure 3.2.8. Boxplot of whole body weight for female frogs at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the 

interquartile range, the circle is the mean, and the horizontal line is the 

median. Gender is based on genotypic sex. ..........................................................33

Figure 3.2.9. Boxplot of snout-to-vent length for male frogs at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the 

interquartile range, the circle is the mean, and the horizontal line is the 

median. Gender is based on genotypic sex. ..........................................................33

Figure 3.2.10. Boxplot of snout-to-vent length for female frogs at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the 

interquartile range, the circle is the mean, and the horizontal line is the 

median. Gender is based on genotypic sex. ..........................................................34
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Figure 3.2.11. Boxplot of the proportion of male frogs having nuptial pads at the 

termination of the Xenopus laevis 4-tert-octylphenol exposure. The box 

represents the interquartile range, the circle is the mean, and the horizontal 

line is the median. Gender is based on genotypic sex. ..........................................35

Figure 3.2.12. Boxplot of the proportion of female frogs having nuptial pads at the 

termination of the Xenopus laevis 4-tert-octylphenol exposure. The box 

represents the interquartile range, the circle is the mean, and the horizontal 

line is the median. Gender is based on genotypic sex. ..........................................36

Figure 3.2.13. Boxplot of liver weight:whole body weight for male frogs at the termination 

of the Xenopus laevis 4-tert-octylphenol exposure. The box represents the 

interquartile range, the circle is the mean, and the horizontal line is the 

median. Gender is based on genotypic sex. ..........................................................37

Figure 3.2.14. Boxplot of liver weight:whole body weight for female frogs at the 

termination of the Xenopus laevis 4-tert-octylphenol exposure. The box 

represents the interquartile range, the circle is the mean, and the horizontal 

line is the median. Gender is based on genotypic sex. ..........................................38

Figure 3.6.1. Boxplot of plasma thyroxine (T4) concentration for NF Stage 62 larvae 

during the Xenopus laevis 4-tert-octylphenol exposure. The box represents 

the interquartile range, the circle is the mean, and the horizontal line is the 

median. ...................................................................................................................41

Figure 3.6.2. Boxplot of plasma thyroxine concentration for male frogs at the termination 

of the Xenopus laevis 4-tert-octylphenol exposure. The box represents the 

interquartile range, the circle is the mean, the asterisk is an outlier, and the 

horizontal line is the median. Gender is based on genotypic sex. ........................41

Figure 3.6.3. Boxplot of plasma thyroxine concentration for female frogs at the 

termination of the Xenopus laevis 4-tert-octylphenol exposure. The box 

represents the interquartile range, the circle is the mean, and the horizontal 

line is the median. Gender is based on genotypic sex. ..........................................42
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LIST OF APPENDICES

Page

APPENDIX A - CERTIFICATES OF ANALYSIS ......................................................................49

APPENDIX B - DILUTION WATER CHARACTERIZATION .................................................52

APPENDIX C - PROTOCOL, AMENDMENTS, AND DEVIATION ........................................55

APPENDIX D – GENDER REPORT ...........................................................................................97

APPENDIX E – STATISTICAL REPORT .................................................................................146

APPENDIX F – ANALYTICAL DATA SUMMARY ...............................................................175

APPENDIX G - REPLICATE SURVIVAL DATA ....................................................................180

APPENDIX H – NF STAGE 62 DATA ......................................................................................188

APPENDIX I – NF STAGE 66 CULL DATA ............................................................................200

APPENDIX J – TERMINATION DATA – GROWTH AND GENDER ...................................208

APPENDIX K – TERMINATION DATA – NUPTIAL PAD AND LIVER WEIGHT .............216

APPENDIX L – WATER QUALITY DATA .............................................................................224

APPENDIX M – PLASMA VITELLOGENIN DATA ..............................................................234

APPENDIX N – PLASMA T4 DATA ........................................................................................248
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EXECUTIVE SUMMARY

A 113-day flow-through test with African clawed frog (Xenopus laevis) tadpoles exposed to 

sublethal concentrations (i.e., 6.25, 12.5, 25.0, and 50.0 μg a.i/L nominal concentrations) of 

4-tert-octylphenol was conducted from 03 May to 24 August 2012 to assess the potential effects 

of the test substance on survival, development and growth, and secondary mechanistic biomarker 

responses. The test was initiated with Nieuwkoop and Faber (NF) stage 9 Xenopus laevis 

embryos obtained from an in-house culture of Xenopus laevis adults. 

Overall mean measured concentrations of 4-tert-octylphenol in the 6.25, 12.5, 25, and 50 μg/L

nominal test substance treatments were 4.97, 10.3, 19.5, and 38.0 μg/L, respectively, 

representing 76 to 82% of the nominal treatment concentrations. Following high recoveries from 

all test substance treatments at study initiation (ranging from 136-150% of nominal), measured 

concentrations in test substance treatment solutions ranged from 62 to 134% of the overall mean 

measured concentrations. Coefficient of variation values ranged from 22 to 30%, which 

exceeded the acceptability criterion stated in the study protocol. No residues of the 

4-tert-octylphenol were detected in the controls above the MQL of 3.08 ng/mL. Recoveries from 

the 4-tert-octylphenol quality control fortifications ranged from 88 to 118% of the nominal 

concentrations. Because the mean measured concentrations of 4-tert-octylphenol were not all 

within 20% of nominal concentrations during the exposure, the biological response results were 

reported based upon the mean of the measured concentrations. 

All water quality parameters, with the exception of temperature, remained within acceptable 

levels throughout the exposure. Dissolved oxygen concentrations ranged from 4.0 to 8.7 mg/L, 

representing 47 to 102% saturation. Test solution pH ranged from 7.3 to 8.5 during the test. The 

temperature of all test solutions ranged from 19.9 to 21.9°C during the test. With the exception of 

the following study days (0, 6, 8, 12, 19, 21, 32, 39, 50, 60, 97, 106, 112, and 113), test solution 

temperature differences among treatments on a given day exceeded 0.5oC (n = 22). Temperature 

differences exceeding 0.5 oC among treatments ranged from 0.6 – 1.0oC with the following 

exceptions (differences of 1.3, 1.2, 1.2 oC on day 41, 85, and 88, respectively). The continuous 

temperature data collected from a centrally located test chamber indicated that, with two exceptions

(05July12 and 30July12), temperature remained within the range of 21 ± 1°C specified by the 

protocol. On 05July12, test solution temperature was outside the acceptable range for 

approximately 3 hours and reached a maximum of 22.2oC. On 30July12, test solution 

temperature was outside the acceptable range for approximately 45 minutes and reached a 

minimum of 19.5oC. These exceptions had no adverse effect on study integrity. 

With several exceptions as noted above, this study satisfied the acceptability and performance 

criteria stated in the study protocol. The no-observable-effect concentration and lowestobservable-effect concentration were calculated as mean measured concentrations of 38.0 μg/L 

4-tert-octylphenol and >38.0 μg/L 4-tert-octylphenol, respectively, for each Xenopus laevis

parameter. The below table presents the results for each parameter evaluated.
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Parameter No-Observable-Effect 

Concentration (μg/L)

Lowest-ObservableEffect Concentration 

(μg/L)

NF stage 62

Time to Metamorphosis (Replicate Analysis) 38.0 >38.0

Time to Metamorphosis (Kaplan-Meier) 38.0 >38.0

Whole Body Weight 38.0 >38.0

Snout-to-Vent Length 38.0 >38.0

Plasma Thyroxine Level 38.0 >38.0

Plasma Vitellogenin Level NR 1 NR

NF stage 66 Cull

Whole Body Weight 38.0 >38.0

Snout-to-Vent Length 38.0 >38.0

Frogs at Termination

Whole Body Weight (Male) 38.0 >38.0

Whole Body Weight (Female) 38.0 >38.0

Snout-to-Vent Length (Male) 38.0 >38.0

Snout-to-Vent Length (Female) 38.0 >38.0

Liver Weight:Whole Body Weight (Male) 38.0 >38.0

Liver Weight:Whole Body Weight (Female) 38.0 >38.0

Nuptial Pad Development (Male) 38.0 >38.0

Nuptial Pad Development (Female) 38.0 >38.0

Plasma Thyroxine Level (Male) 38.0 >38.0

Plasma Thyroxine Level (Female) 38.0 >38.0

Plasma Vitellogenin Level (Male, Female) NR NR

1

 NR = no result. 
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1.0 INTRODUCTION

The U.S. Environmental Protection Agency (U.S. EPA) is implementing the Endocrine 

Disruptor Screening Program (EDSP). To support this program, U.S. EPA has contracted with 

Battelle to provide ecotoxicological testing services, including chemical, analytical, statistical,

and quality assurance/quality control support to assist the U.S. EPA in developing, 

standardizing, and validating an ecotoxicological test for identifying and characterizing 

endocrine effects through exposure to pesticides, industrial chemicals, and environmental 

contaminants. The study conducted for Task Order 31 was subcontracted to ABC Laboratories 

for obtaining technical support for the Larval Amphibian Growth and Development Assay 

being considered for the Endocrine Disruptor Screening Program to chemicals suspected to be 

endocrine disrupting in vertebrates. 

This Task Order 31 study evaluated the Larval Amphibian Growth and Development Assay for 

use in examining adverse responses in amphibians exposed to vertebrate-relevant endocrinedisrupting compounds. The chemical 4-tert-octylphenol is a known endocrine-disruptor and was 

used for this study. The measurements of survival, development and growth, and secondary 

mechanistic biomarker responses should be indicative of potential adverse effects on population 

relevant parameters and potential endocrine disrupter activity for this species. The data 

generated was used to statistically determine the no-observed-effect concentration (NOEC) and 

the lowest-observed-effect concentration (LOEC) values for the individual biological endpoints. 

2.0 MATERIALS AND METHODS

2.1 Test Substance and Reference Substances 

Pre-loaded saturator columns containing the test substance, 4-tert-octylphenol (Lot No.: 

05130KD), were received from the U.S. EPA, Duluth, Minnesota, on 31 May 2011 and were

assigned ABC reference numbers MM-2628-00001 and MM-2628-00002. The expiration date

for these saturator columns was 31 May 2013. An additional pre-loaded saturator column 

containing the test substance (Lot No.: 05130KD) was received from Fort Environmental 

Laboratories, Stillwater, Oklahoma, on 08 February 2012 and was assigned ABC reference 

number MM-4939. A copy of the chain-of-custody form accompanied the saturator column. 

The do not use beyond date for this saturator column was 08 February 2014. A sample of the 

test substance (Lot No.: 05130KD) was received from the U.S. EPA, Duluth, Minnesota, on 

31 May 2011 and was assigned ABC reference number MM-2631. The do not use beyond date 

assigned to the test substance was 31 May 2013. This sample of the test substance was used to 

prepare additional saturator columns for the exposure. The certificate of analysis reported the 

purity of the test substance as 99.3% (Appendix A). A sample of the test substance (Lot No.: 

08118jhv) was received from the Battelle Marine Science Laboratory, Sequim, Washington on 

13 June 2012 and was assigned ABC reference number MM-5729. The expiration date assigned 

to the test substance was 30 November 2015. This sample of the test substance was used to 

prepare an additional saturator column for the exposure. The certificate of analysis reported the 

purity of the test substance as 98.0% (Appendix A). The saturator columns and test substance 

samples were stored at room temperature. The test substance was described by the Material 

Safety Data Sheet as a white solid and stable under recommended storage conditions. The test 

substance was used to prepare all matrix spiking solutions as well as analytical reference 
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standards. Any remaining test substance will be returned to the EDSP Chemical Repository. A 

retention sample of the test substance (Lot No.: 08118jhv) was archived at the U.S. EPA 

Chemical Repository, Battelle Marine Science Laboratory, Sequim, Washington. 

2.2 Test Species

The test species for this study was the African clawed frog, Xenopus laevis. Xenopus laevis

embryos were obtained from an in-house culture of adults. The adults exhibited no signs of 

disease nor were any prophylactic or therapeutic treatments utilized during the 14-day period 

prior to pairing. The adults were maintained under flow-through conditions and fed a diet of 

sinking frog food (i.e., frog brittle; NASCO; Fort Atkinson, Wisconsin) supplemented with a 

commercial fish food (i.e., salmon starter; Rangen Inc.; Buhl, Idaho). 

Eggs were collected from breeding aquaria and examined microscopically to determine embryo 

viability and developmental stage. All adult frogs in culture were maintained on a 16-hr light: 

8 hr dark photoperiod. Adult frogs were moved to an area of darkness when bred for the 

generation of embryos at the appropriate testing stage (i.e., Nieuwkoop and Faber (NF) stage 9).

2.3 Dilution Water 

The dilution water was laboratory freshwater prepared by blending naturally hard well water 

with well water that was demineralized by reverse osmosis. These waters were blended to yield 

a total hardness of approximately 140 to 160 mg CaCO3/L. The water was then passed through a 

sediment filter prior to use. The contaminant screen for a representative sample of the base 

blended water is provided in Appendix B. Analysis of a dilution water sample collected prior to 

initiation of the study in March 2012 contained 12 μg iodine/L. Data for the March 2012 

analysis is maintained as a facility record. Analysis of a dilution water sample collected while 

the study was in-life indicated a similar iodine concentration (i.e., 17 μg/L). 

2.4 Test Methods

Test procedures followed the ABC test protocol entitled “4-tert-Octylphenol: Larval Amphibian 

Growth and Development Assay using Xenopus laevis,” with amendments and deviation 

(Appendix C). This protocol was based upon the draft proposal for a new OECD guideline 

“Larval Amphibian Growth and Development Assay (LAGDA)” (1). 

2.4.1 Saturator Solution Preparations 

4-tert-octylphenol is a chemical that is not soluble enough in water to directly prepare a suitable 

stock solution. Therefore, a saturator column was used for the preparation of the 

4-tert-octylphenol solution. Initially, columns prepared by the U.S. EPA were used. Additional 

saturator columns were prepared by ABC Laboratories on 26 April, 01 June, 15 June, and 22 

June 2012 as follows. 

Saturator columns were clean Pyrex glass tubes approximately 1.2 meters in length and 2.54 cm 

in outer diameter, bent into a U-shape. The bent section of the column was open and the walls 

did not touch each other. The two “legs” of the U were approximately equal in length, parallel, 

and 10 cm apart. Each column was packed tightly with glass wool using a dowel rod, until an 8 
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to 10 cm void remained at the top of each leg. The packed column was rinsed by drawing 

acetone through the column, followed by clean air to dry. A column-coating solution was 

prepared by dissolving approximately 5 g of 4-tert-octylphenol in 100 mL of acetone. The 

solution was divided into two equal aliquots, one for coating each leg of the saturator column. 

One aliquot was added to each leg and drawn down with vacuum pressure until it nearly reached 

the bottom of the leg. The vacuum was reversed and the solution was drawn back up to just 

below the head of the column. This process was repeated until the solution was no longer 

visible. The other leg of the column was coated in the same fashion. The solution was not 

allowed to collect in the bottom of the column to ensure all of the test substance solution was 

distributed to the glass wool. Care was taken not to draw the solution past the glass wool on the 

outlet side of the column to avoid accumulation of pure compound particles that may pass into 

the diluter system, causing erratic exposure concentrations. After the acetone was completely 

evaporated from within the column, the stoppers from the column ends were removed and a 

small plug of clean glass wool was placed at each end to prevent coated wool from escaping the 

column. If not used immediately, the columns were stored at room temperature with solid 

stoppers on each end and wrapped with aluminum to prevent photodegradation. 

Prior to use on the test system, saturator columns were flushed to waste at a rate of 

approximately 20 mL/minute for approximately 18 to 24 hours prior to the collection phase. 

After the flush to waste period, the flow rate was adjusted to a rate of 4 mL/minute. Based upon 

Porter et al. (2), this process was expected to generate solution outflow with a concentration of 

approximately 8.5 to 11 mg/L of 4-tert-octylphenol. The concentration of the saturator column 

outflow was analytically verified prior to placing the saturator column on the diluter system. In 

an attempt to maximize 4-tert-octylphenol concentrations in test solutions relative to target 

nominal concentrations, saturator columns outflow was added directly to the chemical mixing 

box of the diluter system. Initially, a single saturator column was used on the test system. A 

second saturator column was subsequently added to improve recoveries relative to target nominal 

concentrations. Saturator columns were replaced as needed based upon results from analytical 

sampling. 

2.4.2 Exposure System

A 2-L proportional equal solvent diluter system, similar to that described by Mount and Brungs 

(3), equipped with FMI metering pump dispenser was used for the preparation of test solutions 

and intermittent introduction of the solutions to the test chambers. The nominal exposure 

concentrations selected by the U.S. EPA were 0 (dilution-water control), 6.25, 12.5, 25.0, and 

50.0 μg a.i./L. The diluter system was constructed from plate glass glued together with silicone 

adhesive. Each mixing/flow-splitting cell divided each 2-L volume four ways resulting in a 

volume of approximately 500 mL being delivered to each test chamber with each cycle of the 

diluter system. The accuracy of the delivery of the test solution to the test chambers was verified 

by volumetric measurement before test initiation and adjusted such that the delivered volume 

was ±10% of the target volume. Proper operation of the proportional diluter and all mechanical 

systems was verified twice each day during the definitive test. The diluter system was labeled 

with the study number. 

Addition of the test substance to the diluter system was initiated on 28 February 2012. The FMI 

metering pump introduced approximately 20-mL volumes of saturator solution outflow to the 
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diluter system where it was mixed with approximately 3.9 L of dilution water to generate the 

highest test substance treatment solution. The three lower test substance treatment solutions 

were generated by mixing appropriate volumes of the highest test substance treatment solution 

with dilution water at each cycle of the proportional diluter system. The target volume of 

saturator outflow delivered to the chemical mixing box was subsequently adjusted to improve 

test substance recoveries relative to target nominal concentrations. The diluter system was 

initially set to a cycle rate of 5.0 cycles per hour. The cycle rate was increased on 17 May 2012 

(study day 14) to approximately 5.8 cycles per hour to increase dissolved oxygen levels after 

data collected on 15 May 12 (study day 12) indicated potentially low levels. The cycle rate was 

increased again on 29 May 2012 (study day 26) to approximately 7.6 cycles per hour in an 

attempt to improve test substance recoveries from test solutions relative to target nominal 

concentrations. The diluter delivered approximately 60 L of dilution water control and test

substance treatment solution to each chamber daily at 5.0 cycles per hour, 70 L at 5.8 cycles per 

hour, and 91 L at 7.6 cycles per hour, which was sufficient to provide approximately 6.0, 7.0, 

and 9.1 volume additions, respectively, in a 24-hour period. 

The test chambers were glass aquaria measuring approximately 22 cm wide by 40 cm long by 

22 cm high with a test solution depth of 12 cm. These dimensions yielded a test solution volume 

of approximately 10 L. During the definitive test, the control was replicated eight times and each 

test substance treatment was replicated four times. All chambers were individually drained 

through the side of the chamber to a floor drain. Chamber drains were covered with stainless 

steel screen to prevent the tadpoles from escaping. Starting on study day 75, test chambers were 

covered with stainless steel screens to minimize the possibility of juvenile escape. Test 

chambers were arranged in a water bath using a computer-generated random number table. The 

temperature-controlled water bath was maintained such that the control and test substance 

treatment solutions were maintained at 21 ± 1°C during the exposure. Test chambers were 

labeled with the treatment and replicate.

Electronically controlled fluorescent lights illuminated all aquaria and provided a 12-hour light: 

12-hour dark photoperiod with 30-minute simulated dawn and dusk transition periods. Light 

intensity on day 111, as measured with a LI-COR Model LI-189 light meter equipped with a 

photometric sensor, ranged from 608 to 613 lux. 

2.4.3 Biological Methods 

Twelve male-female pairs of adult Xenopus laevis were artificially stimulated to breed by 

injecting each male with 300 IU hCG and each female with 400 IU hCG. After spawning of 

adults, embryos were collected, treated with cysteine, and examined for viability. Eggs from the 

single spawn producing the greatest number of eggs with ≥70% viability were used to initiate the 

study. Eggs were maintained in dilution water at approximately 21°C during the screening 

process. Approximately 4 hours after initial screening of eggs, all viable embryos approaching 

NF stage 9 were segregated for initiation. Prior to egg placement into test chambers, all 

impartially selected groups of 25 eggs were reexamined to confirm that all eggs were at 

developmental NF stage 9. 
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2.4.3.1 Test Initiation

The test was initiated on 03 May 2012 when 25 NF stage 9 embryos were added to each of eight 

test chambers for the dilution water control and each of four test chambers for each test 

substance treatment, yielding 200 embryos for the control and 100 embryos per treatment group. 

Embryos were impartially selected and distributed, five at a time, to a set of labeled containers. 

Embryos within a container were then released into the appropriate test chamber as determined 

by a random number table. Observations of mortality, moribundity, malformations, and behavior 

were recorded for all treatments daily. Dead animals were removed from the test tank as soon as 

observed and were categorized by developmental stage as either pre-NF 58 (i.e., pre-forelimb 

emergence), NF58-NF62, NF63-NF66 (between NF63 and complete tail absorption), or postNF66 (post-larval) (4).

2.4.3.2 Larval Sub-Sampling

When some of the larvae in a particular replicate attained NF stage 62, the test chamber was split 

with a glass partition to separate the NF stage 62 larvae from less developed individuals. One 

larva from every four that attained NF stage 62 was selected for larval sub-sampling. Subsampled individuals were anesthetized by immersion in a MS-222 solution that had been 

buffered to approximately pH 7.0. Blotted wet weight (to the nearest 0.001 g) and snout-to-vent 

length (to the nearest mm) of each sub-sampled individual were recorded and any gross 

morphological abnormalities or signs of toxicity were noted (e.g., scoliosis, petechiae, 

hemorrhage) prior to dissection. 

Blood for thyroxine (T4) and vitellogenin analysis was obtained by exposing the heart, snipping 

a major blood vessel, and collecting blood in a heparinized capillary tube. Immediately 

following blood collection, each individual was euthanized by immersion in a 2000 mg/L MS222 solution that had been buffered to approximately pH 7.0. The upper torso from just below 

the forelimbs was then removed and the lower body discarded. The trimmed carcass was placed 

in an individually labeled container with a sufficient amount of Davidson’s fixative to allow for 

free circulation of the fixative around the tissues (e.g., at least 10 times the approximated 

volumes of the tissues). At least 48 but no more than 96 hours after tissues were placed in 

Davidson’s fixative, they were rinsed in tap water and stored in 10% neutral buffered formalin 

(NBF). Tissue was shipped to Experimental Pathology Laboratories, Inc., Sterling, Virginia, and 

the results of these analyses are reported separately.

The sealed tubes with blood were placed on ice prior to centrifugation. After centrifugation, the 

plasma was expelled into a microcentrifuge tube coated with aprotinin. The volume of plasma 

obtained from each individual was recorded to facilitate pooling of samples prior to subsequent 

analyses. The plasma samples were stored at approximately -80oC following collection and 

remained in storage until analyzed. Prior to quantification of T4 and vitellogenin levels in the 

plasma, an equal volume of plasma from all sacrificed larvae from a given replicate test chamber 

was pooled. The amount of plasma contributed by each larva was equal the lowest individual 

volume collected. Sample(s) that yield an extremely low plasma volume were excluded from 

this pooling process. Portions of the pooled plasma sample will be used to perform quantification 

of T4 and vitellogenin levels. The ELISA kit Total T4 (Thyroxine) EIA (cat# 07BC1007) by 
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MP Biomedical was used to quantify Xenopus T4 in pooled plasma samples. Vitellogenin was 

quantified using EPA Duluth methodology. 

2.4.3.3 End of the Larval Exposure 

The larval portion of the experiment concluded on study day 70. A cull of surviving individuals 

was performed to reduce animal density to 10 individuals per test chamber. An additional 

juvenile was inadvertently removed from the control, replicate C test chamber at the juvenile cull 

(i.e., nine individuals were present after the cull was complete). Excess individuals were 

randomly identified and euthanized by immersion in a buffered MS-222 solution. The excess 

individuals were weighed (i.e., blotted wet weight), measured (i.e., snout-to-vent length), and 

phenotypic sex (i.e., male, female, or indeterminate) determined for each individual based on 

gonad morphology. The glass partition was removed to avoid future over-crowding resulting 

from juvenile growth. 

2.4.3.4 Juvenile Sampling

Juvenile sampling was completed over a two day period, with half of the replicates from the 

control and each test substance treatment randomly selected for termination on each day. On 

study day 112 and 113, ten weeks after the median NF stage 62 date (i.e., 15 June 2012; study 

day 43) among control larvae, remaining juveniles were anesthetized by an intraperitoneal 

injection of a buffered 10% MS-222 solution. Blotted wet weight and snout-to-vent length of 

each juvenile were recorded, and any gross morphological abnormalities or signs of toxicity were 

noted (e.g., scoliosis, petechiae, hemorrhage). Blood was collected by exposing the heart, 

snipping a major vessel and collecting blood in a heparinized capillary tube that was immediately 

placed on ice. Following blood collection, the forelimbs were examined under 

10x magnification for the presence/absence of nuptial pads, and the liver was removed and 

retained while the stomach and intestines were removed and discarded. The phenotypic sex was 

determined by examining gonad morphology. One hind limb was removed during dissection, 

placed in a labeled tube and stored in a cooler with ice until transfer on the same day to a -20oC 

freezer. The frozen hind limbs were shipped to Battelle-Pacific Northwest Division, Sequim, 

Washington, for gender genotyping. The retained liver was weighed (to nearest 0.0001 g), fixed 

in Davidson’s fixative, and preserved in 10% NBF as outlined in Section 2.4.3.2. The remaining 

carcass with gonads was also fixed in Davidson’s fixative and preserved in 10% NBF as outlined 

in Section 2.4.3.2. The livers and carcass with gonads were shipped to Experimental Pathology 

Laboratories, Inc., Sterling, Virginia, and the results of these analyses are reported separately.

The sealed tubes with blood were kept on ice until centrifugation. After centrifugation, the 

plasma was expelled into a microcentrifuge tube that was coated with aprotinin and stored at 

approximately -80oC. Vitellogenin and T4 levels were subsequently quantified as described in 

Section 2.4.3.7 (VTG) and 2.4.3.8 (T4). 

2.4.3.5 Water Chemistry

Temperature, pH, and dissolved oxygen concentration were measured in all replicate chambers at 

test initiation and then twice weekly throughout the test, including test termination. Temperature 

was measured with a WTW pH 330i pH meter, a WTW OXi 330 dissolved oxygen meter, or 

WTW Multi 3500i or 3430 multimeter. Solution pH was measured with a WTW pH 330i 
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pH meter, WTW Multi 3500i or 3430 multimeter. Dissolved oxygen concentration was 

measured with a WTW OXi 330 dissolved oxygen meter or WTW Multi 3500i or 3430 

multimeter. No aeration was provided to any control or test substance treatment chamber during 

the test. A continuous recording of temperature in replicate B of the 12.5 μg/L treatment was

made using a data logger and thermistor probe. Alkalinity, hardness, and conductivity were 

measured in the dilution water at initiation and on study days 28, 57, and 89. Conductivity was 

measured with a WTW Cond 3300i conductivity meter and total hardness and total alkalinity were 

measured using titrimetric methods adapted from Standard Methods (5).

2.4.3.6 Feeding

Larvae received a diet consisting of Sera Micron® solution beginning when larvae were freeswimming, with live brine shrimp added to the diet starting on study day 22. The total amount of 

food offered per animal per day varied depending on the study day as follows: free-swimming 

stage - day 7, 40 mg/larvae/day; day 8-10, 50 mg/larvae/day; day 11-14, 70 mg/larvae/day; 

day 15-21, 80 mg/larvae/day; day 22-70, 80 mg/larvae/day plus 1 mL of live brine shrimp. The 

total amount of food was divided into equal portions and fed three times daily. The amount of 

Sera Micron added to each tank was initially calculated based on 25 larvae and was adjusted for 

changing larval density. Once animals completed metamorphosis (NF stage 66), the portion of 

the test chamber containing juveniles received 0.05 mL of sinking frog food (e.g., Frog Brittle) 

powder per juvenile once daily in addition to Sera Micron solution and brine shrimp. The amount 

and consistency of food received by juveniles changed after the median metamorphosis date 

among controls as follows: weeks 0-1, 0.05 mL powder + 0.05 mL crushed pellets; weeks 2-3, 

0.2 mL crushed pellets; weeks 4-5, 0.3 mL crushed pellets; weeks 6-9, 0.4 mL crushed pellets. 

Coarse chunks of Frog Brittle (i.e., crushed Frog Brittle) were used in place of whole pellets to 

improve the accuracy of food measurement. Once all animals had metamorphosed and the cull 

had occurred, juveniles were fed only sinking frog food with each tank initially receiving the 

amount appropriate for 10 individuals. 

2.4.3.7 Vitellogenin Analysis

Vitellogenin (VTG) analysis of plasma samples was conducted according to a method provided 

by EPA-Duluth, which involved coating of microplates and mixing of required solutions and 

antigens. For this purpose, X. laevis VTG standards, “anti-X. tropicalis VTG” antibody, as well 

as positive control plasma were provided by EPA-Duluth. To ensure consistency between plates, 

each day the assay was performed a fortified interassay quality control sample was analyzed. The 

fortified QC sample was freshly prepared for each analysis by spiking pooled male non-study 

plasma with VTG from the same source as was used to prepare standards for the calibration 

curve. The target nominal concentration of the fortified QC sample was 2 μg/dL. The fortified 

QC and study samples were analyzed at least in duplicate. The results of spiked Xenopus

samples were to be 70 to 150% of the nominal concentration and the coefficient of variation 

(CV) of QC samples between days was not to be greater than 20% to be acceptable. For each 

analysis, the calibration curve had an R2 of 0.99 or greater and the back calculated standard 

concentrations of the standards used to prepare the standard curve were between 70 and 150% of 

the nominal concentration. 
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Plates were coated with coating solution containing VTG standard and coating buffer (50 mM 

carbonate buffer pH 9.6), and incubated at approximately 4°C overnight. The coating solution 

was aspirated (Molecular Devices AquaMax 2000 plate washer) and blocking solution (2.5% 

normal goat serum in coating buffer) was added followed by a 2 hour incubation at 

approximately 37°C. Pre-adsorbed primary antibody serum was diluted, combined with samples 

as well as standards in microcentrifuge tubes and incubated at 37°C for one hour. Plates were 

washed three times with washing buffer (PBS + 0.05% Tween-20 pH 7.4), and after all buffer 

was removed, appropriate wells were loaded with diluted standards and samples and incubated at 

approximately 37°C. After 1 hour, plates were washed three times with washing buffer and 

diluted secondary antibody was added for another incubation at 37°C for 1 hour. After 

incubation the plates were washed three times with washing buffer, the TMB substrate solution 

was added to all wells, and the plates were incubated in the dark, at room temperature, for at 

least 10 minutes. The reaction was stopped with 1M H3PO4 and the absorbance read by a 

spectrophotometer (Molecular Devices SpectraMax 190) at a wavelength of 450 nm. All 

readings were collected electronically and analyzed using Molecular Devices SoftMax Pro GxP 

5.3 software. 

The reported results, expressed as ng/mL, were based on sample runs that had standard curve R2

values >0.990. The curves for the reported data all were based upon the four-parameter logistic 

model equation: y = ((A – D)/(1+ ((x/C)^B))) + D. If the VTG value for a particular sample was 

below the standard curve, the result was reported as less than the dilution factor for that sample 

multiplied by the lowest acceptable standard for a given run. If the VTG value for a particular 

sample was above the curve, the sample was re-diluted to fall within the standard curve. If 

measured VTG concentrations were below the standard curve but still calculated by the software, 

1/2 of lowest standard concentration of that particular run was reported as the VTG 

concentration. When measured VTG concentrations were below the standard curve and could 

not be calculated by the software, the concentration of VTG was set equal to 0.

2.4.3.8 Thyroxine (T4) Analysis

The ELISA kit Total T4 (Thyroxine) EIA (cat# 07BC1007) by MP Biomedical was used to 

quantify Xenopus T4 in plasma samples. Thyroxine (lot no. BCBD0567V; expiration date: 

August 2013) obtained from Sigma was used to prepare T4 standards. A 1x Tris-buffer (0.01M 

Tris, 0.15M NaCl) pH-adjusted to approximately 7.62 was prepared as dilution buffer for 

standards and samples. For each analysis, the calibration curve had an R2 of 0.99 or greater and 

the back calculated concentrations of the standards used to prepare the calibration curve were 

between 70 and 150% of the nominal concentration. A minimum of five standards in the 

functional range were utilized to ensure accuracy of the calibration curve. Standards were 

discarded if the CV between replicates was >20% or if the back calculated concentrations were 

not within 70 to 150% of the nominal concentration. Each day the assay was performed a 

fortified QC sample was analyzed to ensure consistency between plates. The fortified 

QC sample was freshly prepared for each analysis by spiking pooled non-study plasma with T4 

from the same source as was used to prepare standards for the calibration curve. The target 

nominal concentration of the fortified QC sample was 2 μg/dL. The fortified QC and study 

samples were analyzed at least in duplicate. The concentration of the fortified QC sample, as 

derived from the calibration curve, was to be 70 to 150% of the nominal concentration and the 

CV between replicates was to be ≤20% for the plate results to be considered acceptable unless 
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other evidence indicated the data were reliable. Fluctuations in calibration curves between plates 

did not invalidate a plate, but other criteria (e.g., acceptable standard curve and QC recoveries) 

within each individual plate were considered before not reporting results obtained from the 

plate(s).

Dilution buffer, diluted standards, and plasma were added to the appropriate wells on the precoated plates. Conjugate reagent was added to each well, plates were gently mixed for 

30 seconds, and the plates were incubated at approximately 22oC for one hour. The plates were 

then washed (Molecular Devices AquaMax 2000 plate washer) five times with water. TMB 

reagent was added to the wells, plates were gently mixed for five seconds, and then incubated 

again at approximately 22oC for approximately 30 minutes in the dark. After incubation, the 

reaction was stopped with 1M HCl and the plate contents were gently mixed for 30 seconds. 

Within 15 minutes after stopping the reaction, the absorbance was read using a 

spectrophotometer (Molecular Devices SpectraMax 190) at a wavelength of 450 nm. All 

readings were collected electronically and analyzed using Molecular Devices SoftMax Pro GxP 

5.3 software. 

The reported results, expressed as ng/mL, were based on sample runs that had standard curve R2

values >0.99. The curves for the reported data all were based upon the four-parameter logistic 

model equation: y = ((A - D)/(1+ ((x/C)^B))) + D. If the T4 value for a particular sample was 

below the range of the standard curve, the result was reported 0.5 multiplied by the minimum 

quantitation limit for a given run. If the T4 value for a sample was above the range of the 

standard curve, the sample was re-diluted to fall within the standard curve and then re-analyzed. 

2.5 Analytical Method 

Test solutions were analyzed for the concentration of 4-tert-octylphenol using high performance 

liquid chromatography with ultraviolet detection (HPLC-UV). Details of the sample collection, 

preparation, and method of analysis are described below. 

2.5.1 Preparation of Analytical Standard and Matrix Spiking Solutions 

Primary stock solutions of 4-tert-octylphenol were prepared in methanol on 17 August 2011 and 

23 July 2012 at concentrations of 0.614 and 0.765 mg/mL, respectively. Subsequent dilutions of 

these stock solutions were prepared in 50:50 methanol:water for use as analytical reference 

standards. 

Primary stock solutions of 4-tert-octylphenol were prepared in methanol on 17 August 2011 and 

23 July 2012 at concentrations of 0.562 and 0.626 mg/mL, respectively. Subsequent dilutions of 

this stock solution was prepared in methanol and used to prepare quality control (QC) samples.

All analytical standard and matrix spiking solutions were stored refrigerated between uses. 

2.5.2 Test Solution Analysis 

The concentrations of 4-tert-octylphenol were measured in test solution samples collected from 

the control and each treatment one day prior to initiation (day –N), day 0 (initiation), and at least 

weekly throughout the definitive test. Samples collected on day -1 were from one of the four 
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replicates for all treatments. All replicates of all treatments were sampled on day 0 and at 

termination. A single alternating replicate (e.g., replicate A on day 3, replicate B on day 7, etc.) 

sample was collected from each treatment at the remaining time points. The concentration of 

4-tert-octylphenol in the saturator outflow solution samples was determined on the same days as 

the test solution samples. 

A 10-mL sample was collected from the control, each test substance treatment solution, and the 

saturator outflow solution(s). The samples were placed into appropriately sized culture tubes. 

Samples were diluted with methanol and further diluted, if necessary, with 50:50 methanol:water

to a concentration within the analytical standard concentration range (i.e., 1.54 to 61.4 ng/mL). 

The samples were analyzed by a HPLC system equipped with a UV detector. 4-tert-octylphenolfortified samples were also prepared for analysis at each sampling point.

2.5.3 Instrument Conditions

Sample analysis was performed with a HPLC (Agilent model 1100 or equivalent) equipped with 

an UV detector using the following analytical parameters:

Column: Alltima C18, 4.6 mm × 150 mm, 5 μm 

Temperature: 40°C 

Mobile Phase: 75% methanol: 25% water (Isocratic)

Flow Rate: 1.1 mL/minute

Injection Volume: 300 μL

Wavelength: 220 nm 

Detector: UV 

Stop Time: 10 minutes

Attenuation: 500 mAU 

The injection volumes were modified from the conditions specified by the protocol to optimize 

the chromatographic conditions. 

2.5.4 Calculations for 4-tert-octylphenol

Calculation of 4-tert-octylphenol concentrations in test samples was performed by the external 

standard analysis function of Empower 2 software. The concentration of 4-tert-octylphenol from 

each sample was determined directly from the standard curve by the equation:

μg/L sample volume mLin

mLin

analysis volume

standard curve

fromμg/L

=

































Example calculation for the 6.25 μg/L nominal treatment (Level 1 D) on day 0: 

The standard curve equation is of the form: y = mx + b 

where:
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y = peak area units

m = slope of the standard curve 

x = μg 4-tert octylphenol/L

b = y-intercept of the standard curve 

Standard Curve: y = 549.7048x – 29.69535 

Sample Peak Area: = 2,301 

μg/L from Standard Curve = 4.239 μg/L

The concentration of 4-tert-octylphenol in the sample was calculated by the equation:

( ) 8.48 μg a.i./L

5 mL

 μg 239.4( a.i./L) 10 mL =

Recovery of 6.25 μg/L nominal treatment, replicate D, sample on day 0: 

136% 100x

μg/L 6.25

a.i./L μg 8.48 =

The minimum quantifiable limit (MQL) was determined from the equation:

MQL expressed as μg/L 

sampled (mL)

volume

analysis(mL)

forvolume

concentrat /(ion L)

lowest standard

=

















































μg

Lowest standard concentration: 1.54 μg/L

Volume for analysis: 10 mL

Volume of sample: 5 mL

therefore:

MQL = a.i./L μg 3.08 (5 mL)

 (10 μg/L) (1.54 mL) =

2.6 Genetic Sexing

DNA samples from the juvenile frogs were obtained from the hind foot removed during 

dissection. The hind foot from each specimen was stored at -20°C until DNA isolation. Testing 

for the presence or absence of a marker was done by PCR as described in Battelle’s method 

number EDSP.C5-046-00 (Genetic Sexing of Xenopus laevis Using PCR Followed by Gel 

Separation). The method consisted of two stages. In stage one, the DNA was extracted from a 

small tissue sample obtained from an individual Xenopus laevis. In stage two, the DNA sample 

was employed in a PCR reaction to determine whether the sex determining DM-W gene marker 

was present (ZW type, or female) or absent (ZZ type, or male). Each PCR reaction included 
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primers for the DMRT1 gene as a positive control to be amplified in both ZW and ZZ types. 

Because the DM-W gene marker is only present in genotypic females, true quantification was 

not necessary, only verification of this sequence within the DNA sample was needed. Therefore, 

the samples normally used to generate a standard curve for quantitation were omitted from the 

96-well plate. The genetic sexing report from Battelle is presented in Appendix D. 

2.7 Statistical Analyses 

Parametric assumptions were tested using the residuals from a one-way analysis of variance 

(ANOVA) of the treatment means of the raw variable or following a transformation. Deviations 

from normality were tested with the Shapiro-Wilks’ test (α= 0.05) and homogeneity of within 

class means were tested with Levene’s test (α=0.05). Count data were transformed to the square 

root, proportional data were transformed to the arcsine square root, and continuous data were

transformed to the natural logarithm. If the responses were monotonic, the William’s test of 

ordered alternatives was used to estimate the lowest-observed-effects concentration (LOEC) and 

the no-observed-effects concentration (NOEC) when the assumptions of normality and 

homogeneity of within class variances were met, otherwise the step-down Jonckheere-Terpstra 

nonparametric test of ordered alternatives was used. If the responses were not monotonic, 

Analysis of Variance (ANOVA) followed by Dunnett's test was used to estimate the 

LOEC/NOEC when the assumptions of normality and homogeneity of within class variances 

were met. When these assumptions were not met, the Kruskal-Wallis (K-W) test followed by a 

step-down Dunn’s test was used. Statistical outliers were defined as those values outside the 

bounds determined by Q1 – 1.5*IQD and Q3 + 1.5*IQD where Q1 and Q3 are the first and third 

quartiles of the data and IQD is the difference between Q3 and Q1. 

No variables met the assumptions for the William’s test of ordered alternatives. The JonckheereTerpstra nonparametric test of ordered alternatives was run, however, because this test does not 

account for the difference in the number of replicates between the control (n=8) and the test 

treatments (n=4), the LOEC/NOEC was estimated from a K-W multiple comparisons test of the 

control median compared to the test medians. ANOVA followed by Dunnett’s test was used to 

estimate the LOEC/NOEC for non-monotonic responses when the assumptions of normality and 

homogeneity of within class variances were met. When the ANOVA (a comparison among all 

treatments) was significant, the Dunnett’s Method was used to compare the mean responses 

between the control and treatment doses. The K-W test was used when assumptions of normality 

and homogeneity of within class variances were not met. When the K-W (a comparison among 

all treatments) was significant, a K-W multiple comparison test was used to evaluate the median 

responses between the control and treatment doses and to estimate the LOEC/NOEC. No 

identified outliers were removed from the analyses. Time to metamorphosis at NF stage 62 was 

analyzed by following the statistical procedures used for the other study parameters and by a 

Kaplan-Meier product-limit estimator. A binomial probability was calculated on the incidence 

of nuptial pads with the expected proportion of nuptial pad occurrence calculated from the mean 

of the control replicates. Significance was based on α = 0.05. Descriptive statistics included the 

mean, standard deviation (StDev), and the coefficient of variation (CV) of replicates within 

treatments. The mean and standard deviation of the treatment-related responses for each 

biological parameter are provided in the results tables. However, statistical differences detected 

by the K-W test or the K-W multiple comparisons test were between median values. When frog 

sex is mentioned in statistical analyses, the genotypic sex was used. Samples CE6-T and CE7-T 
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produced weak bands and the genetic results are equivocal. Individual CE6-T was a phenotypic 

and genotypic male; individual CE7-T was a phenotypic male but a genotypic female. Analyses 

of all NF stage 62 parameters and both NF stage 66 cull parameters were not separated by frog 

sex because there is no expectation that these parameters would vary by sex at the particular 

times of development at which they were measured. Statistics were done and graphs prepared in 

Minitab v16, except for the analytical chemistry data and nuptial pad binomial probability, which 

were analyzed in Microsoft Excel. Table mean or median values for Time to NF stage 62 were 

rounded to the nearest whole number; however boxplot values were not because Minitab creates 

the plots by using the values it calculates. The statistical report from Battelle is presented in 

Appendix E. 

3.0 RESULTS AND DISCUSSION

3.1 Analytical Results

Measured 4-tert-octylphenol concentrations in the 6.25, 12.5, 25.0, and 50.0 μg/L nominal test 

substance treatments at day –1 were 4.24, 8.35, 15.8, and 32.1 μg/L, respectively, representing 

63 to 68% of the nominal treatment concentrations. Measured 4-tert-octylphenol concentrations 

on day 0 ranged from 136 to 150% of the nominal treatment concentrations. Overall mean 

measured concentrations of 4-tert-octylphenol in the 6.25, 12.5, 25.0, and 50.0 μg/L nominal test 

substance treatments were 4.97, 10.3, 19.5, and 38.0 μg/L, respectively, which represented 76 to 

82% of the nominal treatment concentrations (Table 3.1.1). Following high recoveries from all 

test substance treatments at study initiation, measured concentrations were 80 to 120% of the 

overall mean measured concentrations with the following exceptions: 1) day 3, 28, 32, 39, 54, 

77, 91, and 97 samples from the 6.25 μg/L nominal treatment were 126, 65, 66, 62, 129, 126, 

134, and 123%, respectively, 2) day 18, 28, 32, and 39 samples from the 12.5 μg/L nominal 

treatment were 77, 70, 78, and 72%, respectively, 3) day 3, 28, 32, 77 and 81 samples from the 

25.0 μg/L nominal treatment were 133, 79, 70, 133 and 124%, respectively, and 4) day 3, 28, 32, 

47, 61 and 77 samples from the 50.0 μg/L nominal treatment were 130, 76, 66, 123, 69, and 

130%, respectively. The measured 4-tert-octylphenol concentration in saturator overflow

solution samples during the exposure ranged from 64 to 149% of the nominal concentration. No 

residues of the 4-tert-octylphenol were detected in the controls above the MQL of 3.08 ng/mL. 

Recoveries from the 4-tert-octylphenol quality control fortifications ranged from 88 to 118% of 

the nominal concentrations. Because the mean measured concentrations of 4-tert-octylphenol 

were not within 20% of nominal concentrations during the exposure, the biological response 

results were reported based upon the mean of the measured concentrations. An overall summary 

of the analyses at each sampling event are presented in Appendix F. 

On the morning of 03 July 2012 (study day 61), the drive belt for one of the two FMI metering 

pumps delivering saturator solution to the chemical mixing box was discovered broken. As a 

result, no saturator solution was delivered from one of the two saturator columns for an 

undetermined amount of time. A new belt was installed on the metering pump and saturator 

solution delivery was resumed as soon as possible. Analysis of samples collected soon after the 

above pump malfunction was addressed indicated recoveries ranging from 52-73% of nominal. 
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Table 3.1.1. Summary of analytical results for the Xenopus laevis 4-tert-octylphenol in-life exposure.

Nominal 

Concentration 

(μg/L)

Mean Measured

Concentration (μg/L) StDev CV Recovery

0 <MQL a NA NA NA

6.25 4.97 1.48 30% 80%

12.5 10.3 2.22 22% 82%

25.0 19.5 4.72 24% 78%

50.0 38.0 10.4 27% 76%

a MQL = Minimum Quantifiable Limit = 3.08 μg/L

3.2 Biological Results

At termination of the exposure, mean survival was 93% in the control and 84, 85, 93, and 92% in 

the 4.97, 10.3, 19.5, and 38.0 μg/L test substance treatments, respectively. See Appendix G for 

detailed survival data throughout the exposure. Two replicates (i.e., replicate B of the 4.79 and 

10.3 μg/L treatments) were compromised (i.e., >20% mortality) but were not excluded from 

statistical analyses. Sublethal observations and/or abnormalities observed prior to study day 11 

included the following: a) white coloration of eggs in the control (n = 9), the 4.97 μg/L treatment 

(n = 12), the 10.3 μg/L treatment (n = 7), the 19.5 μg/L treatment (n = 1), and the 38.0 μg/L 

treatment (n = 1); b) larvae on the bottom of the test chamber in the control (n = 1), the 10.3 μg/L 

treatment (n = 3), and the 19.5 μg/L treatment (n = 2); c) larvae swimming erratically in the 

control (n = 2), and the 10.3 μg/L treatment (n = 1); and d) a larva with spinal curvature in the 

10.3 μg/L test substance treatment. The predominant sublethal observation throughout the 

remainder of the study was spinal curvature. This abnormality began to occur on study day 19 

and was observed in the control and all test substance treatments. Spinal curvature was no 

longer observed after study day 57, with the exception of study days 70, 112, and 113. Sublethal 

observations including one or two larvae surfacing, discolored, swimming erratically, displaying 

edema, or on the bottom of the test chamber were observed infrequently in the control and test 

substance treatments from study day 12 through 48. No sublethal observations and/or 

abnormalities were observed from study day 71 to study day 111. 

NF Stage 62 Survival: 

Survival through NF stage 62 was 94% for the control treatment. Thirteen of 158 controltreatment larvae died before reaching NF stage 62; one larva was inadvertently killed during test 

chamber cleaning and one larva did not achieve NF stage 62 prior to the end of the larval 

exposure (i.e., day 70). These individuals were not counted in the total number of controltreatment larvae. Total survival in the 4-tert-octylphenol treatments ranged from 85 to 94% 

(Appendix G-2). 

Time to NF Stage 62: 

The median time to NF stage 62 was 43 days in the control treatment. The average time for 

Xenopus laevis larvae to reach NF stage 62 based on the within-replicate average analysis was 
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43 days in the control treatment and ranged from 43 to 44 days in the 4-tert-octylphenol 

treatments (Table 3.2.1, Figure_3.2.1). No significant differences among treatments were 

detected at α = 0.05. The average time to NF stage 62 based on the Kaplan-Meier product-limit 

estimator analysis was 42 days in the control treatment and ranged from 42 to 44 days in the 

4-tert-octylphenol treatments ( Table 3.2.1, Figure_3.2.2). No significant differences among 

treatments were detected at α = 0.05. A comparative evaluation of the two methods of 

estimating time to NF stage 62 is provided in Appendix 1 of the statistical report. The number of 

days until NF stage 62 was reached for each NF stage 62 larva sampled is presented in Appendix 

H.

Table 3.2.1. Mean (and standard deviation) time to NF stage 62 during the Xenopus laevis

4-tert-octylphenol exposure.

Mean Measured

Concentration (μg/L)

Time to NF stage 62 (d)

Within Replicate Average Kaplan-Meier Estimate

<MQL 43 (1.6) 42 (1.8)

4.97 43 (1.1) 42 (1.5)

10.3 44 (2.0) 44 (2.0)

19.5 44 (1.1) 43 (1.6)

38.0 44 (1.0) 43 (0.9)

p-value 0.472 0.491

Test: 1-way ANOVA K-W

<MQL 4.97 10.3 19.5 38.0

48

47

46

45

44

43

42

Mean Measured Dose (μg/L)

Time to NF Stage 62 (d)

Figure 3.2.1. Boxplot of time to NF stage 62 during the Xenopus laevis 4-tert-octylphenol exposure based 

on the replicate analysis. The box represents the interquartile range, the circle is the mean,

the asterisk is an outlier, and the horizontal line is the median.
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<MQL 4.97 10.3 19.5 38.0

47

46

45

44

43

42

41

40

Mean Measured Dose (μg/L)

Time to NF Stage 62 (d)

Figure 3.2.2. Boxplot of time to NF stage 62 during the Xenopus laevis 4-tert-octylphenol exposure based 

on the Kaplan-Meier product-limit estimator analysis. The box represents the interquartile 

range, the circle is the mean, the asterisk is an outlier, and the horizontal line is the median.

NF Stage 62 Growth: 

The whole body (wet) weight and snout-to-vent length for all NF stage 62 larvae measured are 

presented in Appendix H. Mean whole body (wet) weight of NF stage 62 larvae was 2.07 g in 

the control treatment and ranged from 2.04 to 2.19 g in the 4-tert-octylphenol treatments 

(Table 3.2.2; Figure 3.2.3). No significant differences in whole body weight among treatments 

were detected at α = 0.05. Mean snout-to-vent length of NF stage 62 larvae in the control 

treatment was 23 mm and ranged from 23 to 24 mm in the 4-tert-octylphenol treatments 

(Table 3.2.2; Figure 3.2.4). No significant differences in snout-to-vent length among treatments 

were detected at α = 0.05. 

Table 3.2.2. Mean (and standard deviation) whole body weight and snout-to-vent length for NF stage 62 

larvae during the Xenopus laevis 4-tert-octylphenol exposure.

Mean Measured

Concentration (μg/L)

Whole Body Weight 

(g)

Snout-to-Vent Length 

(mm)

<MQL 2.07 (0.173) 23 (0.5)

4.97 2.04 (0.131) 24 (0.5)

10.3 2.17 (0.125) 23 (0.8)

19.5 2.11 (0.241) 23 (0.8)

38.0 2.19 (0.113) 24 (0.9)

p-value 0.614 0.220

Test: K-W 1-way ANOVA
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<MQL 4.97 10.3 19.5 38.0

2.4

2.3

2.2

2.1

2.0

1.9

1.8

1.7

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 3.2.3. Boxplot of whole body weight for NF stage 62 larvae during the Xenopus laevis

4-tert-octylphenol exposure. The box represents the interquartile range, the circle is the 

mean, and the horizontal line is the median.

<MQL 4.97 10.3 19.5 38.0

25.0

24.5

24.0

23.5

23.0

22.5

22.0

Mean Measured Dose (μg/L)

Snout-to-Vent Length (mm)

Figure 3.2.4. Boxplot of snout-to-vent length for NF stage 62 larvae during the Xenopus laevis

4-tert-octylphenol exposure. The box represents the interquartile range, the circle is the 

mean, the asterisk is an outlier, and the horizontal line is the median.

Juvenile Survival: 

The survival of juveniles following the NF stage 66 cull was 100% for the control and all 

4-tert-octylphenol treatments. Following the NF stage 62 cull, one frog in the control and 

4.97 μg/L treatments escaped during routine daily activities. These individuals were not counted 

as exposure-related mortalities. Cumulative mean survival from Day 0 to Day 113 was 
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93 percent in the control treatment and ranged from 84 to 93 percent in the 4-tert-octylphenol 

treatments. Two replicates had less than 80 percent survival; 72 percent in replicate B of the 

4.97 μg/L treatment and 76 percent in replicate B of the 10.3 μg/L treatment. 

Growth of NF Stage 66 Culled Juveniles: 

The whole body (wet) weight, snout-to-vent length, and phenotypic sex of all NF stage 66 culled 

frogs is presented in Appendix I. Mean whole body (wet) weight of frogs measured at the NF 

stage 66 cull was 3.71 g in the control treatment and ranged from 3.44 to 4.02 g in the 4-tertoctylphenol treatments (Table 3.2.3; Figure 3.2.5). No significant differences in whole body 

weight among treatments were detected for these frogs at α = 0.05. Mean snout-to-vent length of 

frogs measured at the NF stage 66 cull was 30 mm in the control treatment and ranged from 30 

to 31 mm in the 4-tert-octylphenol treatments (Table 3.2.3; Figure 3.2.6). No significant 

differences in snout-to-vent length among treatments were detected for these frogs at α = 0.05. 

Table 3.2.3. Mean (and standard deviation) whole body weight and snout-to-vent length at the NF 

stage 66 cull of the Xenopus laevis 4-tert-octylphenol exposure.

Mean Measured

Concentration (μg/L)

Whole Body Weight 

(g)

Snout-to-Vent Length 

(mm)

<MQL 3.71 (0.303) 30 (0.8)

4.97 4.02 (0.295) 31 (0.7)

10.3 3.44 (0.075) 30 (0.8)

19.5 3.66 (0.325) 30 (1.7)

38.0 3.61 (0.435) 30 (1.1)

p-value 0.078 0.208

Test: K-W K-W

<MQL 4.97 10.3 19.5 38.0

4.4

4.2

4.0

3.8

3.6

3.4

3.2

3.0

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 3.2.5. Boxplot of whole body weight for frogs at the NF stage 66 cull of the Xenopus laevis

4-tert-octylphenol exposure. The box represents the interquartile range, the circle is the 

mean, the asterisk is an outlier, and the horizontal line is the median.
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<MQL 4.97 10.3 19.5 38.0

32

31

30

29

28

27

Mean Measured Dose (μg/L)

Snout-to-Vent Length (mm)

Figure 3.2.6. Boxplot of snout-to-vent length for frogs at the NF stage 66 cull of the Xenopus laevis

4-tert-octylphenol exposure. The box represents the interquartile range, the circle is the 

mean, the asterisk is an outlier, and the horizontal line is the median.

Growth at Termination: 

The whole body (wet) weight, snout-to-vent length, phenotypic sex, and genotypic sex of all 

frogs at termination are presented in Appendix J. Mean whole body (wet) weight of male frogs 

measured at the termination of the 4-tert-octylphenol exposure was 10.0 g in the control 

treatment and ranged from 9.3 to 10.8 g in the 4-tert-octylphenol treatments (Table 3.2.4; 

Figure 3.2.7). Mean whole body (wet) weight of female frogs measured at the termination of the 

4-tert-octylphenol exposure was 10.7 g in the control treatment and ranged from 9.8 to 11.0 g in 

the 4-tert-octylphenol treatments (Table 3.2.4; Figure 3.2.8). No significant differences among 

treatments were detected in whole body weight for male or female frogs at the termination of the 

exposure at α = 0.05. Mean snout-to-vent length of male frogs measured at the termination of 

the 4-tert-octylphenol exposure was 41 mm in the control treatment and ranged from 41 to 

44 mm in the 4-tert-octylphenol treatments (Table 3.2.4; Figure 3.2.9). Mean snout-to-vent 

length of female frogs measured at the termination of the 4-tert-octylphenol exposure was 

42 mm in the control treatment and ranged from 42 to 44 mm in the 4-tert-octylphenol 

treatments (Table 3.2.4; Figure 3.2.10). No significant differences in snout-to-vent length among 

treatments were detected for male or female frogs at the termination of the exposure at α = 0.05. 
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Table 3.2.4. Mean (and standard deviation) whole body weight and snout-to-vent length at the

termination of the Xenopus laevis 4-tert-octylphenol exposure.

Mean Measured

Concentration 

(μg/L)

Male Whole Body 

Weight (g)

Female Whole 

Body Weight (g)

Male Snout-toVent Length (mm)Female Snout-toVent Length (mm)

<MQL 10.0 (1.25) 10.7 (1.43) 41 (2.1) 42 (1.9)

4.97 10.7 (0.34) 10.8 (0.96) 42 (0.9) 43 (1.0)

10.3 10.8 (0.33) 9.8 (1.01) 44 (1.8) 42 (0.4)

19.5 9.3 (1.16) 10.8 (1.24) 41 (2.6) 44 (1.5)

38.0 10.2 (1.14) 11.0 (2.83) 42 (0.8) 43 (4.0)

p-value 0.362 0.708 0.337 0.327

Test: K-W K-W K-W K-W

Note: Gender is based on genotypic sex. 

<MQL 4.97 10.3 19.5 38.0

12

11

10

9

8

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 3.2.7. Boxplot of whole body weight for male frogs at the termination of the Xenopus laevis

4-tert-octylphenol exposure. The box represents the interquartile range, the circle is the 

mean, and the horizontal line is the median. Gender is based on genotypic sex. 
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<MQL 4.97 10.3 19.5 38.0

14

13

12

11

10

9

8

7

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 3.2.8. Boxplot of whole body weight for female frogs at the termination of the Xenopus laevis

4-tert-octylphenol exposure. The box represents the interquartile range, the circle is the 

mean, and the horizontal line is the median. Gender is based on genotypic sex. 

<MQL 4.97 10.3 19.5 38.0

46

45

44

43

42

41

40

39

38

37

Mean Measured Dose (μg/L)

Snout-to-Vent Length (mm)

Figure 3.2.9. Boxplot of snout-to-vent length for male frogs at the termination of the Xenopus laevis 4-tertoctylphenol exposure. The box represents the interquartile range, the circle is the mean, and 

the horizontal line is the median. Gender is based on genotypic sex. 
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<MQL 4.97 10.3 19.5 38.0

47

46

45

44

43

42

41

40

39

38

Mean Measured Dose (μg/L)

Snout-to-Vent Length (mm)

Figure 3.2.10. Boxplot of snout-to-vent length for female frogs at the termination of the Xenopus laevis 4-

tert-octylphenol exposure. The box represents the interquartile range, the circle is the mean, 

and the horizontal line is the median. Gender is based on genotypic sex. 

Nuptial Pad Development at Termination: 

The status of nuptial pads (i.e., present or absent) for each frog at termination is presented in 

Appendix K. The mean proportions of male and female frogs in the control treatment that had 

nuptial pads at the termination of the exposure were 0.23 and 0.19, respectively. The mean 

proportions of male and female frogs in the 4-tert-octylphenol treatments that had nuptial pads at 

the termination of the exposure ranged from 0.25 to 0.57 and from 0.35 to 0.47, respectively. 

(Table 3.2.5; Figure 3.2.11; Figure 3.2.12). No significant differences among treatment medians 

were detected for females or males at α = 0.05. 

In addition to the Kruskal-Wallis test of the null hypothesis of equal medians for all exposure 

concentrations, a binomial probability was also calculated on the incidence of nuptial pads for 

females and males (Table 3.2.6). The expected proportion of nuptial pad occurrence was 

calculated for males and females from the mean proportion from the control replicates 

(probability of nuptial pads occurring by chance = 0.22 for males, 0.19 for females; n = 8). The 

probability of obtaining the total number of occurrences out of 39 or 40 frogs (the number in 

each treatment) from chance alone was calculated independently for each exposure 

concentration. The proportion of males in the 10.3-μg/L 4-tert-octylphenol concentration having 

nuptial pads was 0.57, which was considered significantly greater than chance (p < 0.001). The 

proportions of females in the 4.97- and 38.0-μg/L 4-tert-octylphenol concentrations having 

nuptial pads were 0.47 and 0.50, respectively, which were considered significantly greater than 

chance (p = 0.008 and 0.005). 
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Table 3.2.5. Mean (standard deviation) proportion of female and male frogs and total number of frogs 

having nuptial pads at the termination of the Xenopus laevis 4-tert-octylphenol exposure.

Mean Measured

Concentration (μg/L)

Nuptial Pads 

(proportion of females)

Nuptial Pads 

(proportion of males)

Total Number of 

Frogs with Nuptial 

Pads

<MQL 78 0.23 (0.24) 0.19 (0.16)

4.97 39 0.25 (0.18) 0.47 (0.19)

10.3 40 0.57 (0.18) 0.36 (0.33)

19.5 39 1 0.26 (0.19) 0.35 (0.25)

38.0 40 0.28 (0.28) 0.45 (0.33)

p-value – 0.184 0.288

Test: – 1-way ANOVA K-W 

1 One female in replicate C was missing forelimbs

Note: Gender is based on genotypic sex. 

<MQL 4.97 10.3 19.5 38.0

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Mean Measured Dose (μg/L)

Proportion of Males with Nuptial Pads

Figure 3.2.11. Boxplot of the proportion of male frogs having nuptial pads at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the interquartile range, the 

circle is the mean, and the horizontal line is the median. Gender is based on genotypic sex. 
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<MQL 4.97 10.3 19.5 38.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Mean Measured Dose (μg/L)

Proportion of Females with Nuptial Pads

Figure 3.2.12. Boxplot of the proportion of female frogs having nuptial pads at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the interquartile range, the 

circle is the mean, and the horizontal line is the median. Gender is based on genotypic sex. 

Table 3.2.6. Proportion of total frogs within each treatment having nuptial pads at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. 

Mean Measured

Concentration 

(μg/L) N

Proportion of Total 

Males with Nuptial 

Pads

Binomial 

Probability

Proportion of 

Total Females 

with Nuptial Pads

Binomial 

Probability

<MQL 78 0.22 – 0.19 –

4.97 39 0.27 0.349 0.47 0.008

10.3 40 0.57 <0.001 0.35 0.085

19.5 39 1 0.28 0.359 0.33 0.085

38.0 40 0.29 0.262 0.50 0.005 1 One female in replicate C was missing forelimbs

Note: Gender is based on genotypic sex. 

Liver Weight at Termination: 

The liver (wet) weight for each frog at termination is presented in Appendix K. Mean liver 

weight:whole body weight ratios of frogs measured for the control treatment at the termination of 

the 4-tert-octylphenol exposure were 0.058 for males and 0.059 for females. The ratio for males 

ranged from 0.057 to 0.059 in the 4-tert-octylphenol treatments (Table 3.2.7; Figure 3.2.13). 

The ratio for females ranged from 0.056 to 0.061 in the 4-tert-octylphenol treatments 

(Table 3.2.7; Figure 3.2.14). No significant differences among treatments were detected in liver 

weight:whole body weight for male or female frogs at the termination of the exposure at 

α = 0.05. 
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Table 3.2.7. Mean (and standard deviation) liver weight:whole body weight for male and female 

frogs at the termination of the Xenopus laevis 4-tert-octylphenol exposure.

Mean Measured

Concentration (μg/L)

Liver 

Weight:Whole Body 

Weight (Males)

Liver 

Weight:Whole Body 

Weight (Females)

<MQL 0.058 (0.003) 0.059 (0.006)

4.97 0.057 (0.003) 0.058 (0.002)

10.3 0.059 (0.005) 0.061 (0.005)

19.5 0.059 (0.003) 0.060 (0.005)

38.0 0.058 (0.004) 0.056 (0.002)

p-value 0.906 0.322

Test: 1-way ANOVA K-W

 Note: Gender is based on genotypic sex. 

<MQL 4.97 10.3 19.5 38.0

0.064

0.062

0.060

0.058

0.056

0.054

0.052

Mean Measured Dose (μg/L)

Liver Weight:Whole Body Weight

Figure 3.2.13. Boxplot of liver weight:whole body weight for male frogs at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the interquartile 

range, the circle is the mean, and the horizontal line is the median. Gender is based 

on genotypic sex. 
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<MQL 4.97 10.3 19.5 38.0

0.070

0.065

0.060

0.055

Mean Measured Dose (μg/L)

Liver Weight:Whole Body Weight

Figure 3.2.14. Boxplot of liver weight:whole body weight for female frogs at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the interquartile range, the 

circle is the mean, and the horizontal line is the median. Gender is based on genotypic sex. 

3.3 Genotypic/Phenotypic Sex Ratios 

Detailed results of genetic sexing of the juvenile frogs using PCR followed by gel separation are 

presented in Appendix D and Appendix J. The phenotypic and genotypic sex ratios at the 

termination differed within each dose (Table 3.3.1). The genotypic sex determinations of 

45 individuals differed from their phenotypic determinations. 

Table 3.3.1. Frog phenotypic and genotypic sex ratios at the termination of the Xenopus laevis 4-tertoctylphenol exposure.

Mean Measured

Concentration (μg/L) N

Phenotype Genotype Number of 

Mismatches 1 Females Males Females Males

<MQL 78 33 45 37 41 4

4.97 39 21 18 17 22 10

10.3 40 18 22 17 23 13

19.5 40 18 22 22 18 16

38.0 40 18 22 16 24 2

 1 The number of occurrences per treatment that the genotype did not match the phenotype

3.4 Water Quality 

All water quality parameters, with the exception of temperature, remained within acceptable 

limits throughout the course of the 113-day exposure (Table 3.4.1). The dissolved oxygen 

concentrations ranged from 4.0 to 8.7 mg/L, representing 47 to 102% saturation. Test solution 

pH ranged from 7.5 to 8.5 during the test. The temperature of all test solutions ranged from 19.9 

to 21.9°C during the test. The continuous temperature data collected from a centrally located test 
ABC Study No. 66086 

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chamber indicated that, with two exceptions (05July12 and 30July12), the temperature remained 

within the temperature range of 21 ± 1°C specified by the protocol. On 05July12, test solution 

temperature was outside the acceptable range for approximately 3 hours and reached a maximum 

of 22.2 oC. On 30July12, test solution temperature was outside the acceptable range for 

approximately 45 minutes and reached a minimum of 19.5 oC. These exceptions to solution 

temperature had no adverse effect on study integrity. Hardness, alkalinity, and conductivity in the 

dilution water ranged from 136 to 154 mg CaCO3/L, from 150 to 160 mg CaCO3/L, and from 

340 to 357 μS, respectively. Individual water parameter measurements for temperature, 

dissolved oxygen concentration, and pH during the 113-day exposure are presented in 

Appendix L. 

Table 3.4.1. Ranges for Water Quality Measurements During the 113-Day Exposure with the African 

Clawed Frog, Xenopus laevis, Exposed to 4-tert-octylphenol

Mean Measured 

Concentration

(μg/L)

Temperature

(°C) a

Dissolved

Oxygen

(mg/L) a,b pH a

Conductivity

(μS) a

Alkalinity

(mg/L) a

Hardness

(mg/L) a

Control 20.1 – 21.8 4.01 – 8.70 7.47 – 8.46 340 – 357 150 – 160 136 – 154

4.97 20.2 – 21.7 4.33 – 8.72 7.59 – 8.26 340 – 357 152 – 160 140 – 150

10.3 20.2 – 21.7 4.55 – 8.71 7.62 – 8.28 -- -- --

19.5 20.5 – 21.9 4.31 – 8.69 7.63 – 8.24 -- -- --

38.0 19.9 – 21.7 4.38 – 8.67 7.33 – 8.46 -- -- --

a Values based upon individual replicate measurements for each treatment level.

b 100% dissolved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21 and 22°C, respectively.

3.5 Vitellogenin Results

All plasma vitellogenin (VTG) concentrations for NF stage 62 larvae were less than the 

minimum quantifiable limit or could not be calculated from the standard curve (Appendix M). 

No statistical analyses were conducted for this parameter.

The measured VTG concentrations in plasma of the juveniles at study termination are presented 

in Appendix M. Plasma VTG concentrations for 117 of 128 males and 103 of 109 females from 

all treatments at termination were less than the minimum quantifiable limit or could not be 

calculated from the standard curve. No analyses were performed on VTG concentrations.

3.6 Plasma Thyroxine (T4) Results

The mean plasma thyroxine (T4) concentration for NF stage 62 larvae was 227 ng/mL in the 

control treatment and ranged from 203 to 287 ng/mL in the 4-tert-octylphenol treatments

(Table 3.6.1; Figure 3.6.1). No significant differences in plasma T4 concentration among 

treatments were detected. The measured plasma T4 concentrations in pooled NF stage 62 

samples in presented in Appendix N. 
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The mean plasma T4 concentrations for control-treatment male and female frogs at the 

termination of the 4-tert-octylphenol exposure were 61.0 and 76.8 ng/mL, respectively

(Table 3.6.2; Figure 3.6.2). Plasma T4 concentrations for frogs in the 4-tert-octylphenol 

treatments ranged from 55.4 to 79.4 ng/mL for males and from 57.2 to 72.6 ng/mL for females

(Table 3.6.2; Figure 3.6.3). No significant differences in plasma T4 concentrations among 

treatments were detected for males or females. The measured plasma T4 concentrations in 

samples from juveniles at study termination are presented in Appendix N. 

Table 3.6.1. Mean (and standard deviation) plasma thyroxine (T4) for NF Stage 62 frogs during the 

Xenopus laevis 4-tert-octylphenol exposure.

Mean Measured

Concentration (μg/L)

Plasma Thyroxine (T4) 

(ng/mL)

<MQL 227 (72.6)

4.97 283 (125)

10.3 203 (94.4)

19.5 280 (171)

38.0 287 (84.8)

p-value 0.739

Test: K-W

Table 3.6.2. Mean (and standard deviation) plasma thyroxine (T4) for frogs at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure.

Mean Measured

Concentration (μg/L)

Male Plasma 

Thyroxine (T4) 

(ng/mL)

Female Plasma 

Thyroxine (T4) 

(ng/mL)

<MQL 61.0 (24.0) 76.8 (28.6)

4.97 66.2 (19.3) 60.1 (14.2)

10.3 79.4 (23.5) 57.2 (21.6)

19.5 59.1 (20.5) 61.6 (15.6)

38.0 55.4 (12.1) 72.6 (11.6)

p-value 0.544 0.550

Test: 1-Way ANOVA K-W

 Note: Gender is based on genotypic sex.
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0.0 4.97 10.3 19.5 38.0

500

400

300

200

100

Mean Measured Dose (μg/L)

Thyroxine Concentration (ng/mL)

Figure 3.6.1. Boxplot of plasma thyroxine (T4) concentration for NF Stage 62 larvae during the Xenopus 

laevis 4-tert-octylphenol exposure. The box represents the interquartile range, the circle is 

the mean, and the horizontal line is the median. 

0.0 4.97 10.3 19.5 38.0

110

100

90

80

70

60

50

40

30

Mean Measured Dose (μg/L)

Thyroxine Concentration (ng/mL)

Figure 3.6.2. Boxplot of plasma thyroxine concentration for male frogs at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the interquartile range, the 

circle is the mean, the asterisk is an outlier, and the horizontal line is the median. Gender 

is based on genotypic sex.
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0.0 4.97 10.3 19.5 38.0

120

100

80

60

40

20

Mean Measured Dose (μg/L)

Thyroxine Concentration (ng/mL)

Figure 3.6.3. Boxplot of plasma thyroxine concentration for female frogs at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. The box represents the interquartile range, the 

circle is the mean, and the horizontal line is the median. Gender is based on genotypic sex.

3.7 Validity and Performance Criteria

With several exceptions, validity and performance criteria stated in the study protocol were met 

for this study. Water quality characteristics remained within the tolerance limits outlined in the 

protocol with the exception of temperature. The test solution temperature deviated from the 

acceptable range (i.e., 21 ± 1 oC) on 05July12 and 30July12 (study day 63 and 88, respectively). On 

05July12, test solution temperature was outside the acceptable range for approximately 3 hours 

and reached a maximum of 22.2 oC. On 30July12, test solution temperature was outside the 

acceptable range for approximately 45 minutes and reached a minimum of 19.5 oC. With the 

exception of the following study days (0, 6, 8, 12, 19, 21, 32, 39, 50, 60, 97, 106, 112, and 113), test 

solution temperature differences among treatments on a given study day exceeded the acceptable 

level of variation stated in the protocol (i.e., 0.5 oC). Temperature differences exceeding 0.5 oC 

among treatments ranged from 0.6 – 1.0 oC with the following exceptions (differences of 1.3, 1.2, 

1.2 oC on day 41, 85, and 88, respectively). Delivery of chemical to the test organisms (e.g., 

concentrations of the chemical in test water) was not maintained at ≤20% CV over the entire test

(CV values ranged from 22 to 30% for the test substance treatments). Survival of test organisms 

was ≥90% in all control replicates and mean survival was ≥84% in all test substance treatments. 

Two test replicates (replicate B of the 4.79 and 10.3 μg/L treatments) were compromised (i.e., 

>20% mortality). This number of compromised replicates did not exceed the acceptable number 

as per the study protocol. Time to NF stage 62 among control larvae ranged from 38 to 44 days. 

The mean weight of culled NF stage 66 control larvae was 3.71 g.

4.0 CONCLUSIONS

With the above noted exceptions, this study satisfied acceptability and performance criteria 

stated in the study protocol. The no-observable-effect concentration and lowest-observableeffect concentration were calculated as mean measured concentrations of 38.0 μg/L 4-tert-
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octylphenol and >38.0 μg/L 4-tert-octylphenol, respectively, for each Xenopus laevis parameter. 

(Table 4.0.1).

Table 4.0.1. Calculated No-Observable-Effect Concentration and Lowest-Observable-Effect 

Concentration based on mean measured 4-tert-octylphenol concentrations for parameter 

measured for Xenopus laevis exposed to 4-tert-octylphenol.

Parameter No-Observable-Effect 

Concentration (μg/L)

Lowest-ObservableEffect Concentration 

(μg/L)

NF stage 62

Time to Metamorphosis (Replicate Analysis) 38.0 >38.0

Time to Metamorphosis (Kaplan-Meier) 38.0 >38.0

Whole Body Weight 38.0 >38.0

Snout-to-Vent Length 38.0 >38.0

Plasma Thyroxine Level 38.0 >38.0

Plasma Vitellogenin Level NR 1 NR

NF stage 66 Cull

Whole Body Weight 38.0 >38.0

Snout-to-Vent Length 38.0 >38.0

Frogs at Termination

Whole Body Weight (Male) 38.0 >38.0

Whole Body Weight (Female) 38.0 >38.0

Snout-to-Vent Length (Male) 38.0 >38.0

Snout-to-Vent Length (Female) 38.0 >38.0

Liver Weight:Whole Body Weight (Male) 38.0 >38.0

Liver Weight:Whole Body Weight (Female) 38.0 >38.0

Nuptial Pad Development (Male) 38.0 >38.0

Nuptial Pad Development (Female) 38.0 >38.0

Plasma Thyroxine Level (Male) 38.0 >38.0

Plasma Thyroxine Level (Female) 38.0 >38.0

Plasma Vitellogenin Level (Male, Female) NR NR

1

 NR = no result. 

5.0 RECORDS

The original raw data and the study plan were provided to the U.S. EPA with the final report. 

Copies of all data in support of this report were retained at ABC Laboratories, Inc., along with 

facility records and a copy of the final report and the study plan. 
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PROTOCOL DEVIATIONS 

Item 1 Protocol Section: 11.0 Exposure System 

Deviation: Only a single diluter function check was performed on 23May12. 

Reason for 

Deviation:

Technical oversight.

Effect on Study: None. Diluter function checks were performed twice daily throughout the 

remainder of the study.

Item 2 Section 13.0 Temperature

Deviation: Test solution temperature deviated from the acceptable range (i.e., 21 ± 1oC) in 

two instances during the course of the test (05July12 and 30July12). 

Reason for 

Deviation:

On 05July12, the chiller units used to regulate water bath (and therefore test 

solution) temperature were inadequate to counteract occasional fluctuations 

in the temperature of the ambient dilution water supplied to the test system.

On 30July12, a diluter malfunction caused the diluter to not cycle for 

approximately 80-85 minutes. During this malfunction, no fresh test solution 

was supplied to chambers and test solution temperature declined to a 

temperature similar to that of the water bath. 

Effect on Study: No adverse effect on study integrity. On 05July12, test solution temperature 

was outside the acceptable range for approximately 3 hours and reached a 

maximum of 22.2 oC. On 30July12, test solution temperature was outside the 

acceptable range for approximately 45 minutes and reached a minimum of 

19.5 oC. 

Item 3 Section 13.0 Temperature

Deviation: With the exception of the following study days (0, 6, 8, 12, 19, 21, 32, 39, 50, 

60, 97, 106, 112, and 113), test solution temperature differences among 

treatments on a given study day exceeded 0.5 oC (n = 22). Temperature 

differences exceeding 0.5 oC among treatments ranged from 0.6 – 1.0 oC with 

the following exceptions (differences of 1.3, 1.2, 1.2 oC on day 41, 85, and 88, 

respectively). 

Reason for 

Deviation:

Efforts made to limit test solution temperature variation to ≤0.5 oC across 

treatments within a given day (e.g., appropriate placement of re-circulating 

pumps in the water bath relative to chiller inflow/outflow tubes) were not 

sufficient to achieve this criterion throughout the course of the study. 
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Effect on Study: No adverse effect on study integrity. With the above noted exceptions, test 

solution temperatures across treatments on a given day were consistent (i.e., 

≤1.0 oC variation) throughout the test. 

Item 4 Section 15.0 Physical Parameters Monitored

Deviation: The alkalinity, hardness, and conductivity were measured in one replicate of 

the control and one concentration of the test substance at test initiation 

(03May12) and monthly thereafter (31May12, 27Jun12, 31Jul12) with the 

following exception: these parameters were not measured during the month of 

August.

Reason for 

Deviation:

Technical oversight

Effect on Study: None. Available data indicates that these parameters were consistent 

throughout the study (conductivity = 340-357 μS, alkalinity = 150-160, 

hardness = 136-154).

Item 5 Protocol Section: 17.0 Experimental Procedure

Deviation: Test Initiation

Embryos at Nieuwkoop-Faber (NF) stage 9 were randomly distributed into 

exposure treatment tanks. 

End of Larval Exposure

At the end of the larval exposure, one additional NF stage 66 frog (i.e., six in 

total) was inadvertently removed from the control, replicate C test chamber (a 

total of nine animals were present in this chamber at completion of the cull).

Reason for 

Deviation: Test Initiation

Communication with EPA Duluth indicated that examining embryos at a later 

development stage may help to minimize the number of non-viable/abnormal 

embryos selected for test initiation. In an attempt to improve larval hatch, 

embryos were examined as they approached NF stage 9 prior to distributing 

embryos to exposure treatment tanks.

End of Larval Exposure

Technical oversight. 
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Effect on Study: Test Initiation

None. No meaningful change in test exposure period resulted from using NF 

stage 9 embryos to initiate the test.

End of Larval Exposure

No adverse effect on study integrity. The above noted error was discovered 

on study day 78 (i.e., eight days after the end of the larval exposure) and the 

volume of feed added to the control, replicate C test chamber was corrected 

for this day and throughout the remainder of the study (i.e., study day 78-

112). While an additional 10% of feed was added to the above mentioned 

test chamber for seven days, data collected at test termination indicates that 

this did not have a meaningful effect on growth related endpoints (the range 

of data values for control, replicate C growth related endpoints at test 

termination are similar to those of other control replicates). 

Item 6 Protocol Section: 18.0 Diet

Deviation Whole Frog Brittle pellets were ground briefly with a mortar and pestle to 

produce coarse chunks of feed. 

During weeks 2-3 after the median metamorphosis date among controls, 

juveniles were fed 0.2 mL of sinking frog food/animal/day as per the protocol. 

The total amount of sinking frog food offered was calculated by multiplying 

the above feeding rate by the numbers of juveniles (i.e., NF stage 66 animals) 

present in a particular test chamber on a given day. In several instances 

(below), the incorrect volume of sinking frog food was offered to juveniles. 

a) 6.25 μg a.i./L nominal treatment, replicate A: Instead of 3.0 and 3.2 mL of 

food, 3.2 and 3.4 mL were offered on study days 57-59 and 60-61, respectively 

(representing approximately 7 and 6% of additional food, respectively).

b) 12.5 μg a.i./L nominal treatment, replicate C: Instead of 3.0 and 3.2 mL of 

food, 3.2 and 3.4 mL were offered on study days 57 and 58-61, respectively 

(representing approximately 7 and 6% of additional food, respectively).

c) 50 μg a.i./L nominal treatment, replicate B: Instead of 3.0 and 3.2 mL of 

food, 3.4 and 3.6 mL were offered on study days 57 and 58-61, respectively 

(representing approximately 13% of additional food).

Reason for

Deviation:

Coarse chunks of Frog Brittle were used in place of whole pellets to improve 

the accuracy of food measurement and therefore achieve the post-NF stage 62 

feeding rates stated in protocol section 18.0.

Incorrect feeding volumes were offered as noted above because of calculation 

error.
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Effect on Study: No adverse effect on study integrity. Frog Brittle was provided at the 

feeding rate stated in protocol section 18.0.

The above described incorrect feeding volumes did not have any adverse 

effect on study integrity because of the short duration of the feeding error 

(i.e., 5 days) relative to the duration of the juvenile exposure (from 46 to 

69 days).
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REFERENCES

1. Organization for Economic Co-operation and Development. 2010. Guideline for the 

Testing of chemicals; Draft – The Larval Amphibian Growth and Development Assay. 28 pp. 

October 2010. 

2. Porter, K.L., A.W. Olmstead, D.M. Kumsher, W.E. Dennis, R.L. Sprando., G.W. Holcombe, 

J.J. Korte, A. Lindberg-Livingston, and S.J. Degitz. 2011. Effects of 4-tert-octylphenol on 

Xenopus tropicalis in a long term exposure, Aquatic Toxicology 103, PP 159-169 

3. Mount, D.I. and W.A. Brungs. 1967. A Simplified Dosing Apparatus for Fish 

Toxicological Studies. Water Res. 1: 21-29. 

4. Nieuwkoop,P.D. & Faber,J. (1994) Normal Table of Xenopus laevis. Garland Publishing, 

New York

5. American Public Health Association. 1998. Methods 2320 and 2340 In: Standard 

Methods for the Examination for Water and Wastewater. 20th ed. Washington, D.C.
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APPENDIX A - CERTIFICATES OF ANALYSIS
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APPENDIX B - DILUTION WATER CHARACTERIZATION
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Chemical Characteristics of ABC Well Water Used by

ABC Laboratories' Chemical Services Group

February 2012 ABC Well Water Screen (non-GLP)

Organophosphate 

(μg/L) 2012

Historical 

Range 

1998-2012 1 Elements (mg/L) 2012

Historical Range 

1998-2012 1

Azinphos ethyl5, 7, 9 --- <1.0 Aluminum4 <0.0500 <0.0500

Azinphos-methyl3 <1 <0.200-<1 Antimony4 <0.0500 <0.0500

Bolstar4 <1 <0.200-<1 Arsenic <0.0030 <0.0030-<0.050

Chloropyrifos4 <1 <0.200-<1 Barium4 0.0188 0.0188-0.0206

Coumaphos4 <1 <0.400-<1 Beryllium4 <0.0010 <0.0010

Demeton, Total --- <0.200-<1.0 Boron6 0.396 0.370-0.432

Demeton-O8 <1 <1 Cadmium <0.0020 <0.0020-<0.0050

Demeton-S8 <1 <1 Calcium2 65.2 52.0-83.1

Diazinon <1 <0.200-<1 Chromium <0.0100 <0.010

Dichlorvos4 <1 <0.200-<1 Cobalt4 <0.0100 <0.0100

Dimethoate4 <1 <1 Copper <0.0100 <0.010

Disulfoton <1 <0.200-<1 Iron 0.107 <0.0059-0.16

EPN4 <1 <0.200-<1 Lead <0.0020 <0.0020-0.0400

Ethion5, 7, 9 --- <1.0 Magnesium2 30.3 27.0-33.3

Ethoprop4 <1 <0.200-<1 Manganese4 <0.0050 <0.0050

Famphur6 <1 <1 Molybdenum4 <0.0100 <0.0100

Fensulfothion4 <1 <1 Mercury5 <0.00020 <0.00020-

<0.00060

Fenthion4 <1 <0.200-<1 Nickel <0.0100 <0.0100-<0.020

Malathion <1 <0.200-<1 Potassium2 7.99 6.6-8.42

Merphos4, 7 <1 <0.200-<1 Selenium <0.0500 <0.050

Mevinphos4 <1 <1 Silver <0.0100 <0.010

Monocrotophos4, 7 <1 <1 Sodium2 29.5 27.0-32.2

Naled4, 7 <1 <1-<2.00 Thallium4 <0.0500 <0.0500

O,O,O-Triethyl 

phosphorothioate6 

<1 <1 Tin4 <0.0200 <0.0200

Vanadium4 <0.0100 <0.0100

Parathion5, 7, 9 --- <1.0 Zinc 0.0975 0.0118-0.0975

Parathion, ethyl3 <1 <0.200-<1 Polychlorinated 

Parathion, methyl Biphenyls (μg/L) 3 <1 <0.200-<1

Phorate4 <1 <0.200-<1 Aroclor 1016 <1.00 <1.00

Ronnel4 <1 <0.200-<1 Aroclor 1221 <1.00 <1.00

Stirophos4 <1 <0.200-<1 Aroclor 1232 <1.00 <1.00

Sulfotepp4 <1 <0.200-<1 Aroclor 1242 <1.00 <1.00

TEPP4, 7 <1 <0.200-<1 Aroclor 1248 <1.00 <1.00

Thionazin6 <1 <1 Aroclor 1254 <1.00 <1.00

Tokuthion4 <1 <0.200-<1 Aroclor 1260 <1.00 <1.00

Trichloronate4 <1 <0.200-<1
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Chemical Characteristics of ABC Well Water Used by

ABC Laboratories' Chemical Services Group (continued)

February 2012 ABC Well Water Screen (non-GLP)

Chlorinated 

Herbicides (μg/L) 2012

Historical 

Range 

1998-2012 1

Chlorinated 

Hydrocarbons (μg/L) 2012

Historical 

Range 

1998-2012 1

2,4,5-TP (Silvex) <0.200 <0.200-<50 4,4’-DDD <0.05 <0.04-<0.05

2,4-D <0.200 <0.200-<250 4,4’-DDE <0.05 <0.04-<0.05

4,4’-DDT <0.05 <0.04-<0.05

Aldrin <0.05 <0.04-<0.05

Miscellaneous (mg/L)

α-BHC <0.05 <0.04-<0.05

β-BHC <0.05 <0.04-<0.05

∆-BHC <0.05 <0.04-<0.05

Nitrite N <0.05 <0.01-≤0.05 Chlordane <0.05 <0.05-<0.48

Nitrate N 0.307 <0.110-0.329 Dieldrin <0.05 <0.04-<0.05

Total Phosphorus as P <0.050 <0.050-0.640 Endosulfan I <0.05 <0.04-<0.05

Endosulfan II <0.05 <0.04-<0.05

Endosulfan sulfate <0.05 <0.04-<0.05

Endrin <0.05 <0.04-<0.05

Endrin aldehyde <0.05 <0.04-<0.05

Endrin ketone <0.05 <0.04-<0.05

γ-BHC <0.05 <0.04-<0.05

Heptachlor <0.05 <0.04-<0.05

Heptachlor epoxide <0.05 <0.04-<0.05

Methoxychlor <0.05 <0.04-<0.095

Toxaphene <0.50 <0.50-<3.80

1 Data supporting these values are on file at ABC Laboratories. Less than (<) values indicate recovery was below 

the greatest limit of detection during these analyses. 2 Historical Range is from 2003.

3 Historical Range is from 2008. 4 Historical Range is from 2009. 5 Historical Range does not include 2009.

6 Historical Range is from 2011. 7 Historical Range does not include 2011 and 2012.

8 Historical Range is from 2012.

9

 Historical Range does not include 2012

Note: ABC well water is the base water for ABC Aged-Blended Water.
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APPENDIX C - PROTOCOL, AMENDMENTS, AND DEVIATION
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APPENDIX D – GENDER REPORT
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APPENDIX E – STATISTICAL REPORT
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APPENDIX F – ANALYTICAL DATA SUMMARY
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Table F-1. Measured Concentrations of 4-tert-octylphenol During the Larval Growth and Development Assay with the African 

Clawed Frog, Xenopus laevis 

Control Level 1 Level 2 Level 3 Level 4

(0 μg/L) Nominal (μg/L): 6.25 Nominal (μg/L): 12.5 Nominal (μg/L): 25.0 Nominal (μg/L): 50.0

Sample Measured Measured % of % of Measured % of % of Measured % of % of Measured % of % of

Day / Rep. (μg/L) (μg/L) Nominal Mean (μg/L) Nominal Mean (μg/L) Nominal Mean (μg/L) Nominal Mean

Study 

Initiation 

Day 0 

Rep. A <MQL a 8.98 144 181 18.0 144 175 36.1 144 185 74.9 150 197

Rep. B <MQL a 9.04 145 182 17.5 140 170 35.8 143 184 74.2 148 195

Rep. C <MQL a 8.49 136 171 18.0 144 175 33.9 136 174 74.3 149 196

Rep. D <MQL a 8.48 136 171 17.4 139 169 36.9 148 189 73.9 148 194

Rep. E <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. F <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. G <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. H <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Daily Mean <MQL a 8.75 140 176 17.7 142 172 35.7 143 183 74.3 149 196

3 <MQL a 6.25 100 126 12.0 96 117 26.0 104 133 49.4 99 130

7 <MQL a 4.77 76 96 8.87 71 86 19.1 76 98 36.5 73 96

13 <MQL a 5.15 82 104 8.91 71 87 15.6 62 80 32.4 65 85

18 <MQL a 4.09 65 82 7.97 64 77 16.7 67 86 32.3 65 85

28 <MQL a 3.21 51 65 7.26 58 70 15.5 62 79 28.7 57 76

32 <MQL a 3.27 52 66 8.00 64 78 13.6 54 70 24.9 50 66

39 <MQL a 3.10 50 62 7.44 60 72 16.4 66 84 31.7 63 83

47 <MQL a 5.51 88 111 11.3 90 109 20.9 84 107 46.9 94 123

54 <MQL a 6.42 103 129 11.1 89 108 20.2 81 104 44.2 88 116

60 <MQL a 4.26 68 86 10.9 87 106 18.9 76 97 36.8 74 97

61 <MQL a 4.58 73 f 92 9.50 76 f 92 15.7 63 f 81 26.2 52 f 69

64 <MQL a 5.12 82 103 10.4 83 101 22.8 91 117 40.4 81 106

a

 MQL =[ Low STD Conc. * Analysis Volume (mL)] / Sample Volume (mL) = (1.54 ng/mL * 20 mL) / 10 mL = 3.08 ng/mL b

 Not Applicable c The higher measured concentrations are suspected to have resulted from the animals being removed prior to the collection of the samples.

d The actual concentration for Day 88, Level 1 was <MQL. 1/2 MQL (1.54 ng/mL) was used for calculating the Overall Mean.

e When applicable, the Daily Mean was used for calculating the Overall Mean

f Due to a pump malfunction, solution was delivered to the chemical mixing box from one of two 4-tert-octylphenol saturator columns prior to collection of day 61. This 

malfunction may have contributed to lower test substance recoveries from some of the samples. 
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Table F-1. Measured Concentrations of 4-tert-octylphenol During the Larval Growth and Development Assay with the African 

Clawed Frog, Xenopus laevis (continued)

Control Level 1 Level 2 Level 3 Level 4

(0 μg/L) Nominal (μg/L): 6.25 Nominal (μg/L): 12.5 Nominal (μg/L): 25.0 Nominal (μg/L): 50.0

Sample Measured Measured % of % of Measured % of % of Measured % of % of Measured % of % of

Day / Rep. (μg/L) (μg/L) Nominal Mean (μg/L) Nominal Mean (μg/L) Nominal Mean (μg/L) Nominal Mean

70 <MQL a 5.31 85 107 10.7 86 104 17.1 68 88 33.8 68 89

73 <MQL a 5.11 82 103 9.17 73 89 16.8 67 86 34.2 68 90

77 <MQL a 5.46 87 110 11.0 88 107 17.8 71 91 31.1 62 82

81 <MQL a 5.01 80 101 11.7 94 114 24.1 96 124 38.8 78 102

88 <MQL a 1.54 d 25 b 31 b 8.71 70 85 17.6 70 90 32.8 66 86

91 <MQL a 6.68 107 134 12.1 97 117 21.8 87 112 44.4 89 117

97 <MQL a 6.10 98 123 11.0 88 107 20.6 82 106 45.4 91 119

104 <MQL a 4.48 72 90 10.3 82 100 17.8 71 91 35.4 71 93

Rep. A <MQL a 4.46 71 90 10.30 82 c 100 19.3 77 99 36.5 73 96

Study 

Termination 

Days 112 and 

113

Rep. B <MQL a 6.62 106 c 133 9.65 77 94 16.3 65 84 39.3 79 103

Rep. C <MQL a 5.60 90 c 113 9.66 77 94 18.9 76 97 36.0 72 95

Rep. D <MQL a 3.77 60 76 9.25 74 90 18.4 74 94 32.9 66 87

Rep. E <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. F <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. G <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. H <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Daily 

Mean <MQL a 5.11 82 103 9.72 78 95 18.2 73 94 36.2 73 95

Overall

Mean e <MQL a 4.97 80 10.3 82 19.5 78 38.0 76

SD 1.479 2.21 4.72 10.44

a

 MQL =[ Low STD Conc. * Analysis Volume (mL)] / Sample Volume (mL) = (1.54 ng/mL * 20 mL) / 10 mL = 3.08 ng/mL b

 Not Applicable c The higher measured concentrations are suspected to have resulted from the animals being removed prior to the collection of the samples. d The actual concentration for Day 88, Level 1 was <MQL. 1/2 MQL (1.54 ng/mL) was used for calculating the Overall Mean.

e When applicable, the Daily Mean was used for calculating the Overall Mean

f Due to a pump malfunction, solution was delivered to the chemical mixing box from one of two 4-tert-octylphenol saturator columns prior to collection of day 61. This 

malfunction may have contributed to lower test substance recoveries from some of the samples. 
ABC Study No. 66086 

Page 178 of 261 

Table F-2. Measured Concentrations of 4-tert-octylphenol from the Saturator Column 

Outflows During the Larval Growth and Development Assay with the African 

Clawed Frog, Xenopus laevis

Sample Day

Measured Concentration Expressed as μg/L

(Percent of Nominal Concentration)

Saturator Outflow A

(11,000) a 

Saturator Outflow B

(11,000) a 

0 12,500 (114) NA

3 13,200 (120) NA

7 12,400 (113) NA

13 10,800 (98) NA

18 10,800 (98) NA

28 7,050 (64) 12,600 (115) a

32 7,990 (73) 15,000 (136) 

39 12,200 (111) 12,100 (110)

47 12,200 (111) 11,700 (106)

54 12,000 (109) 11,800 (107)

60 11,200 (102) 12,500 (114)

64 12,700 (115) 11,200 (102)

70 12,600 (115) 9,750 (89)

73 11,200 (102) 9270 (84) 

77 9,570 (87) 12,900 (117)

81 9,230 (84) 14,500 (132)

88 10,800 (98) 12,500 (114)

91 12,700 (115) 12,600 (115)

97 11,600 (105) 13,000 (118)

104 13,800 (125) 16,400 (149)

112 11,600 (105) 9,650 (88)

113 10,900 (99) 9,450 (86)

NA = Not applicable a A second saturator column was placed on the diluter system on day 26 in an attempt to improve test substance

recoveries relative to target nominal concentrations.
ABC Study No. 66086 

Page 179 of 261 

Table F-3. Measured Concentrations of 4-tert-octylphenol Quality Control Samples During 

the Larval Growth and Development Assay with the African Clawed Frog, 

Xenopus laevis

Sample Day

Measured Concentration Expressed as μg/L

(Percent of Nominal Concentration)

Low Spike

(5.06)

a 

High Spike

(61.8)

a 

0 4.72 (93) 68.1 (110)

3 4.43 (88) 61.6 (100)

7 5.31 (105) 67.4 (109)

13 5.10 (101) 65.9 (107)

18 b 5.24 (104) 63.2 (102)

18 c 5.32 (105) 66.3 (107)

28 5.24 (104) 62.2 (101)

32 5.98 (118) 67.1 (109)

39 5.44 (108) 68.2 (110)

47 5.82 (115) 65.0 (105)

54 5.18 (102) 68.3 (111)

60 5.43 (107) 66.2 (107)

61 5.17 (102) 65.4 (106)

64 5.24 (104) 65.7 (106)

70 5.45 (108) 66.7 (108)

73 5.09 (101) 65.3 (106)

77 5.15 (102) 65.4 (106)

81 5.71 (113) 65.6 (106)

88 5.77 (114) 65.6 (106)

91 5.13 (101) 65.8 (106)

97 5.64 (111) 72.9 (118)

104 5.22 (103) 65.8 (106)

112 5.09 (100) 62.8 (102)

113 5.48 (108) 60.9 (99)

a Nominal concentration as μg/L. b Non-centrifuged. c Centrifuged. 
ABC Study No. 66086 

Page 180 of 261 

APPENDIX G - REPLICATE SURVIVAL DATA
ABC Study No. 66086 

Page 181 of 261 

Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to 4-tert-octylphenol for 113-Days

Mean 

Measured 

Concentration

(μg/L)

R 

e 

p

Day 0 

Initial 

Number

Day 5 Day 12 Day 14

Number 

Hatched

Cumulative

Number 

Dead

Not 

Found a

% 

Survival

Number 

Surviving

Cumulative

Number 

Dead % Survival

Number 

Surviving

Cumulative

Number 

Dead % Survival

0 (Control) A 25 25 0 0 100 25 0 100 25 0 100

B 25 24 1 0 96 24 1 96 24 1 96

C 25 24 0 1 96 25 0 100 25 0 100

D 25 23 2 0 92 23 2 92 23 2 92

E 25 25 0 0 100 25 0 100 25 0 100

F 25 24 1 0 96 23 2 92 23 2 92

G 25 24 1 0 96 23 2 92 23 2 92

H 25 22 2 1 88 22 3 88 22 3 88

4.97 A 25 22 3 0 88 22 3 88 22 3 88

B 25 18 6 1 72 18 7 72 18 7 72

C 25 24 1 0 96 24 1 96 24 1 96

D 25 25 0 0 100 23 2 92 23 2 92

10.3 A 25 21 4 0 84 21 4 84 21 4 84

B 25 21 3 1 84 21 4 84 21 4 84

C 25 24 1 0 96 23 2 92 23 2 92

D 25 23 2 0 92 23 2 92 23 2 92

19.5 A 25 24 0 1 96 24 1 96 24 1 96

B 25 24 0 1 96 25 0 100 25 0 100

C 25 25 0 0 100 25 0 100 25 0 100

D 25 23 2 0 92 23 2 92 23 2 92

38.0 A 25 23 0 2 92 24 1 96 24 1 96

B 25 21 3 1 84 21 4 84 21 4 84

C 25 24 1 0 96 24 1 96 24 1 96

D 25 23 1 1 92 24 1 96 24 1 96

a All "Not Found" considered dead on study day 9 and added to cumulative number dead. 
ABC Study No. 66086 

Page 182 of 261 

Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to 4-tert-octylphenol for 113-Days 

(continued)

Mean 

Measured 

Concentration

(μg/L)

R 

E 

P

Day 0 Initial 

Number

Day 21 a Day 28 Day 35

Number 

Surviving

Cumulative

Number 

Dead

% 

Survival

Number 

Surviving

Cumulative

Number 

Dead

% 

Survival

Number 

Surviving

Cumulative

Number 

Dead

% 

Survival

0 (Control) A 25 25 0 100 25 0 100 25 0 100

B 25 24 1 96 24 1 96 24 1 96

C 25 24 0 100 23 1 96 23 1 96

D 25 23 2 92 23 2 92 23 2 92

E 25 25 0 100 25 0 100 25 0 100

F 25 23 2 92 23 2 92 23 2 92

G 25 23 2 92 23 2 92 23 2 92

H 25 22 3 88 22 3 88 22 3 88

4.97 A 25 21 4 84 21 4 84 21 4 84

B 25 18 7 72 18 7 72 18 7 72

C 25 24 1 96 24 1 96 24 1 96

D 25 22 3 88 22 3 88 22 3 88

10.3 A 25 21 4 84 21 4 84 21 4 84

B 25 21 4 84 20 5 80 20 5 80

C 25 23 2 92 23 2 92 23 2 92

D 25 23 2 92 23 2 92 23 2 92

19.5 A 25 24 1 96 24 1 96 24 1 96

B 25 24 1 96 24 1 96 24 1 96

C 25 24 1 96 24 1 96 24 1 96

D 25 22 3 88 22 3 88 22 3 88

38.0 A 25 24 1 96 24 1 96 24 1 96

B 25 21 4 84 21 4 84 21 4 84

C 25 24 1 96 24 1 96 24 1 96

D 25 24 1 96 24 1 96 24 1 96

a One larva in the control, replicate C chamber was inadvertently killed during cleaning on study day 17 and was not counted as an exposure related mortality. 
ABC Study No. 66086 

Page 183 of 261 

Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to 4-tert-octylphenol for 113-Days 

(continued) 

Mean 

Measured 

Concentration

(μg/L)

R 

E 

P

Day 0 

Initial 

Number

Number 

Removed 

for 

Sampling

Day 56 Day 63 Day 70

Number 

Alive

Cumulative

Number 

Dead

% 

Survival

Number 

Alive

Cumulative

Number 

Dead

% 

Survival

Number 

Alive

Cumulative

Number 

Dead

% 

Survival

0 (Control) A 25 5 20 0 100 20 0 100 20 0 100

B 25 5 19 1 96 19 1 96 19 1 96

C 25 5 17 2 92 17 2 92 17 2 92

D 25 5 17 3 88 17 3 88 17 3 88

E 25 5 20 0 100 20 0 100 20 0 100

F 25 5 17 3 88 17 3 88 17 3 88

G 25 5 18 2 92 18 2 92 18 2 92

H 25 5 17 3 88 17 3 88 17 3 88

4.97 A 25 5 16 4 84 16 4 84 16 4 84

B 25 5 13 7 72 13 7 72 13 7 72

C 25 5 19 1 96 19 1 96 19 1 96

D 25 5 16 4 84 16 4 84 16 4 84

10.3 A 25 5 15 5 80 15 5 80 15 5 80

B 25 5 14 6 76 14 6 76 14 6 76

C 25 5 18 2 92 18 2 92 18 2 92

D 25 5 18 2 92 18 2 92 18 2 92

19.5 A 25 5 19 1 96 19 1 96 19 1 96

B 25 5 19 1 96 19 1 96 19 1 96

C 25 5 19 1 96 19 1 96 19 1 96

D 25 5 17 3 88 17 3 88 16 4 84

38.0 A 25 5 19 1 96 19 1 96 19 1 96

B 25 5 16 4 84 16 4 84 16 4 84

C 25 5 19 1 96 19 1 96 19 1 96

D 25 5 18 2 92 18 2 92 18 2 92
ABC Study No. 66086 

Page 184 of 261 

Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to 4-tert-octylphenol for 113-Days 

(continued) 

Mean 

Measured 

Concentration

(μg/L)

R 

E 

P

Day 0 Initial 

Number

Number 

Removed at 

juvenile cull

Day 77 a Day 84

Number 

Alive

Cumulative

Number Dead

% 

Survival

Number 

Alive

Cumulative

Number Dead

% 

Survival

0 (Control) A 25 10 10 0 100 10 0 100

B 25 8 10 1 b 96 10 1 96

C 25 8 9 2 92 9 2 92

D 25 7 10 3 88 10 3 88

E 25 10 10 0 100 10 0 100

F 25 7 10 3 88 10 3 88

G 25 8 10 2 92 10 2 92

H 25 7 10 3 88 10 3 88

4.97 A 25 6 10 4 84 10 4 84

B 25 3 10 7 72 10 7 72

C 25 9 10 1 96 10 1 96

D 25 6 10 4 84 10 4 84

10.3 A 25 5 10 5 80 10 5 80

B 25 4 10 6 76 10 6 76

C 25 7 10 2 b 92 10 2 92

D 25 8 10 2 92 10 2 92

19.5 A 25 9 10 1 96 10 1 96

B 25 9 10 1 96 10 1 96

C 25 9 10 1 96 10 1 96

D 25 6 10 4 84 10 4 84

38.0 A 25 9 10 1 96 10 1 96

B 25 6 10 4 84 10 4 84

C 25 9 10 1 96 10 1 96

D 25 8 10 2 92 10 2 92

a One extra juvenile in the control, replicate C chamber was inadvertently removed at the juvenile cull. This was not counted as an exposure related mortality.

b

 One larva did not attain NF stage 62 prior to the day 70. This individual was removed at the juvenile cull and was not counted as an exposure related mortality.
ABC Study No. 66086 

Page 185 of 261 

Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to 4-tert-octylphenol for 113-Days 

(continued) 

Mean 

Measured 

Concentration

(μg/L)

R 

E 

P

Day 0 

Initial 

Number

Day 91 Day 98

Number 

Alive

Cumulative

Number Dead

% 

Survival

Number 

Alive

Cumulative

Number Dead

% 

Survival

0 (Control) A 25 10 0 100 10 0 100

B 25 10 1 96 10 1 96

C 25 9 2 92 9 2 92

D 25 10 3 88 10 3 88

E 25 10 0 100 10 0 100

F 25 10 3 88 10 3 88

G 25 10 2 92 10 2 92

H 25 10 3 88 10 3 88

4.97 A 25 10 4 84 10 4 84

B 25 10 7 72 10 7 72

C 25 10 1 96 10 1 96

D 25 10 4 84 10 4 84

10.3 A 25 10 5 80 10 5 80

B 25 10 6 76 10 6 76

C 25 10 2 92 10 2 92

D 25 10 2 92 10 2 92

19.5 A 25 10 1 96 10 1 96

B 25 10 1 96 10 1 96

C 25 10 1 96 10 1 96

D 25 10 4 84 10 4 84

38.0 A 25 10 1 96 10 1 96

B 25 10 4 84 10 4 84

C 25 10 1 96 10 1 96

D 25 10 2 92 10 2 92
ABC Study No. 66086 

Page 186 of 261 

Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to 4-tert-octylphenol for 113-Days 

(continued) 

Mean 

Measured 

Concentration

(μg/L)

R 

E 

P

Day 0 

Initial 

Number

Day 105 Termination

Number 

Alive

Cumulative

Number Dead a

% 

Survival

Number 

Alive

Cumulative

Number Dead

% 

Survival

0 (Control) A 25 10 0 100 10 0 100

B 25 10 1 96 10 1 96

C 25 9 2 92 9 2 92

D 25 9 3 88 9 3 88

E 25 10 0 100 10 0 100

F 25 10 3 88 10 3 88

G 25 10 2 92 10 2 92

H 25 10 3 88 10 3 88

4.97 A 25 10 4 84 10 4 84

B 25 10 7 72 10 7 72

C 25 10 1 96 10 1 96

D 25 9 4 83 9 4 83

10.3 A 25 10 5 80 10 5 80

B 25 10 6 76 10 6 76

C 25 10 2 92 10 2 92

D 25 10 2 92 10 2 92

19.5 A 25 10 1 96 10 1 96

B 25 10 1 96 10 1 96

C 25 10 1 96 10 1 96

D 25 10 4 84 10 4 84

38.0 A 25 10 1 96 10 1 96

B 25 10 4 84 10 4 84

C 25 10 1 96 10 1 96

D 25 10 2 92 10 2 92

a One animal escaped from Rep. D of the control and 4.97 μg/L treatment during routine daily activities and was not counted as an exposure related mortality.
ABC Study No. 66086 

Page 187 of 261 

Appendix G-2. Survival Summary for the African Clawed Frog, Xenopus laevis, Exposed to 4-tert-octylphenol for 113-Days

Mean 

Measured 

Concentration

(μg/L)

R

E

P

Day 0 

Initial 

number of 

Organisms

Number 

removed d

Day 71 

initial 

number

Day 113

NF Stage 

62 

survivors 

NF Stage 62 

% Survival

Treatment 

mean

Cumulative number 

juveniles dead e

% 

survival

Treatment 

mean juvenile 

% survival

Control A 25 25 100 94 15 10 0 100 100

B 25 23 a 96 14 10 0 100

C 25 23 b 96 13 9 0 f 100

D 25 23 92 12 10 0 g 100

E 25 25 100 15 10 0 100

F 25 22 88 12 10 0 100

G 25 23 92 13 10 0 100

H 25 22 88 12 10 0 100

4.97 A 25 21 84 85 11 10 0 100 100

B 25 18 72 8 10 0 100

C 25 24 96 14 10 0 100

D 25 22 88 11 10 0 g 100

10.3 A 25 20 80 85 10 10 0 100 100

B 25 19 c 76 9 10 0 100

C 25 22 a 92 13 10 0 100

D 25 23 92 13 10 0 100

19.5 A 25 24 96 94 14 10 0 100 100

B 25 24 96 14 10 0 100

C 25 24 96 14 10 0 100

D 25 22 88 11 10 0 100

38.0 A 25 24 96 93 14 10 0 100 100

B 25 21 84 11 10 0 100

C 25 24 96 14 10 0 100

D 25 24 96 13 10 0 100

a One larva did not attain NF stage 62 prior to the day 70. This individual was removed at the juvenile cull and was not counted as an exposure related mortality. b One larva was inadvertently killed during test chamber cleaning on study day 17. This individual was not counted as an exposure related mortality. c Included three larvae for which the date achieved NF stage 62 was not documented. d Five NF stage 62 larvae were removed for larval sub-sampling and remainder were removed at cull to 10 animals on study day 70. e Indicates exposure-related mortalities following juvenile cull. f One extra juvenile was inadvertently removed at the juvenile cull. This individual was not counted as an exposure related mortality. g One juvenile escaped during routine daily activities. This individual was not counted as an exposure related mortality.
ABC Study No. 66086 

Page 188 of 261 

APPENDIX H – NF STAGE 62 DATA
ABC Study No. 66086 

Page 189 of 261 

Appendix H-1. Replicate Data for Number of Days to NF Stage 62, Weight and Length 

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 Achieved Weight (g) Snout-to-Vent Length (mm)

0 (Control) A 1 44 1.483 20

0 (Control) A 2 44 2.328 22

0 (Control) A 3 44 1.893 23

0 (Control) A 4 44 2.35 25

0 (Control) A 5 44 2.462 25

0 (Control) B 1 39 1.842 21

0 (Control) B 2 40 2.097 24

0 (Control) B 3 40 2.578 25

0 (Control) B 4 41 2.666 25

0 (Control) B 5 41 2.121 23

0 (Control) C 1 40 2.044 22

0 (Control) C 2 41 1.882 23

0 (Control) C 3 42 1.584 21

0 (Control) C 4 42 1.614 22

0 (Control) C 5 42 1.405 22

0 (Control) D 1 38 1.990 23

0 (Control) D 2 40 2.161 23

0 (Control) D 3 40 2.396 25

0 (Control) D 4 40 1.484 22

0 (Control) D 5 40 1.817 23

0 (Control) E 1 39 1.935 22

0 (Control) E 2 40 2.132 23

0 (Control) E 3 40 2.135 24

0 (Control) E 4 40 1.680 23

0 (Control) E 5 41 2.189 23

0 (Control) F 1 40 1.884 22

0 (Control) F 2 40 1.851 23

0 (Control) F 3 41 2.342 25

0 (Control) F 4 41 2.112 23

0 (Control) F 5 41 2.502 23

0 (Control) G 1 40 2.224 23

0 (Control) G 2 40 2.289 24

0 (Control) G 3 40 2.076 23

0 (Control) G 4 40 2.352 25

0 (Control) G 5 40 2.018 22

0 (Control) H 1 40 1.632 22

0 (Control) H 2 40 1.930 24

0 (Control) H 3 40 2.760 25

0 (Control) H 4 40 2.348 24

0 (Control) H 5 40 2.067 23
ABC Study No. 66086 

Page 190 of 261 

Appendix H-1. Replicate Data for Number of Days to NF Stage 62, Weight and Length 

(continued) 

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 

Achieved Weight (g) Snout-to-Vent 

Length (mm)

4.97 A 1 40 1.983 23

4.97 A 2 41 1.868 23

4.97 A 3 41 1.622 23

4.97 A 4 41 1.834 23

4.97 A 5 43 2.629 26

4.97 B 1 40 2.098 25

4.97 B 2 40 2.336 25

4.97 B 3 40 1.709 23

4.97 B 4 40 1.987 24

4.97 B 5 41 2.243 24

4.97 C 1 40 1.992 24

4.97 C 2 40 2.295 24

4.97 C 3 40 2.774 24

4.97 C 4 41 1.754 22

4.97 C 5 41 2.211 24

4.97 D 1 40 1.674 21

4.97 D 2 40 1.783 23

4.97 D 3 40 2.127 24

4.97 D 4 40 1.932 24

4.97 D 5 40 1.980 23

10.3 A 1 40 1.893 23

10.3 A 2 40 1.670 23

10.3 A 3 41 2.542 27

10.3 A 4 41 2.982 25

10.3 A 5 41 2.443 24

10.3 B 1 40 2.097 22

10.3 B 2 40 1.562 22

10.3 B 3 41 1.411 21

10.3 B 4 41 1.743 22

10.3 B 5 41 3.263 27

10.3 C 1 44 2.085 24

10.3 C 2 44 2.295 24

10.3 C 3 44 1.972 23

10.3 C 4 45 2.103 23

10.3 C 5 45 2.668 24

10.3 D 1 41 2.105 23

10.3 D 2 41 1.980 23

10.3 D 3 41 2.588 23

10.3 D 4 41 2.508 24

10.3 D 5 41 1.496 21
ABC Study No. 66086 

Page 191 of 261 

Appendix H-1. Replicate Data for Number of Days to NF Stage 62, Weight and Length 

(continued) 

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 

Achieved Weight (g) Snout-to-Vent 

Length (mm)

19.5 A 1 40 1.893 21

19.5 A 2 40 2.038 23

19.5 A 3 43 2.071 24

19.5 A 4 43 1.927 22

19.5 A 5 43 1.814 22

19.5 B 1 41 1.864 23

19.5 B 2 41 2.370 23

19.5 B 3 43 2.404 24

19.5 B 4 44 2.403 24

19.5 B 5 44 2.695 25

19.5 C 1 40 2.216 22

19.5 C 2 40 2.485 23

19.5 C 3 40 1.697 23

19.5 C 4 41 2.663 26

19.5 C 5 41 2.352 25

19.5 D 1 40 2.173 24

19.5 D 2 40 1.830 22

19.5 D 3 41 1.589 22

19.5 D 4 41 1.700 20

19.5 D 5 41 2.017 24

38.0 A 1 38 1.890 23

38.0 A 2 40 1.805 25

38.0 A 3 40 2.475 24

38.0 A 4 41 2.238 25

38.0 A 5 41 2.233 25

38.0 B 1 40 1.856 21

38.0 B 2 40 2.639 24

38.0 B 3 40 2.474 25

38.0 B 4 41 2.607 25

38.0 B 5 42 1.331 20

38.0 C 1 42 2.335 26

38.0 C 2 42 2.526 25

38.0 C 3 42 2.281 24

38.0 C 4 42 2.394 25

38.0 C 5 43 2.202 25

38.0 D 1 41 2.063 24

38.0 D 2 41 2.117 24

38.0 D 3 42 2.016 24

38.0 D 4 42 2.006 23

38.0 D 5 42 2.263 24
ABC Study No. 66086 

Page 192 of 261 

Appendix H-2. Replicate Data for Number of Days to NF Stage 62

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 Achieved Tadpole# Study Day NF 62 Achieved

0 (Control) A 1 44 14 47

0 (Control) A 2 44 15 48

0 (Control) A 3 44 16 48

0 (Control) A 4 44 17 48

0 (Control) A 5 44 18 48

0 (Control) A 6 44 19 48

0 (Control) A 7 45 20 49

0 (Control) A 8 45 21 49

0 (Control) A 9 46 22 49

0 (Control) A 10 46 23 50

0 (Control) A 11 47 24 51

0 (Control) A 12 47 25 56

0 (Control) A 13 47

0 (Control) B 1 39 14 43

0 (Control) B 2 40 15 43

0 (Control) B 3 40 16 43

0 (Control) B 4 41 17 43

0 (Control) B 5 41 18 43

0 (Control) B 6 41 19 44

0 (Control) B 7 42 20 45

0 (Control) B 8 42 21 45

0 (Control) B 9 42 22 45

0 (Control) B 10 42 23 45

0 (Control) B 11 42 24 --

0 (Control) B 12 43 25 --

0 (Control) B 13 43

0 (Control) C 1 40 14 43

0 (Control) C 2 41 15 43

0 (Control) C 3 42 16 43

0 (Control) C 4 42 17 44

0 (Control) C 5 42 18 44

0 (Control) C 6 42 19 45

0 (Control) C 7 42 20 45

0 (Control) C 8 42 21 46

0 (Control) C 9 43 22 47

0 (Control) C 10 43 23 48

0 (Control) C 11 43 24 --

0 (Control) C 12 43 25 --

0 (Control) C 13 43

‘--’ denotes a mortality that occurred before NF stage 62. 
ABC Study No. 66086 

Page 193 of 261 

Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued) 

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 Achieved Tadpole# Study Day NF 62 Achieved

0 (Control) D 1 38 14 41

0 (Control) D 2 40 15 42

0 (Control) D 3 40 16 42

0 (Control) D 4 40 17 43

0 (Control) D 5 40 18 43

0 (Control) D 6 40 19 43

0 (Control) D 7 40 20 45

0 (Control) D 8 41 21 47

0 (Control) D 9 41 22 48

0 (Control) D 10 41 23 54

0 (Control) D 11 41 24 --

0 (Control) D 12 41 25 --

0 (Control) D 13 41

0 (Control) E 1 39 14 44

0 (Control) E 2 40 15 44

0 (Control) E 3 40 16 44

0 (Control) E 4 40 17 44

0 (Control) E 5 41 18 44

0 (Control) E 6 41 19 44

0 (Control) E 7 41 20 45

0 (Control) E 8 42 21 45

0 (Control) E 9 42 22 45

0 (Control) E 10 42 23 47

0 (Control) E 11 43 24 47

0 (Control) E 12 43 25 48

0 (Control) E 13 44

0 (Control) F 1 40 14 43

0 (Control) F 2 40 15 43

0 (Control) F 3 41 16 43

0 (Control) F 4 41 17 44

0 (Control) F 5 41 18 44

0 (Control) F 6 41 19 44

0 (Control) F 7 41 20 44

0 (Control) F 8 42 21 44

0 (Control) F 9 42 22 47

0 (Control) F 10 42 23 --

0 (Control) F 11 42 24 --

0 (Control) F 12 43 25 --

0 (Control) F 13 43

‘--’ denotes a mortality that occurred before NF stage 62. 
ABC Study No. 66086 

Page 194 of 261 

Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued) 

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 Achieved Tadpole# Study Day NF 62 Achieved

0 (Control) G 1 40 14 43

0 (Control) G 2 40 15 44

0 (Control) G 3 40 16 44

0 (Control) G 4 40 17 44

0 (Control) G 5 40 18 45

0 (Control) G 6 40 19 45

0 (Control) G 7 41 20 45

0 (Control) G 8 41 21 49

0 (Control) G 9 41 22 49

0 (Control) G 10 41 23 49

0 (Control) G 11 41 24 --

0 (Control) G 12 42 25 --

0 (Control) G 13 43

0 (Control) H 1 40 14 42

0 (Control) H 2 40 15 42

0 (Control) H 3 40 16 42

0 (Control) H 4 40 17 43

0 (Control) H 5 40 18 43

0 (Control) H 6 41 19 43

0 (Control) H 7 41 20 44

0 (Control) H 8 41 21 45

0 (Control) H 9 42 22 45

0 (Control) H 10 42 23 --

0 (Control) H 11 42 24 --

0 (Control) H 12 42 25 --

0 (Control) H 13

4.97 A 1 40 14 45

4.97 A 2 41 15 45

4.97 A 3 41 16 45

4.97 A 4 41 17 47

4.97 A 5 43 18 47

4.97 A 6 43 19 47

4.97 A 7 43 20 47

4.97 A 8 43 21 54

4.97 A 9 43 22 --

4.97 A 10 44 23 --

4.97 A 11 45 24 --

4.97 A 12 45 25 --

4.97 A 13 45

‘--’ denotes a mortality that occurred before NF stage 62. 
ABC Study No. 66086 

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Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued) 

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 Achieved Tadpole# Study Day NF 62 Achieved

4.97 B 1 40 14 44

4.97 B 2 40 15 44

4.97 B 3 40 16 44

4.97 B 4 40 17 46

4.97 B 5 41 18 46

4.97 B 6 41 19 --

4.97 B 7 41 20 --

4.97 B 8 41 21 --

4.97 B 9 41 22 --

4.97 B 10 41 23 --

4.97 B 11 41 24 --

4.97 B 12 42 25 --

4.97 B 13 42

4.97 C 1 40 14 42

4.97 C 2 40 15 43

4.97 C 3 40 16 43

4.97 C 4 41 17 43

4.97 C 5 41 18 43

4.97 C 6 41 19 44

4.97 C 7 41 20 44

4.97 C 8 42 21 45

4.97 C 9 42 22 45

4.97 C 10 42 23 45

4.97 C 11 42 24 46

4.97 C 12 42 25 47

4.97 C 13 42

4.97 D 1 40 14 44

4.97 D 2 40 15 44

4.97 D 3 40 16 44

4.97 D 4 40 17 44

4.97 D 5 40 18 45

4.97 D 6 42 19 45

4.97 D 7 42 20 47

4.97 D 8 42 21 49

4.97 D 9 42 22 49

4.97 D 10 43 23 --

4.97 D 11 43 24 --

4.97 D 12 44 25 --

4.97 D 13 44

‘--’ denotes a mortality that occurred before NF stage 62. 
ABC Study No. 66086 

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Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued) 

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 Achieved Tadpole# Study Day NF 62 Achieved

10.3 A 1 40 14 44

10.3 A 2 40 15 44

10.3 A 3 41 16 44

10.3 A 4 41 17 45

10.3 A 5 42 18 48

10.3 A 6 42 19 49

10.3 A 7 42 20 52

10.3 A 8 42 21 --

10.3 A 9 43 22 --

10.3 A 10 43 23 --

10.3 A 11 43 24 --

10.3 A 12 43 25 --

10.3 A 13 43

10.3 B 1 40 14 45

10.3 B 2 40 15 48

10.3 B 3 41 16 52

10.3 B 4 41 17 -

a

10.3 B 5 41 18 -

a

10.3 B 6 41 19 -

a

10.3 B 7 42 20 --

10.3 B 8 42 21 --

10.3 B 9 43 22 --

10.3 B 10 43 23 --

10.3 B 11 44 24 --

10.3 B 12 44 25 --

10.3 B 13 44

10.3 C 1 44 14 48

10.3 C 2 44 15 48

10.3 C 3 44 16 48

10.3 C 4 45 17 49

10.3 C 5 45 18 50

10.3 C 6 45 19 50

10.3 C 7 46 20 50

10.3 C 8 46 21 51

10.3 C 9 46 22 54

10.3 C 10 47 23 --

10.3 C 11 47 24 --

10.3 C 12 47 25 --

10.3 C 13 47

‘--’ denotes a mortality that occurred before NF stage 62. a Date NF62 attained not recorded.
ABC Study No. 66086 

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Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued) 

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 Achieved Tadpole# Study Day NF 62 Achieved

10.3 D 1 41 14 44

10.3 D 2 41 15 45

10.3 D 3 41 16 45

10.3 D 4 41 17 45

10.3 D 5 41 18 45

10.3 D 6 42 19 45

10.3 D 7 42 20 46

10.3 D 8 42 21 47

10.3 D 9 42 22 47

10.3 D 10 42 23 47

10.3 D 11 43 24 --

10.3 D 12 44 25 --

10.3 D 13 44

19.5 A 1 40 14 44

19.5 A 2 40 15 45

19.5 A 3 43 16 45

19.5 A 4 43 17 45

19.5 A 5 43 18 45

19.5 A 6 43 19 45

19.5 A 7 43 20 45

19.5 A 8 43 21 46

19.5 A 9 43 22 46

19.5 A 10 43 23 47

19.5 A 11 43 24 49

19.5 A 12 44 25 --

19.5 A 13 44

19.5 B 1 41 14 47

19.5 B 2 41 15 47

19.5 B 3 43 16 47

19.5 B 4 44 17 47

19.5 B 5 44 18 48

19.5 B 6 44 19 48

19.5 B 7 44 20 48

19.5 B 8 44 21 49

19.5 B 9 45 22 49

19.5 B 10 45 23 49

19.5 B 11 45 24 54

19.5 B 12 46 25 --

19.5 B 13 46

‘--’ denotes a mortality that occurred before NF stage 62. 
ABC Study No. 66086 

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Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued) 

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 Achieved Tadpole# Study Day NF 62 Achieved

19.5 C 1 40 14 43

19.5 C 2 40 15 44

19.5 C 3 40 16 44

19.5 C 4 41 17 44

19.5 C 5 41 18 44

19.5 C 6 41 19 44

19.5 C 7 41 20 45

19.5 C 8 41 21 47

19.5 C 9 42 22 47

19.5 C 10 42 23 56

19.5 C 11 42 24 62

19.5 C 12 43 25 --

19.5 C 13 43

19.5 D 1 40 14 44

19.5 D 2 40 15 44

19.5 D 3 41 16 44

19.5 D 4 41 17 44

19.5 D 5 41 18 44

19.5 D 6 41 19 44

19.5 D 7 42 20 46

19.5 D 8 42 21 46

19.5 D 9 42 22 62

19.5 D 10 42 23 --

19.5 D 11 42 24 --

19.5 D 12 43 25 --

19.5 D 13 44

38.0 A 1 38 14 42

38.0 A 2 40 15 42

38.0 A 3 40 16 43

38.0 A 4 41 17 43

38.0 A 5 41 18 43

38.0 A 6 41 19 43

38.0 A 7 41 20 43

38.0 A 8 42 21 44

38.0 A 9 42 22 45

38.0 A 10 42 23 45

38.0 A 11 42 24 46

38.0 A 12 42 25 --

38.0 A 13 42

‘--’ denotes a mortality that occurred before NF stage 62. 
ABC Study No. 66086 

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Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued) 

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62 Achieved Tadpole# Study Day NF 62 Achieved

38.0 B 1 40 14 45

38.0 B 2 40 15 45

38.0 B 3 40 16 45

38.0 B 4 41 17 46

38.0 B 5 42 18 47

38.0 B 6 42 19 47

38.0 B 7 43 20 48

38.0 B 8 43 21 52

38.0 B 9 43 22 --

38.0 B 10 44 23 --

38.0 B 11 44 24 --

38.0 B 12 44 25 --

38.0 B 13 44

38.0 C 1 42 14 45

38.0 C 2 42 15 45

38.0 C 3 42 16 45

38.0 C 4 42 17 45

38.0 C 5 43 18 45

38.0 C 6 43 19 46

38.0 C 7 43 20 46

38.0 C 8 43 21 46

38.0 C 9 43 22 47

38.0 C 10 44 23 47

38.0 C 11 44 24 48

38.0 C 12 44 25 --

38.0 C 13 44

38.0 D 1 41 14 44

38.0 D 2 41 15 44

38.0 D 3 42 16 44

38.0 D 4 42 17 44

38.0 D 5 42 18 45

38.0 D 6 42 19 45

38.0 D 7 42 20 46

38.0 D 8 43 21 46

38.0 D 9 43 22 46

38.0 D 10 43 23 48

38.0 D 11 43 24 50

38.0 D 12 43 25 --

38.0 D 13 44

‘--’ denotes a mortality that occurred before NF stage 62. 
ABC Study No. 66086 

Page 200 of 261 

APPENDIX I – NF STAGE 66 CULL DATA 
ABC Study No. 66086 

Page 201 of 261 

Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull

Treatment

(μg/L) Replicate

Juvenile

Code Weight (g)

Snout-to-Vent 

Length (mm) Phenotypic Sex

0 (Control) A CA1-66 4.825 33 Female

0 (Control) A CA2-66 3.683 30 Female

0 (Control) A CA3-66 2.575 28 Female

0 (Control) A CA4-66 3.870 31 Male

0 (Control) A CA5-66 2.603 27 Male

0 (Control) A CA6-66 1.906 26 Male

0 (Control) A CA7-66 2.732 27 Female

0 (Control) A CA8-66 2.367 27 Female

0 (Control) A CA9-66 4.532 31 Female

0 (Control) A CA10-66 1.184 23 Indeterminate

0 (Control) B CB1-66 4.877 32 Female

0 (Control) B CB2-66 4.470 33 Female

0 (Control) B CB3-66 2.224 25 Male

0 (Control) B CB4-66 3.747 30 Male

0 (Control) B CB5-66 4.230 31 Male

0 (Control) B CB6-66 3.405 29 Female

0 (Control) B CB7-66 3.499 28 Male

0 (Control) B CB8-66 4.258 31 Male

0 (Control) B CB9-66 -- -- --

0 (Control) B CB10-66 -- -- --

0 (Control) C CC1-66 2.939 28 Male

0 (Control) C CC2-66 4.678 31 Male

0 (Control) C CC3-66 4.251 31 Female

0 (Control) C CC4-66 4.396 32 Female

0 (Control) C CC5-66 3.785 31 Male

0 (Control) C CC6-66 3.485 30 Male

0 (Control) C CC7-66 5.243 35 Female

0 (Control) C CC8-66 3.704 30 Male

0 (Control) C CC9-66 -- -- --

0 (Control) C CC10-66 -- -- --

0 (Control) D CD1-66 5.086 33 Female

0 (Control) D CD2-66 3.571 31 Female

0 (Control) D CD3-66 2.085 26 Male

0 (Control) D CD4-66 5.216 33 Male

0 (Control) D CD5-66 2.532 26 Female

0 (Control) D CD6-66 2.928 28 Male

0 (Control) D CD7-66 4.140 32 Male

0 (Control) D CD8-66 -- -- --

0 (Control) D CD9-66 -- -- --

0 (Control) D CD10-66 -- -- --

‘--’ denotes a juvenile was not available for this sample. 
ABC Study No. 66086 

Page 202 of 261 

Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull 

(continued) 

Treatment

(μg/L) Replicate

Juvenile

Code Weight (g)

Snout-to-Vent 

Length (mm) Phenotypic Sex

0 (Control) E CE1-66 4.276 32 Male

0 (Control) E CE2-66 3.735 30 Male

0 (Control) E CE3-66 3.215 29 Female

0 (Control) E CE4-66 3.705 30 Male

0 (Control) E CE5-66 3.498 30 Male

0 (Control) E CE6-66 3.956 31 Female

0 (Control) E CE7-66 2.628 26 Female

0 (Control) E CE8-66 3.382 29 Male

0 (Control) E CE9-66 3.820 30 Female

0 (Control) E CE10-66 4.722 37 Male

0 (Control) F CF1-66 4.158 30 Male

0 (Control) F CF2-66 3.793 31 Male

0 (Control) F CF3-66 3.250 29 Female

0 (Control) F CF4-66 3.777 30 Female

0 (Control) F CF5-66 2.979 28 Male

0 (Control) F CF6-66 4.925 33 Female

0 (Control) F CF7-66 4.224 31 Female

0 (Control) F CF8-66 -- -- --

0 (Control) F CF9-66 -- -- --

0 (Control) F CF10-66 -- -- --

0 (Control) G CG1-66 3.831 31 Female

0 (Control) G CG2-66 3.774 30 Male

0 (Control) G CG3-66 4.012 31 Indeterminate

0 (Control) G CG4-66 4.101 31 Female

0 (Control) G CG5-66 3.455 29 Female

0 (Control) G CG6-66 3.248 30 Female

0 (Control) G CG7-66 5.591 35 Female

0 (Control) G CG8-66 2.370 27 Female

0 (Control) G CG9-66 -- -- --

0 (Control) G CG10-66 -- -- --

0 (Control) H CH1-66 3.312 29 Male

0 (Control) H CH2-66 3.574 31 Male

0 (Control) H CH3-66 3.711 30 Male

0 (Control) H CH4-66 2.932 29 Female

0 (Control) H CH5-66 4.987 33 Female

0 (Control) H CH6-66 3.607 29 Female

0 (Control) H CH7-66 4.164 31 Female

0 (Control) H CH8-66 -- -- --

0 (Control) H CH9-66 -- -- --

‘--’ denotes a juvenile was not available for this sample. 
ABC Study No. 66086 

Page 203 of 261 

Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull 

(continued) 

Treatment

(μg/L) Replicate

Juvenile

Code Weight (g)

Snout-to-Vent 

Length (mm) Phenotypic Sex

Control H CH10-66 -- -- --

4.97 A L1A1-66 5.936 35 Female

4.97 A L1A2-66 2.836 27 Indeterminate

4.97 A L1A3-66 4.133 32 Female

4.97 A L1A4-66 4.756 33 Female

4.97 A L1A5-66 4.522 33 Male

4.97 A L1A6-66 4.051 32 Female

4.97 A L1A7-66 -- -- --

4.97 A L1A8-66 -- -- --

4.97 A L1A9-66 -- -- --

4.97 A L1A10-66 -- -- --

4.97 B L1B1-66 3.922 31 Female

4.97 B L1B2-66 4.153 31 Male

4.97 B L1B3-66 4.327 33 Female

4.97 B L1B4-66 -- -- --

4.97 B L1B5-66 -- -- --

4.97 B L1B6-66 -- -- --

4.97 B L1B7-66 -- -- --

4.97 B L1B8-66 -- -- --

4.97 B L1B9-66 -- -- --

4.97 B L1B10-66 -- -- --

4.97 C L1C1-66 4.728 33 Female

4.97 C L1C2-66 3.790 31 Male

4.97 C L1C3-66 5.706 35 Female

4.97 C L1C4-66 3.502 31 Female

4.97 C L1C5-66 3.603 30 Female

4.97 C L1C6-66 3.600 30 Male

4.97 C L1C7-66 2.325 27 Male

4.97 C L1C8-66 1.937 25 Male

4.97 C L1C9-66 4.140 32 Male

4.97 C L1C10-66 -- -- --

4.97 D L1D1-66 5.251 34 Female

4.97 D L1D2-66 5.422 34 Male

4.97 D L1D3-66 3.722 30 Female

4.97 D L1D4-66 2.483 28 Female

4.97 D L1D5-66 3.263 29 Male

4.97 D L1D6-66 3.062 30 Male

4.97 D L1D7-66 -- -- --

4.97 D L1D8-66 -- -- --

 ‘--’ denotes a juvenile was not available for this sample. 
ABC Study No. 66086 

Page 204 of 261 

Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull 

(continued) 

Treatment

(μg/L) Replicate

Juvenile

Code Weight (g)

Snout-to-Vent 

Length (mm) Phenotypic Sex

4.97 D L1D9-66 -- -- --

4.97 D L1D10-66 -- -- --

10.3 A L2A1-66 2.391 24 Female

10.3 A L2A2-66 3.623 31 Female

10.3 A L2A3-66 3.247 29 Female

10.3 A L2A4-66 4.456 32 Male

10.3 A L2A5-66 3.841 30 Female

10.3 A L2A6-66 -- -- --

10.3 A L2A7-66 -- -- --

10.3 A L2A8-66 -- -- --

10.3 A L2A9-66 -- -- --

10.3 A L2A10-66 -- -- --

10.3 B L2B1-66 2.891 28 Female

10.3 B L2B2-66 4.952 34 Male

10.3 B L2B3-66 3.594 31 Female

10.3 B L2B4-66 2.465 28 Female

10.3 B L2B5-66 -- -- --

10.3 B L2B6-66 -- -- --

10.3 B L2B7-66 -- -- --

10.3 B L2B8-66 -- -- --

10.3 B L2B9-66 -- -- --

10.3 B L2B10-66 -- -- --

10.3 C L2C1-66 4.636 32 Female

10.3 C L2C2-66 1.434 23 Female

10.3 C L2C3-66 5.909 35 Female

10.3 C L2C4-66 2.877 29 Female

10.3 C L2C5-66 4.139 37 Female

10.3 C L2C6-66 3.511 31 Female

10.3 C L2C7-66 1.674 29 Male

10.3 C L2C8-66 -- -- --

10.3 C L2C9-66 -- -- --

10.3 C L2C10-66 -- -- --

10.3 D L2D1-66 1.029 21 Female

10.3 D L2D2-66 4.449 32 Male

10.3 D L2D3-66 4.873 34 Male

10.3 D L2D4-66 3.695 32 Male

10.3 D L2D5-66 3.949 31 Male

10.3 D L2D6-66 2.037 26 Female

10.3 D L2D7-66 3.705 31 Male

10.3 D L2D8-66 2.956 28 Female

 ‘--’ denotes a juvenile was not available for this sample. 
ABC Study No. 66086 

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Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull 

(continued) 

Treatment

(μg/L) Replicate

Juvenile

Code Weight (g)

Snout-to-Vent 

Length (mm) Phenotypic Sex

10.3 D L2D9-66 -- -- --

10.3 D L2D10-66 -- -- --

19.5 A L3A1-66 4.102 33 Female

19.5 A L3A2-66 3.184 30 Male

19.5 A L3A3-66 5.172 35 Male

19.5 A L3A4-66 4.020 32 Female

19.5 A L3A5-66 3.918 31 Female

19.5 A L3A6-66 4.104 32 Male

19.5 A L3A7-66 3.244 30 Male

19.5 A L3A8-66 2.583 27 Male

19.5 A L3A9-66 2.243 26 Female

19.5 A L3A10-66 -- -- --

19.5 B L3B1-66 4.074 33 Male

19.5 B L3B2-66 4.525 32 Female

19.5 B L3B3-66 4.479 33 Male

19.5 B L3B4-66 6.279 36 Female

19.5 B L3B5-66 2.902 28 Male

19.5 B L3B6-66 3.375 30 Female

19.5 B L3B7-66 4.588 32 Female

19.5 B L3B8-66 4.815 35 Male

19.5 B L3B9-66 1.630 24 Male

19.5 B L3B10-66 -- -- --

19.5 C L3C1-66 5.394 35 Male

19.5 C L3C2-66 4.531 31 Female

19.5 C L3C3-66 6.042 33 Female

19.5 C L3C4-66 1.862 24 Male

19.5 C L3C5-66 1.107 22 Female

19.5 C L3C6-66 3.691 29 Female

19.5 C L3C7-66 1.088 20 Male

19.5 C L3C8-66 2.092 24 Male

19.5 C L3C9-66 3.716 30 Male

19.5 C L3C10-66 -- -- --

19.5 D L3D1-66 3.676 30 Female

19.5 D L3D2-66 4.297 32 Female

19.5 D L3D3-66 5.333 34 Female

19.5 D L3D4-66 3.681 29 Male

19.5 D L3D5-66 2.775 27 Male

19.5 D L3D6-66 2.265 26 Male

19.5 D L3D7-66 -- -- --

19.5 D L3D8-66 -- -- --

 ‘--’ denotes a juvenile was not available for this sample. 
ABC Study No. 66086 

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Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull 

(continued) 

Treatment

(μg/L) Replicate

Juvenile

Code Weight (g)

Snout-to-Vent 

Length (mm) Phenotypic Sex

19.5 D L3D9-66 -- -- --

19.5 D L3D10-66 -- -- --

38.0 A L4A1-66 3.122 30 Female

38.0 A L4A2-66 3.494 29 Male

38.0 A L4A3-66 2.903 28 Male

38.0 A L4A4-66 3.659 30 Female

38.0 A L4A5-66 5.013 32 Male

38.0 A L4A6-66 1.786 23 Male

38.0 A L4A7-66 4.305 32 Female

38.0 A L4A8-66 4.010 31 Male

38.0 A L4A9-66 3.117 29 Female

38.0 A L4A10-66 -- -- --

38.0 B L4B1-66 4.934 32 Female

38.0 B L4B2-66 3.944 29 Male

38.0 B L4B3-66 4.785 33 Female

38.0 B L4B4-66 4.189 31 Female

38.0 B L4B5-66 3.484 30 Female

38.0 B L4B6-66 3.880 31 Male

38.0 B L4B7-66 -- -- --

38.0 B L4B8-66 -- -- --

38.0 B L4B9-66 -- -- --

38.0 B L4B10-66 -- -- --

38.0 C L4C1-66 5.118 34 Male

38.0 C L4C2-66 2.920 29 Female

38.0 C L4C3-66 1.725 24 Female

38.0 C L4C4-66 3.153 30 Male

38.0 C L4C5-66 4.329 31 Female

38.0 C L4C6-66 4.031 31 Female

38.0 C L4C7-66 3.670 29 Female

38.0 C L4C8-66 4.515 32 Female

38.0 C L4C9-66 2.928 29 Female

38.0 C L4C10-66 -- -- --

38.0 D L4D1-66 3.398 29 Female

38.0 D L4D2-66 2.433 26 Male

38.0 D L4D3-66 4.041 30 Female

38.0 D L4D4-66 3.484 30 Female

38.0 D L4D5-66 2.993 29 Male

38.0 D L4D6-66 3.722 30 Male

38.0 D L4D7-66 1.445 23 Male

‘--’ denotes a juvenile was not available for this sample. 
ABC Study No. 66086 

Page 207 of 261 

Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull 

(continued) 

Treatment

(μg/L) Replicate

Juvenile

Code Weight (g)

Snout-to-Vent 

Length (mm) Phenotypic Sex

38.0 D L4D8-66 3.760 29 Female

38.0 D L4D9-66 -- -- --

38.0 D L4D10-66 -- -- --

‘--’ denotes a juvenile was not available for this sample. 
ABC Study No. 66086 

Page 208 of 261 

APPENDIX J – TERMINATION DATA – GROWTH AND GENDER
ABC Study No. 66086 

Page 209 of 261 

Appendix J-1. Replicate Weight, Length, and Sex Data at Termination 

Treatment

(μg/L) REP JuvenileCode Weight (g) Snout-to-Vent Length (mm) Phenotypic Sex Genotypic Sex

0 (Control) A CA1-T 8.369 40 M M

0 (Control) A CA2-T 10.216 40 F F

0 (Control) A CA3-T 8.302 39 M M

0 (Control) A CA4-T 6.430 36 M M

0 (Control) A CA5-T 14.852 46 F F

0 (Control) A CA6-T 12.185 45 F F

0 (Control) A CA7-T 12.724 46 M M

0 (Control) A CA8-T 7.641 38 F F

0 (Control) A CA9-T 10.427 41 M M

0 (Control) A CA10-T 8.250 37 M M

0 (Control) B CB1-T 8.112 39 M M

0 (Control) B CB2-T 10.430 42 F F

0 (Control) B CB3-T 15.727 51 F F

0 (Control) B CB4-T 6.800 36 M M

0 (Control) B CB5-T 10.295 42 F F

0 (Control) B CB6-T 14.757 44 M M

0 (Control) B CB7-T 7.918 40 M M

0 (Control) B CB8-T 9.079 40 M M

0 (Control) B CB9-T 10.543 42 F F

0 (Control) B CB10-T 13.247 47 F F

0 (Control) C CC1-T 9.904 41 M M

0 (Control) C CC2-T 9.811 40 M M

0 (Control) C CC3-T 9.579 41 M M

0 (Control) C CC4-T 12.010 45 F F

0 (Control) C CC5-T 16.785 48 M M

0 (Control) C CC6-T 8.968 40 F F

0 (Control) C CC7-T 5.727 36 F F

0 (Control) C CC8-T 7.786 37 F F

0 (Control) C CC9-T 10.468 41 M M

0 (Control) C CC10-T -- -- -- --

0 (Control) D CD1-T 11.268 42 F F

0 (Control) D CD2-T 11.920 42 F F

0 (Control) D CD3-T 5.464 35 M M

0 (Control) D CD4-T 7.899 37 M M

0 (Control) D CD5-T 11.617 44 M M

0 (Control) D CD6-T 7.410 39 M M

0 (Control) D CD7-T 12.015 45 M M

0 (Control) D CD8-T 11.805 43 F F

0 (Control) D CD9-T 11.789 42 M M

“--” denotes a juvenile was not available for this sample.
ABC Study No. 66086 

Page 210 of 261 

Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μg/L) REP JuvenileCode Weight (g) Snout-to-Vent Length (mm) Phenotypic Sex Genotypic Sex

0 (Control) D CD10-T -- -- -- --

0 (Control) E CE1-T 8.297 40 F F

0 (Control) E CE2-T 11.321 45 M M

0 (Control) E CE3-T 14.420 47 M M

0 (Control) E CE4-T 12.117 44 M M

0 (Control) E CE5-T 6.819 40 M M

0 (Control) E CE6-T 12.106 47 M M

0 (Control) E CE7-T 6.290 38 M F

0 (Control) E CE8-T 8.824 39 M M

0 (Control) E CE9-T 8.036 37 F F

0 (Control) E CE10-T 10.742 43 F F

0 (Control) F CF1-T 8.240 38 F F

0 (Control) F CF2-T 9.185 41 F F

0 (Control) F CF3-T 10.020 41 M M

0 (Control) F CF4-T 18.080 50 F F

0 (Control) F CF5-T 14.757 47 M M

0 (Control) F CF6-T 5.412 35 F F

0 (Control) F CF7-T 14.354 49 F F

0 (Control) F CF8-T 12.040 46 M M

0 (Control) F CF9-T 9.425 43 M M

0 (Control) F CF10-T 10.380 42 F F

0 (Control) G CG1-T 7.486 38 M M

0 (Control) G CG2-T 8.760 41 F F

0 (Control) G CG3-T 7.158 37 M M

0 (Control) G CG4-T 15.598 47 F F

0 (Control) G CG5-T 9.373 41 M M

0 (Control) G CG6-T 13.189 46 M F

0 (Control) G CG7-T 11.110 43 F F

0 (Control) G CG8-T 8.870 38 M F

0 (Control) G CG9-T 12.122 43 M F

0 (Control) G CG10-T 13.357 47 F F

0 (Control) H CH1-T 13.053 48 M M

0 (Control) H CH2-T 8.177 41 M M

0 (Control) H CH3-T 9.542 42 F F

0 (Control) H CH4-T 11.280 44 M M

0 (Control) H CH5-T 10.628 44 F F

0 (Control) H CH6-T 11.007 42 M M

0 (Control) H CH7-T 14.608 47 F F

0 (Control) H CH8-T 10.807 45 M M

“--” denotes a juvenile was not available for this sample.
ABC Study No. 66086 

Page 211 of 261 

Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μg/L) REP JuvenileCode Weight (g) Snout-to-Vent Length (mm) Phenotypic Sex Genotypic Sex

0 (Control) H CH9-T 8.495 39 M M

0 (Control) H CH10-T 7.517 39 F F

4.97 A L1A1-T 11.733 46 F F

4.97 A L1A2-T 12.736 46 M M

4.97 A L1A3-T 10.561 43 F M

4.97 A L1A4-T 10.194 40 M M

4.97 A L1A5-T 7.088 38 F F

4.97 A L1A6-T 7.677 35 M M

4.97 A L1A7-T 16.235 49 F F

4.97 A L1A8-T 9.815 41 F F

4.97 A L1A9-T 9.865 40 M M

4.97 A L1A10-T 7.881 38 F F

4.97 B L1B1-T 7.763 40 F F

4.97 B L1B2-T 18.255 50 F F

4.97 B L1B3-T 11.119 42 M M

4.97 B L1B4-T 12.503 46 M M

4.97 B L1B5-T 7.117 36 M M

4.97 B L1B6-T 13.321 47 F F

4.97 B L1B7-T 12.234 45 F F

4.97 B L1B8-T 12.515 46 M M

4.97 B L1B9-T 7.827 39 F F

4.97 B L1B10-T 4.262 31 M F

4.97 C L1C1-T 11.386 43 M M

4.97 C L1C2-T 16.404 50 F M

4.97 C L1C3-T 11.801 45 M M

4.97 C L1C4-T 9.136 40 F M

4.97 C L1C5-T 11.392 43 F M

4.97 C L1C6-T 11.827 42 M F

4.97 C L1C7-T 9.459 38 F F

4.97 C L1C8-T 5.973 35 M M

4.97 C L1C9-T 12.992 48 F F

4.97 C L1C10-T 14.448 49 F F

4.97 D L1D1-T 7.404 39 M F

4.97 D L1D2-T 10.226 42 F M

4.97 D L1D3-T 10.794 41 M M

4.97 D L1D4-T 11.822 45 F M

4.97 D L1D5-T 9.087 40 M M

4.97 D L1D6-T 12.459 46 F F

4.97 D L1D7-T 12.368 43 M M 
ABC Study No. 66086 

Page 212 of 261 

Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued) 

Treatment

(μg/L) REP JuvenileCode Weight (g) Snout-to-Vent Length (mm) Phenotypic Sex Genotypic Sex

4.97 D L1D8-T 9.078 40 F M

4.97 D L1D9-T 11.091 41 M M

4.97 D L1D10-T -- -- -- --

10.3 A L2A1-T 9.106 40 F F

10.3 A L2A2-T 9.127 41 M M

10.3 A L2A3-T 5.848 35 M M

10.3 A L2A4-T 17.443 51 M M

10.3 A L2A5-T 11.278 43 M F

10.3 A L2A6-T 9.096 39 M M

10.3 A L2A7-T 12.782 45 M F

10.3 A L2A8-T 14.696 47 F M

10.3 A L2A9-T 10.092 43 M M

10.3 A L2A10-T 11.354 46 M M

10.3 B L2B1-T 10.951 43 M M

10.3 B L2B2-T 8.388 43 F F

10.3 B L2B3-T 9.853 43 F F

10.3 B L2B4-T 12.262 47 M M

10.3 B L2B5-T 13.T7 45 M M

10.3 B L2B6-T 6.624 37 F M

10.3 B L2B7-T 10.513 43 F M

10.3 B L2B8-T 10.370 43 M M

10.3 B L2B9-T 7.502 39 M F

10.3 B L2B10-T 7.792 38 M M

10.3 C L2C1-T 13.906 46 F F

10.3 C L2C2-T 9.497 40 M F

10.3 C L2C3-T 14.139 57 F M

10.3 C L2C4-T 11.173 44 M M

10.3 C L2C5-T 7.121 39 M M

10.3 C L2C6-T 10.769 45 M M

10.3 C L2C7-T 10.954 47 F M

10.3 C L2C8-T 11.449 44 M F

10.3 C L2C9-T 8.443 42 M F

10.3 C L2C10-T 6.585 39 F F

10.3 D L2D1-T 12.535 50 F M

10.3 D L2D2-T 10.945 41 M M

10.3 D L2D3-T 12.673 47 F F

10.3 D L2D4-T 9.825 41 F M

10.3 D L2D5-T 8.601 41 F F

10.3 D L2D6-T 7.026 40 F F

“--” denotes a juvenile was not available for this sample.
ABC Study No. 66086 

Page 213 of 261 

Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μg/L) REP JuvenileCode Weight (g) Snout-to-Vent Length (mm) Phenotypic Sex Genotypic Sex

10.3 D L2D7-T 9.989 42 F F

10.3 D L2D8-T 9.975 42 F F

10.3 D L2D9-T 10.318 42 F F

10.3 D L2D10-T 10.181 42 M M

19.5 A L3A1-T 10.841 41 F M

19.5 A L3A2-T 3.779 32 F M

19.5 A L3A3-T 4.115 31 F M

19.5 A L3A4-T 11.123 43 M F

19.5 A L3A5-T 9.906 44 M F

19.5 A L3A6-T 12.187 46 M M

19.5 A L3A7-T 12.967 42 M F

19.5 A L3A8-T 9.545 43 F F

19.5 A L3A9-T 7.236 38 F F

19.5 A L3A10-T 11.781 47 F F

19.5 B L3B1-T 13.369 48 F F

19.5 B L3B2-T 7.435 41 F M

19.5 B L3B3-T 6.286 39 M F

19.5 B L3B4-T 14.964 47 M F

19.5 B L3B5-T 9.696 41 M F

19.5 B L3B6-T 9.339 42 M M

19.5 B L3B7-T 5.438 37 F F

19.5 B L3B8-T 13.995 49 M M

19.5 B L3B9-T 3.271 31 F M

19.5 B L3B10-T 11.995 46 M M

19.5 C L3C1-T 2.082 38 F F

19.5 C L3C2-T 7.530 38 M M

19.5 C L3C3-T 15.295 52 M M

19.5 C L3C4-T 7.804 40 M F

19.5 C L3C5-T 10.270 41 F M

19.5 C L3C6-T 14.036 48 F F

19.5 C L3C7-T 14.363 50 M F

19.5 C L3C8-T 8.691 41 M M

19.5 C L3C9-T 9.788 42 M F

19.5 C L3C10-T 12.231 46 M F

19.5 D L3D1-T 12.929 45 F F

19.5 D L3D2-T 13.544 49 F F

19.5 D L3D3-T 9.972 44 M M

19.5 D L3D4-T 10.472 43 M M

19.5 D L3D5-T 12.265 43 F F
ABC Study No. 66086 

Page 214 of 261 

Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μg/L) REP JuvenileCode Weight (g) Snout-to-Vent Length (mm) Phenotypic Sex Genotypic Sex

19.5 D L3D6-T 8.619 41 M M 

19.5 D L3D7-T 12.841 47 F F

19.5 D L3D8-T 7.525 40 M M

19.5 D L3D9-T 12.391 47 M M

19.5 D L3D10-T 11.365 45 F F

38.0 A L4A1-T 10.222 40 M M

38.0 A L4A2-T 15.497 48 F F

38.0 A L4A3-T 19.538 55 F F

38.0 A L4A4-T 8.486 42 M M

38.0 A L4A5-T 9.025 41 M M

38.0 A L4A6-T 7.493 42 M M

38.0 A L4A7-T 14.821 48 M M

38.0 A L4A8-T 10.758 43 F F

38.0 A L4A9-T 6.041 37 M M

38.0 A L4A10-T 6.449 37 F F

38.0 B L4B1-T 10.147 41 M M

38.0 B L4B2-T 9.870 42 M M

38.0 B L4B3-T 14.339 46 M M

38.0 B L4B4-T 14.331 48 F F

38.0 B L4B5-T 12.778 46 M M

38.0 B L4B6-T 6.179 42 F F

38.0 B L4B7-T 11.442 45 F F

38.0 B L4B8-T 8.545 41 F F

38.0 B L4B9-T 12.476 44 M M

38.0 B L4B10-T 5.477 31 M M

38.0 C L4C1-T 8.232 38 F F

38.0 C L4C2-T 6.759 36 M M

38.0 C L4C3-T 11.379 44 M M

38.0 C L4C4-T 10.723 42 M M

38.0 C L4C5-T 13.129 48 M M

38.0 C L4C6-T 6.736 38 F F

38.0 C L4C7-T 19.080 50 M M

38.0 C L4C8-T 11.828 45 M M

38.0 C L4C9-T 6.916 35 F M

38.0 C L4C10-T 6.906 37 F F

38.0 D L4D1-T 9.463 43 F M

38.0 D L4D2-T 10.567 44 M M

38.0 D L4D3-T 16.987 51 F F

38.0 D L4D4-T 13.163 47 F F
ABC Study No. 66086 

Page 215 of 261 

Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μg/L) REP JuvenileCode Weight (g) Snout-to-Vent Length (mm) Phenotypic Sex Genotypic Sex

38.0 D L4D5-T 10.850 44 F F

38.0 D L4D6-T 9.537 37 M M

38.0 D L4D7-T 11.891 42 F F

38.0 D L4D8-T 7.040 38 M M

38.0 D L4D9-T 8.596 42 M M

38.0 D L4D10-T 13.680 48 F F
ABC Study No. 66086 

Page 216 of 261 

APPENDIX K – TERMINATION DATA – NUPTIAL PAD AND LIVER WEIGHT
ABC Study No. 66086 

Page 217 of 261 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data 

Juvenile

Code

Phenotypic

Sex

Genotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight 

(g)

Cassette + 

Liver Weight 

(g)

Calculated 

Liver Weight 

(g)

CA1-T M M absent 3.392 3.8291 0.4371

CA2-T F F absent 3.3972 3.9102 0.5130

CA3-T M M absent 3.4099 4.0069 0.5970

CA4-T M M absent 3.3738 3.6852 0.3114

CA5-T F F present 3.4010 4.3996 0.9986

CA6-T F F absent 3.3904 4.1986 0.8082

CA7-T M M present 3.4009 4.1249 0.7240

CA8-T F F absent 3.3963 3.8039 0.4076

CA9-T M M absent 3.3954 3.9459 0.5505

CA10-T M M absent 3.4019 3.8365 0.4346

CB1-T M M absent 3.4028 3.7974 0.3946

CB2-T F F absent 3.4051 4.0654 0.6603

CB3-T F F present 3.3836 4.2982 0.9146

CB4-T M M absent 3.3947 3.7300 0.3353

CB5-T F F absent 3.3986 3.9408 0.5422

CB6-T M M present 3.3989 4.4616 1.0627

CB7-T M M absent 3.3905 3.8233 0.4328

CB8-T M M absent 3.3956 3.9236 0.5280

CB9-T F F absent 3.3837 3.8952 0.5115

CB10-T F F present 3.3898 4.1645 0.7747

CC1-T M M absent 3.3940 3.9602 0.5662 

CC2-T M M absent 3.4015 4.0012 0.5997

CC3-T M M present 3.3946 3.9041 0.5095

CC4-T F F absent 3.3995 4.3122 0.9127

CC5-T M M present 3.3907 4.4649 1.0742

CC6-T F F absent 3.3979 3.6305 0.2326

CC7-T F F absent 3.3940 4.0466 0.6526

CC8-T F F absent 3.4069 3.7947 0.3878

CC9-T M M absent 3.3838 3.9949 0.6111

CC10-T -- -- -- -- -- --

CD1-T F F absent 3.4077 4.1896 0.7819

CD2-T F F absent 3.4013 4.2831 0.8818

CD3-T M M absent 3.4009 3.6417 0.2408

CD4-T M M absent 3.3972 3.9021 0.5049

CD5-T M M absent 3.4000 4.2181 0.8181

CD6-T M M absent 3.4015 3.7779 0.3764

CD7-T M M absent 3.4014 4.2463 0.8449

CD8-T F F absent 3.3981 4.1062 0.7081

“--” denotes a juvenile was not available for this sample.
ABC Study No. 66086 

Page 218 of 261 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Juvenile

Code

Phenotypic

Sex

Genotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight 

(g)

Cassette + 

Liver Weight 

(g)

Calculated 

Liver Weight 

(g)

CD9-T M M absent 3.3998 4.1195 0.7197

CD10-T -- -- -- -- -- --

CE1-T F F absent 3.3971 3.8453 0.4482

CE2-T M M absent 3.4020 4.1695 0.7675

CE3-T M M present 3.3997 4.2736 0.8739

CE4-T M M absent 3.3733 4.2494 0.8761

CE5-T M M absent 3.4052 3.7816 0.3764

CE6-T M M absent 3.3917 4.2030 0.8113

CE7-T M F absent 3.3890 3.7088 0.3198

CE8-T M M absent 3.4015 3.9461 0.5446

CE9-T F F present 3.4097 3.8522 0.4425

CE10-T F F absent 3.3860 3.9249 0.5389

CF1-T F F absent 3.3854 3.8024 0.4170

CF2-T F F absent 3.3883 3.9478 0.5595

CF3-T M M present 3.3896 3.9454 0.5558

CF4-T F F present 3.4001 4.5907 1.1906

CF5-T M M present 3.4090 4.2431 0.8341

CF6-T F F absent 3.3959 3.6573 0.2614

CF7-T F F present 3.4005 4.1791 0.7786

CF8-T M M present 3.4007 4.0775 0.6768

CF9-T M M absent 3.4009 3.9170 0.5161

CF10-T F F absent 3.4071 3.9838 0.5767

CG1-T M M absent 3.3485 3.7334 0.3849

CG2-T F F absent 3.3805 3.9223 0.5418

CG3-T M M absent 3.3954 3.7771 0.3817

CG4-T F F absent 3.3762 4.4591 1.0829

CG5-T M M absent 3.3782 3.9813 0.6031

CG6-T M F absent 3.3647 4.0931 0.7284

CG7-T F F absent 3.3507 4.0327 0.6820

CG8-T M F absent 3.3449 3.8750 0.5301

CG9-T M F absent 3.3684 3.9886 0.6202

CG10-T F F absent 3.3492 4.1987 0.8495

CH1-T M M present 3.3814 4.0755 0.6941

CH2-T M M absent 3.3578 3.7730 0.4152

CH3-T F F absent 3.3800 3.9235 0.5435

CH4-T M M absent 3.3813 4.0287 0.6474

CH5-T F F absent 3.3645 3.9271 0.5626

CH6-T M M absent 3.3787 3.9833 0.6046

“--” denotes a juvenile was not available for this sample.
ABC Study No. 66086 

Page 219 of 261 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Juvenile

Code

Phenotypic

Sex

Genotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight 

(g)

Cassette + 

Liver Weight 

(g)

Calculated 

Liver Weight 

(g)

CH7-T F F present 3.3517 4.1361 0.7844

CH8-T M M absent 3.3505 3.8742 0.5237

CH9-T M M absent 3.3673 3.9230 0.5557

CH10-T F F absent 3.3784 3.7718 0.3934

L1A1-T F F absent 3.3811 4.1898 0.8087

L1A2-T M M present 3.3761 4.0765 0.7004

L1A3-T F M present 3.3619 3.9902 0.6283

L1A4-T M M absent 3.3663 3.9858 0.6195

L1A5-T F F absent 3.3802 3.7608 0.3806

L1A6-T M M absent 3.3736 3.8356 0.4620

L1A7-T F F present 3.3457 4.3554 1.0097

L1A8-T F F absent 3.3644 3.8580 0.4936

L1A9-T M M absent 3.3674 4.0677 0.7003

L1A10-T F F absent 3.3682 3.8246 0.4564

L1B1-T F F absent 3.3690 3.8148 0.4458

L1B2-T F F present 3.3677 4.6705 1.3028

L1B3-T M M absent 3.3649 3.9960 0.6311

L1B4-T M M absent 3.3780 4.0267 0.6487

L1B5-T M M absent 3.3710 3.7808 0.4098

L1B6-T F F present 3.3676 4.1925 0.8249

L1B7-T F F present 3.3784 4.1232 0.7448 

L1B8-T M M absent 3.3673 4.0699 0.7026

L1B9-T F F absent 3.3662 3.8625 0.4963

L1B10-T M F present 3.3663 3.5730 0.2067

L1C1-T M M absent 3.3746 3.9639 0.5893

L1C2-T F M present 3.3684 4.3675 0.9991

L1C3-T M M present 3.3716 4.0283 0.6567

L1C4-T F M absent 3.3711 3.9570 0.5859

L1C5-T F M absent 3.3659 4.0204 0.6545

L1C6-T M F absent 3.3522 3.9878 0.6356

L1C7-T F F absent 3.3825 3.9272 0.5447

L1C8-T M M absent 3.3480 3.6558 0.3078

L1C9-T F F present 3.3765 4.1191 0.7426

L1C10-T F F present 3.3487 4.2130 0.8643

L1D1-T M F absent 3.3675 3.8115 0.4440

L1D2-T F M present 3.3462 3.8721 0.5259

L1D3-T M M absent 3.3795 3.9676 0.5881

L1D4-T F M present 3.3625 4.0079 0.6454

L1D5-T M M absent 3.3510 3.7779 0.4269
ABC Study No. 66086 

Page 220 of 261 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Juvenile

Code

Phenotypic

Sex

Genotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight 

(g)

Cassette + 

Liver Weight 

(g)

Calculated 

Liver Weight 

(g)

L1D6-T F F present 3.3421 4.0248 0.6827

L1D7-T M M absent 3.3637 4.1433 0.7796

L1D8-T F M absent 3.3664 3.9023 0.5359

L1D9-T M M absent 3.3457 3.9176 0.5719

L1D10-T -- -- -- -- -- --

L2A1-T F F absent 3.3452 3.9344 0.5892

L2A2-T M M absent 3.3510 3.7650 0.4140

L2A3-T M M absent 3.3653 3.5798 0.2145

L2A4-T M M present 3.3668 4.3473 0.9805

L2A5-T M F present 3.3580 3.9712 0.6132

L2A6-T M M present 3.3460 3.8352 0.4892

L2A7-T M F present 3.3658 4.0871 0.7213

L2A8-T F M present 3.3580 4.2606 0.9026

L2A9-T M M present 3.3747 3.9487 0.5740

L2A10-T M M present 3.3816 3.9908 0.6092

L2B1-T M M absent 3.3771 4.0713 0.6942

L2B2-T F F absent 3.3678 4.0185 0.6507

L2B3-T F F absent 3.3476 4.0093 0.6617

L2B4-T M M present 3.3778 4.2723 0.8945

L2B5-T M M absent 3.3657 4.3242 0.9585

L2B6-T F M absent 3.3651 3.7719 0.4068

L2B7-T F M present 3.3660 3.9636 0.5976

L2B8-T M M present 3.3728 3.9576 0.5848

L2B9-T M F absent 3.3381 3.7940 0.4559

L2B10-T M M absent 3.3688 3.8967 0.5279

L2C1-T F F present 3.3467 4.1495 0.8028

L2C2-T M F present 3.3642 3.8974 0.5332

L2C3-T F M present 3.3690 4.2301 0.8611

L2C4-T M M absent 3.3781 4.0818 0.7037

L2C5-T M M absent 3.3442 3.7635 0.4193

L2C6-T M M present 3.3804 4.0129 0.6325

L2C7-T F M absent 3.3793 3.9450 0.5657

L2C8-T M F present 3.3677 4.0833 0.7156

L2C9-T M F absent 3.3705 3.7853 0.4148

L2C10-T F F absent 3.3501 3.7669 0.4168

L2D1-T F M present 3.3499 4.2718 0.9219

L2D2-T M M present 3.3519 4.0089 0.6570

L2D3-T F F present 3.3787 4.1412 0.7625

“--” denotes a juvenile was not available for this sample.
ABC Study No. 66086 

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Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Juvenile

Code

Phenotypic

Sex

Genotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight 

(g)

Cassette + 

Liver Weight 

(g)

Calculated 

Liver Weight 

(g)

L2D4-T F M present 3.3691 3.9021 0.5330

L2D5-T F F absent 3.3668 3.8514 0.4846

L2D6-T F F absent 3.3447 3.7092 0.3645

L2D7-T F F absent 3.3680 3.9748 0.6068

L2D8-T F F absent 3.3765 4.0434 0.6669

L2D9-T F F absent 3.3710 4.0218 0.6508

L2D10-T M M absent 3.3739 3.9708 0.5969

L3A1-T F M absent 3.3849 4.1330 0.7481

L3A2-T F M absent 3.3703 3.5589 0.1886

L3A3-T F M absent 3.3650 3.5206 0.1556

L3A4-T M F absent 3.3662 4.0354 0.6692

L3A5-T M F absent 3.3465 3.9212 0.5747

L3A6-T M M absent 3.3468 4.1513 0.8045

L3A7-T M F absent 3.3787 4.2451 0.8664

L3A8-T F F absent 3.3758 4.0568 0.6810

L3A9-T F F absent 3.3512 3.7285 0.3773

L3A10-T F F absent 3.3829 4.2208 0.8379

L3B1-T F F present 3.3806 4.2384 0.8578

L3B2-T F M absent 3.3512 3.8535 0.5023

L3B3-T M F absent 3.3781 3.7575 0.3794

L3B4-T M F present 3.3643 4.2884 0.9241

L3B5-T M F absent 3.3662 4.1090 0.7428

L3B6-T M M present 3.3794 3.9416 0.5622

L3B7-T F F absent 3.3712 3.7038 0.3326

L3B8-T M M absent 3.3612 4.2330 0.8718

L3B9-T F M absent 3.3736 3.5332 0.1596

L3B10-T M M present 3.3692 4.1765 0.8073

L3C1-T F F NA 3.3841 3.4455 0.0614

L3C2-T M M absent 3.3706 3.7837 0.4131

L3C3-T M M present 3.3503 4.1797 0.8294

L3C4-T M F present 3.3568 3.7405 0.3837

L3C5-T F M absent 3.3509 3.9547 0.6038

L3C6-T F F present 3.3769 4.2026 0.8257

L3C7-T M F present 3.3676 4.2239 0.8563

L3C8-T M M absent 3.3626 3.8788 0.5162

L3C9-T M F absent 3.3424 4.0362 0.6938

L3C10-T M F absent 3.3665 4.0599 0.6934

L3D1-T F F absent 3.3669 4.2438 0.8769

L3D2-T F F present 3.3797 4.0986 0.7189
ABC Study No. 66086 

Page 222 of 261 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Juvenile

Code

Phenotypic

Sex

Genotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight 

(g)

Cassette + 

Liver Weight 

(g)

Calculated 

Liver Weight 

(g)

L3D3-T M M absent 3.3499 3.9540 0.6041

L3D4-T M M present 3.3793 4.0085 0.6292

L3D5-T F F absent 3.3649 4.1129 0.7480

L3D6-T M M absent 3.3621 3.8835 0.5214

L3D7-T F F present 3.3789 3.9810 0.6021

L3D8-T M M absent 3.3606 3.9015 0.5409

L3D9-T M M present 3.3836 4.1328 0.7492

L3D10-T F F absent 3.3384 4.0007 0.6623

L4A1-T M M absent 3.3710 3.9629 0.5919

L4A2-T F F present 3.3495 4.2880 0.9385

L4A3-T F F present 3.3414 4.5050 1.1636

L4A4-T M M absent 3.3709 3.8591 0.4882

L4A5-T M M absent 3.3533 3.9469 0.5936

L4A6-T M M absent 3.3669 3.7741 0.4072

L4A7-T M M present 3.3456 4.2056 0.8600

L4A8-T F F absent 3.3853 4.0539 0.6686

L4A9-T M M absent 3.3634 3.6160 0.2526

L4A10-T F F absent 3.3709 3.6733 0.3024

L4B1-T M M present 3.3819 3.9686 0.5867

L4B2-T M M absent 3.3770 3.9426 0.5656

L4B3-T M M present 3.3653 4.2644 0.8991

L4B4-T F F present 3.3535 4.2113 0.8578

L4B5-T M M present 3.3678 4.1542 0.7864

L4B6-T F F absent 3.3506 3.6840 0.3334

L4B7-T F F absent 3.3538 4.0102 0.6564

L4B8-T F F present 3.3705 3.7412 0.3707

L4B9-T M M present 3.3536 4.1148 0.7612

L4B10-T M M absent 3.3687 3.6622 0.2935

L4C1-T F F absent 3.3692 3.8264 0.4572

L4C2-T M M absent 3.3694 3.7519 0.3825

L4C3-T M M absent 3.3619 4.0489 0.6870

L4C4-T M M absent 3.3662 4.0275 0.6613

L4C5-T M M absent 3.3701 4.3592 0.9891

L4C6-T F F absent 3.3684 3.7574 0.3890

L4C7-T M M present 3.3511 4.5865 1.2354

L4C8-T M M present 3.3763 4.1720 0.7957

L4C9-T F M absent 3.3746 3.7426 0.3680

L4C10-T F F absent 3.3702 3.7528 0.3826

L4D1-T F M absent 3.3811 3.8079 0.4268
ABC Study No. 66086 

Page 223 of 261 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Juvenile

Code

Phenotypic

Sex

Genotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight 

(g)

Cassette + 

Liver Weight 

(g)

Calculated 

Liver Weight 

(g)

L4D2-T M M absent 3.3481 4.0127 0.6646

L4D3-T F F present 3.3732 4.3717 0.9985

L4D4-T F F present 3.3792 4.0975 0.7183

L4D5-T F F absent 3.3710 4.0187 0.6477

L4D6-T M M absent 3.3466 3.9003 0.5537

L4D7-T F F present 3.3823 4.0349 0.6526

L4D8-T M M absent 3.3822 3.7380 0.3558

L4D9-T M M absent 3.3812 3.7980 0.4168

L4D10-T F F present 3.3730 4.1125 0.7395
ABC Study No. 66086 

Page 224 of 261 

APPENDIX L – WATER QUALITY DATA
ABC Study No. 66086 

Page 225 of 261 

Table L-1. Test Solution Temperature Measured in Each Replicate Test Chamber

Temperature °C

Control

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 21.2 21.0 21.1 21.1 21.1 20.9 20.9 20.9

6 20.9 20.7 20.8 20.8 20.9 20.7 20.8 20.8

8 21.0 20.9 20.9 20.9 20.9 20.7 20.9 20.9

12 21.1 20.9 21.1 21.1 21.1 20.8 21.0 21.0

15 21.4 21.1 21.3 21.3 21.2 21.1 21.1 21.2

19 21.0 20.8 20.9 20.8 20.9 20.7 20.8 20.8

21 21.2 21.1 21.3 21.2 21.3 21.2 21.1 21.2

27 20.8 20.6 20.8 20.8 20.9 20.6 20.6 20.6

29 20.8 20.5 20.8 20.8 20.9 20.7 20.6 20.7

32 21.1 20.9 21.1 21.2 21.2 20.9 20.9 20.9

34 20.8 20.6 20.8 20.8 20.8 20.6 20.6 20.7

36 20.9 20.6 20.9 20.8 20.9 20.7 20.7 20.7

39 20.9 20.8 20.9 21.0 21.0 20.9 20.7 20.7

41 20.8 20.6 21.1 21.1 21.0 21.0 21.1 21.1

43 21.2 20.9 21.5 21.5 21.5 20.9 20.9 20.9

46 21.6 20.7 20.9 21.0 21.5 20.8 20.6 20.7

50 20.6 20.6 20.7 20.8 20.8 20.7 20.5 20.5

64 21.6 21.5 21.6 21.6 21.8 21.5 21.3 21.4

67 20.7 20.1 20.8 20.3 20.6 20.4 20.1 20.1

71 21.1 21.0 21.2 21.0 21.2 21.0 20.9 21.0

75 21.3 21.2 21.2 21.2 21.3 21.3 21.1 21.1

78 21.4 21.5 21.4 21.1 21.4 21.3 21.4 21.4

82 21.6 21.5 21.5 21.4 21.4 21.7 21.6 21.4

85 20.4 20.5 21.1 21.5 21.1 20.8 20.7 20.6

88 20.7 20.7 21.1 21.0 21.0 20.7 20.7 20.7

92 20.7 20.6 21.1 21.0 21.0 20.8 20.7 20.6

97 20.8 20.8 21.0 20.7 21.0 21.0 20.9 20.9

99 20.4 20.4 20.5 20.6 20.8 20.6 20.6 20.6

102 20.3 20.7 20.8 20.6 20.7 20.5 20.4 20.3

106 20.7 20.6 20.6 20.6 20.4 20.5 20.7 20.7

111 21.1 21.3 21.3 21.3 21.2 21.0 21.0 21.0

112 20.9 -- 20.7 20.8 -- -- -- 20.7

113 -- 20.9 -- -- 20.8 20.7 20.8 --

‘--’ indicates measurement not collected. 
ABC Study No. 66086 

Page 226 of 261 

Table L-1. Test Solution Temperature Measured in Each Replicate Test Chamber (continued)

Temperature °C

Level 1 (4.97 μg/L) Level 2 (10.3 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 20.9 20.9 20.9 21.0 21.0 21.0 20.9 21.0

6 20.7 20.8 20.8 20.8 20.7 20.7 20.6 20.6

8 20.6 20.9 21.0 20.9 20.8 20.8 20.7 20.9

12 20.7 21.1 21.1 21.1 20.9 21.0 20.8 21.0

15 20.9 21.5 21.2 21.3 21.1 21.2 21.0 21.1

19 20.5 20.8 20.9 20.8 20.7 20.7 20.6 20.8

21 20.9 21.3 21.4 21.2 21.2 21.2 21.0 21.1

27 20.4 20.8 20.2 20.8 20.6 20.8 20.6 20.7

29 20.4 20.8 20.7 20.8 20.6 20.7 20.5 20.5

32 20.9 21.3 21.0 21.1 21.0 21.1 20.9 21.0

34 20.4 21.1 20.7 20.8 20.6 20.7 20.5 20.6

36 20.4 21.1 20.8 20.8 20.7 20.7 20.5 20.6

39 20.6 20.9 20.9 20.9 20.8 20.8 20.6 20.7

41 20.8 21.2 21.2 21.2 21.0 21.0 20.6 20.9

43 20.7 21.4 21.2 21.5 20.9 21.5 21.0 21.4

46 20.8 20.7 20.9 20.9 20.8 20.8 20.6 20.9

50 20.4 20.7 20.8 20.7 20.7 20.7 20.4 20.5

64 21.2 21.7 21.5 21.6 21.4 21.4 21.4 21.7

67 20.4 20.5 20.6 20.5 20.5 20.4 20.2 20.5

71 21.1 21.3 21.3 20.5 21.2 21.1 20.9 21.1

75 21.0 21.2 21.2 21.3 21.5 21.2 21.1 21.4

78 21.4 21.5 21.1 21.3 21.3 21.2 21.7 21.4

82 21.4 21.5 21.2 21.5 21.4 21.5 21.5 21.7

85 20.3 20.9 20.8 21.0 20.9 20.9 20.6 20.7

88 20.4 20.8 20.7 21.0 20.9 21.0 20.7 20.8

92 20.5 20.9 20.7 21.1 20.9 20.7 20.8 21.2

97 20.8 21.1 20.8 21.1 20.9 21.0 20.8 20.9

99 20.4 20.7 20.4 20.6 20.4 20.5 20.4 20.5

102 20.4 20.9 20.4 20.8 20.5 20.6 20.6 20.4

106 20.5 20.9 20.5 20.8 20.6 20.6 20.5 20.7

111 20.8 21.5 21.2 21.4 21.3 21.1 21.0 21.1

112 20.5 -- -- 20.8 -- -- 20.6 20.9

113 -- 20.9 20.7 -- 20.7 20.7 -- --

‘--’ indicates measurement not collected. 
ABC Study No. 66086 

Page 227 of 261 

Table L-1. Test Solution Temperature Measured in Each Replicate Test Chamber (continued) 

Temperature °C

Level 3 (19.5 μg/L) Level 4 (38.0 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 21.0 21.0 21.0 21.0 21.0 21.0 21.0 21.0

6 20.8 20.8 20.8 20.8 20.8 20.8 20.8 20.8

8 20.9 21.0 21.0 20.8 21.0 20.9 20.8 20.9

12 21.1 21.1 21.1 21.1 21.0 21.1 21.0 20.9

15 21.3 21.3 21.4 21.1 21.6 21.2 21.2 21.3

19 20.8 20.9 20.8 20.7 20.8 20.8 20.7 20.9

21 21.3 21.4 21.2 21.2 21.2 21.2 21.1 21.3

27 20.9 21.0 20.8 20.7 20.9 20.7 20.7 20.7

29 20.9 20.8 20.9 20.8 21.0 20.7 20.7 20.7

32 21.2 21.2 21.2 21.0 21.3 21.0 21.1 21.1

34 20.8 20.9 20.9 20.6 21.3 20.6 20.6 20.7

36 20.9 21.0 20.9 20.7 21.3 20.7 20.6 20.8

39 20.9 21.0 20.9 20.9 21.1 20.6 20.7 20.9

41 21.2 21.2 21.1 21.1 21.1 19.9 20.7 21.0

43 21.6 21.5 21.0 21.4 20.9 20.9 20.9 20.9

46 20.9 20.9 20.8 20.8 20.8 20.8 20.8 20.9

50 20.8 20.8 20.7 20.7 20.7 20.7 20.7 20.8

64 21.8 21.9 21.8 21.7 21.6 21.7 21.5 21.7

67 20.5 20.6 20.6 20.5 20.4 20.5 20.3 20.5

71 21.3 21.2 21.1 21.0 21.1 21.0 21.0 21.1

75 21.4 21.3 21.2 21.2 21.1 21.2 21.1 21.3

78 21.6 21.4 21.5 21.6 21.2 21.5 21.2 21.1

82 21.8 21.4 21.7 21.6 21.3 21.4 21.1 21.4

85 21.0 20.9 21.0 21.1 20.7 20.9 20.7 20.8

88 21.0 21.1 21.0 21.0 19.9 21.0 21.1 20.8

92 21.2 21.2 20.9 21.0 20.6 20.8 21.0 20.6

97 21.0 21.0 20.9 20.9 20.8 21.1 21.2 20.8

99 20.8 20.9 20.7 20.8 20.1 20.8 20.8 20.5

102 20.6 20.8 20.7 20.9 20.5 20.8 20.8 20.3

106 20.7 20.7 20.7 20.8 20.8 20.6 20.7 20.4

111 21.3 21.3 21.4 21.3 21.3 21.1 21.2 21.1

112 20.9 -- 21.0 -- -- -- 20.9 --

113 -- 20.6 -- 20.9 21.0 20.9 -- --

‘--’ indicates measurement not collected. 
ABC Study No. 66086 

Page 228 of 261 

Table L-2. Test Solution Dissolved Oxygen Concentrations Measured in Each Replicate Test 

Chamber

Dissolved Oxygen (mg/L)

Control

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 8.61 8.67 8.66 8.69 8.65 8.66 8.70 8.70

6 5.59 5.92 6.21 5.57 6.41 6.31 6.13 6.03

8 6.11 6.81 6.77 6.93 7.16 6.82 6.81 6.71

12 5.97 6.63 6.64 6.77 6.60 6.75 6.73 6.50

15 6.29 6.34 6.41 6.38 5.53 6.35 6.23 6.24

19 6.59 6.31 6.46 6.04 6.60 6.20 6.30 6.35

21 5.68 5.48 5.96 5.19 5.70 5.26 5.47 5.48

27 5.87 5.23 5.32 5.26 5.11 5.31 5.25 5.35

29 6.17 5.81 6.20 6.02 5.75 6.04 6.38 5.89

32 5.26 4.89 5.30 5.17 4.89 5.09 4.87 4.90

34 4.94 4.63 5.23 4.93 4.75 4.99 4.83 4.77

36 5.10 4.82 5.21 4.92 4.69 4.74 4.81 5.01

39 5.41 5.43 6.02 5.98 5.17 5.86 5.83 5.82

41 4.01 5.13 5.80 6.30 5.14 5.85 5.67 6.14

43 5.06 5.73 6.69 6.75 6.32 6.06 5.89 6.14

46 5.61 5.53 6.02 5.61 5.25 5.64 5.54 5.95

50 4.87 5.73 5.95 5.64 5.71 6.11 5.91 5.82

53 6.35 5.99 6.95 6.56 6.43 6.89 7.32 6.96

57 6.69 6.96 7.40 7.09 7.05 7.17 7.59 7.32

60 4.18 6.03 5.79 5.35 5.11 5.81 5.68 5.44

64 5.83 6.54 6.46 6.44 6.10 5.53 6.97 6.60

67 6.73 5.71 7.71 6.08 6.17 6.75 6.67 6.49

71 7.21 7.31 7.35 7.27 7.13 7.39 7.41 7.22

75 6.42 6.44 6.46 6.48 6.35 6.43 6.28 6.28

78 6.02 6.23 6.22 8.22 6.34 6.25 6.43 6.40

82 6.63 6.46 7.32 6.70 7.06 6.70 6.93 6.43

85 7.95 6.62 7.35 7.32 7.15 7.08 7.24 7.40

88 6.97 7.05 7.34 7.11 7.03 6.83 6.96 7.05

92 7.39 7.00 7.13 7.66 7.14 6.95 7.04 6.85

97 6.44 6.87 6.39 6.67 6.73 6.70 6.56 6.55

99 6.48 6.48 6.90 6.59 6.58 6.59 6.62 6.64

102 6.76 6.65 7.29 6.87 6.99 6.95 6.75 6.69

106 6.89 6.76 7.19 6.87 7.13 7.16 7.13 6.78

111 6.75 6.78 7.20 7.25 6.78 6.92 6.83 6.68

112 7.90 -- 6.91 7.12 -- -- -- 6.93

113 -- 6.74 -- -- 6.72 6.98 7.09 --

Note: 100% dissolved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21, and 22°C, respectively.

‘--’ indicates measurement not collected. 
ABC Study No. 66086 

Page 229 of 261 

Table L-2. Test Solution Dissolved Oxygen Concentrations Measured in Each Replicate Test 

Chamber (continued) 

Dissolved Oxygen (mg/L)

Level 1 (4.97 μg/L) Level 2 (10.3 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 8.72 8.71 8.69 8.66 8.69 8.71 8.69 8.69

6 5.90 6.39 6.32 6.57 5.92 6.09 5.73 5.85

8 7.09 6.82 6.92 7.16 7.15 6.92 6.09 7.15

12 6.70 6.37 6.54 6.99 6.56 6.21 5.64 6.79

15 6.33 6.45 6.07 6.02 6.34 6.34 6.57 6.42

19 6.38 6.57 6.21 6.63 6.29 6.60 7.02 6.33

21 5.63 5.90 5.43 5.67 5.53 5.96 6.44 5.41

27 5.64 5.64 5.17 5.21 5.61 5.39 6.28 5.25

29 6.31 6.57 6.01 5.79 6.57 6.25 6.57 5.96

32 5.42 5.48 4.80 5.09 5.35 5.64 5.71 4.89

34 5.43 5.51 4.71 5.06 5.16 5.50 5.57 5.00

36 5.09 5.43 4.37 4.88 4.72 5.31 5.31 4.71

39 5.96 6.34 5.51 5.92 5.66 6.17 5.50 5.56

41 5.15 6.40 5.60 5.03 5.55 5.60 4.55 4.78

43 5.73 7.16 6.04 6.34 6.15 6.45 5.27 6.02

46 6.13 6.11 5.52 5.36 5.79 5.75 4.96 5.59

50 5.88 5.95 5.30 5.61 5.57 5.56 5.82 5.92

53 6.46 6.98 6.75 6.84 6.18 6.45 6.66 6.96

57 7.34 7.46 7.41 7.28 7.16 7.39 7.32 7.57

60 5.36 5.37 5.47 4.33 5.50 5.66 5.21 5.42

64 6.29 6.98 6.85 6.77 6.51 6.58 6.69 6.85

67 6.79 6.83 6.81 6.62 6.87 6.89 6.55 6.62

71 7.35 7.23 7.45 6.56 7.11 7.10 6.85 7.36

75 6.25 5.84 6.40 6.81 6.19 5.53 6.34 6.40

78 6.32 6.20 6.22 6.03 6.35 5.53 6.04 6.31

82 6.74 7.89 6.44 5.76 5.91 6.60 6.36 6.82

85 6.84 6.78 6.91 7.32 7.13 7.16 6.77 6.44

88 6.67 6.71 7.21 6.92 6.79 6.93 6.95 6.94

92 6.79 6.81 7.01 6.79 6.89 6.90 6.87 7.05

97 6.57 6.68 6.62 6.33 6.71 6.74 6.80 6.86

99 6.83 6.61 6.67 6.53 6.45 6.75 6.67 6.63

102 6.75 6.58 6.97 7.09 6.90 6.90 6.87 6.98

106 7.09 6.87 6.66 6.89 6.75 7.06 7.05 6.84

111 6.79 6.76 6.83 6.77 6.68 6.92 6.40 6.50

112 6.94 -- -- 6.97 -- -- 6.61 6.88

113 -- 6.71 6.66 -- 6.71 6.83 -- --

Note: 100% dissolved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21, and 22°C, respectively.

‘--’ indicates measurement not collected. 
ABC Study No. 66086 

Page 230 of 261 

Table L-2. Test Solution Dissolved Oxygen Concentrations Measured in Each Replicate Test 

Chamber (continued) 

Dissolved Oxygen (mg/L)

Level 3 (19.5 μg/L) Level 4 (38.0 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 8.69 8.67 8.68 8.69 8.67 8.66 8.64 8.64

6 6.11 6.08 6.23 6.68 5.61 6.35 6.10 5.68

8 6.97 6.73 7.08 7.21 6.86 7.28 7.19 6.78

12 5.97 5.80 6.36 6.70 6.99 5.58 6.74 6.78

15 6.43 6.37 6.19 6.02 6.06 6.32 6.06 5.81

19 6.82 7.01 6.31 6.56 6.39 6.73 6.42 5.53

21 5.96 6.41 5.48 5.64 5.58 5.71 5.40 5.34

27 5.42 5.72 5.20 5.33 5.74 5.34 5.62 4.87

29 5.60 5.91 5.93 5.88 5.66 5.99 5.74 5.56

32 5.04 5.35 4.96 5.31 4.66 5.20 5.06 4.76

34 4.94 4.97 4.92 5.21 4.77 4.94 4.59 4.57

36 4.91 4.90 4.88 5.00 4.53 5.00 4.61 4.38

39 5.32 5.56 5.55 6.16 5.15 5.64 5.37 5.03

41 4.89 4.31 5.76 5.44 5.73 5.09 4.70 4.74

43 6.18 5.23 5.59 6.61 5.34 5.71 5.56 5.40

46 5.52 4.79 5.28 6.01 5.60 5.69 5.60 5.31

50 5.63 5.41 5.06 5.90 5.46 5.40 5.63 5.10

53 6.52 6.64 6.53 6.75 6.08 6.82 6.85 6.21

57 7.21 6.76 6.80 7.18 6.71 7.46 5.45 7.39

60 5.37 4.84 5.15 5.55 4.96 5.39 4.97 4.92

64 6.54 6.64 6.53 6.39 6.40 7.15 6.21 6.32

67 6.59 6.18 6.32 7.02 5.98 6.78 6.21 6.36

71 7.04 6.84 7.03 7.48 6.86 7.13 6.91 7.22

75 6.19 6.22 6.40 6.34 6.20 6.30 6.28 6.28

78 6.63 6.34 6.17 6.17 6.30 6.38 6.12 6.15

82 6.28 6.34 6.32 5.78 6.33 5.92 5.84 6.31

85 6.68 6.78 6.86 7.11 6.22 6.78 6.20 6.58

88 7.10 7.11 6.85 7.19 4.64 7.22 7.21 7.03

92 7.05 7.39 6.68 6.79 6.84 6.69 6.83 6.55

97 6.91 6.53 6.77 6.18 6.52 6.65 6.33 6.54

99 6.59 6.66 6.53 6.86 4.72 6.78 6.76 6.54

102 7.08 7.10 6.77 6.95 6.76 6.79 6.95 6.43

106 7.09 6.91 6.78 6.96 6.64 6.85 6.83 6.54

111 6.95 6.57 6.72 6.87 6.48 6.68 6.66 6.41

112 7.08 -- 6.98 -- -- -- 6.92 6.49

113 -- 6.74 -- 6.83 6.43 6.92 -- --

Note: 100% dissolved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21, and 22°C, respectively.

‘--’ indicates measurement not collected. 
ABC Study No. 66086 

Page 231 of 261 

Table L-3. Test Solution pH Values Measured in Each Replicate Test Chamber

pH

Control

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 8.32 8.32 8.33 8.30 8.31 8.31 8.28 8.27

6 8.07 8.05 8.08 8.01 8.11 8.09 8.06 8.03

8 7.86 7.95 7.95 7.96 8.02 8.00 7.97 7.98

12 8.03 8.06 8.10 8.09 8.09 8.11 8.06 8.09

15 8.15 8.13 8.16 8.14 8.17 8.18 8.15 8.14

19 8.12 8.04 8.14 8.05 8.28 8.11 8.06 8.03

21 8.05 8.00 8.06 7.95 8.01 8.00 7.98 8.00

27 8.25 8.09 8.11 8.05 8.04 8.04 8.03 8.03

29 8.25 8.14 8.19 8.14 8.13 8.14 8.15 8.11

32 7.89 7.87 7.97 7.93 7.90 7.93 7.89 7.89

36 8.46 8.27 8.14 8.07 8.03 7.99 7.98 7.97

41 7.91 7.95 8.03 8.10 7.90 8.04 8.03 8.07

43 7.82 7.94 8.18 8.21 8.12 8.04 8.00 8.02

46 8.01 8.08 8.13 8.05 8.01 8.08 8.04 8.08

50 7.99 8.15 8.19 8.12 8.15 8.22 8.18 8.17

53 8.07 8.02 8.20 8.14 8.14 8.19 8.23 8.19

57 8.13 8.15 8.24 8.20 8.21 8.12 8.07 8.19

60 7.76 7.92 7.91 7.84 7.80 7.85 7.86 7.82

64 7.85 7.90 7.93 7.90 7.88 7.88 7.93 7.95

67 7.47 7.59 7.93 7.71 7.70 7.81 7.77 7.77

71 8.08 8.13 8.14 8.10 8.11 8.14 8.13 8.11

75 7.70 7.98 8.02 7.94 7.96 7.99 7.96 7.96

78 8.06 7.90 7.95 7.86 7.86 7.87 7.82 7.80

82 8.12 8.04 8.05 7.99 8.01 8.04 7.99 7.95

85 7.90 7.85 8.04 8.09 7.97 7.93 7.91 7.91

88 7.66 7.68 7.90 7.78 7.77 7.75 7.73 7.75

92 7.83 7.88 8.04 7.91 7.90 7.95 7.95 7.91

97 7.63 7.68 7.79 7.72 7.73 7.78 7.74 7.74

99 7.60 7.70 7.81 7.77 7.79 7.83 7.79 7.80

102 7.67 7.68 7.78 7.66 7.67 7.66 7.63 7.63

106 7.84 7.81 7.92 7.75 7.85 7.83 7.85 7.79

111 7.76 7.85 7.93 7.90 7.86 7.75 7.76 7.76

112 7.77 -- 7.80 7.77 -- -- -- 7.70

113 -- 7.84 -- -- 7.92 7.93 7.86 --

‘--’ indicates measurement not collected. 
ABC Study No. 66086 

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Table L-3. Test Solution pH Values Measured in Each Replicate Test Chamber (continued)

pH

Level 1 (4.97 μg/L) Level 2 (10.3 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 8.26 8.26 8.24 8.25 8.22 8.23 8.22 8.21

6 8.00 8.07 8.08 8.07 8.02 8.06 7.99 7.99

8 8.02 7.98 7.98 8.00 8.01 8.00 7.92 8.00

12 8.09 8.08 8.07 8.15 8.07 8.05 7.98 8.10

15 8.14 8.13 8.13 8.11 8.17 8.15 8.20 8.18

19 8.11 8.06 8.06 7.99 8.10 8.04 8.06 8.01

21 8.01 8.03 7.99 8.01 8.02 8.04 8.11 8.00

27 8.06 8.07 8.04 8.01 8.05 8.08 8.14 8.01

29 8.13 8.15 8.09 8.06 8.13 8.11 8.14 8.08

32 7.93 7.99 7.91 7.83 7.94 7.98 7.98 7.93

36 7.98 7.99 7.94 7.94 7.93 7.94 7.96 7.91

41 7.98 8.13 8.02 7.97 8.00 8.02 7.92 7.85

43 8.00 8.24 8.11 8.08 8.04 8.11 8.01 8.06

46 8.09 8.12 8.05 8.04 8.07 8.13 7.95 8.02

50 8.19 8.20 8.10 8.15 8.14 8.11 8.12 8.16

53 8.13 8.21 8.19 8.20 8.14 8.16 8.17 8.23

57 8.23 8.21 8.20 8.20 8.14 8.25 8.27 8.28

60 7.84 7.83 7.84 7.74 7.87 7.89 7.82 7.85

64 7.87 8.09 7.98 7.98 7.95 7.96 7.95 7.97

67 7.83 7.86 7.80 7.79 7.82 7.84 7.81 7.77

71 8.17 8.14 8.17 7.96 8.10 8.08 8.07 8.14

75 7.97 7.95 8.01 7.96 7.97 7.96 8.00 8.00

78 7.84 7.86 7.92 7.86 7.85 7.83 7.90 7.89

82 7.94 7.94 7.99 7.90 7.98 7.94 7.95 8.00

85 7.83 7.92 7.97 7.87 7.89 7.87 7.87 7.79

88 7.67 7.69 7.89 7.75 7.74 7.74 7.72 7.74

92 7.86 7.90 7.98 7.89 7.92 7.93 7.86 7.97

97 7.73 7.78 7.76 7.75 7.73 7.73 7.76 7.74

99 7.79 7.76 7.80 7.77 7.80 7.81 7.82 7.80

102 7.59 7.69 7.66 7.69 7.68 7.62 7.64 7.63

106 7.79 7.76 7.71 7.85 7.73 7.78 7.75 7.75

111 7.81 7.80 7.81 7.77 7.74 7.80 7.66 7.70

112 7.75 -- -- 7.76 -- -- 7.62 7.78

113 -- 7.79 7.81 -- 7.79 7.84 -- --

‘--’ indicates measurement not collected. 
ABC Study No. 66086 

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Table L-3. Test Solution pH Values Measured in Each Replicate Test Chamber (continued) 

pH

Level 3 (19.5 μg/L) Level 4 (38.0 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 8.21 8.22 8.20 8.23 8.24 8.23 8.23 8.22

6 8.01 8.02 8.04 8.07 7.98 8.06 8.05 8.01

8 7.97 7.98 8.00 8.00 7.96 8.01 7.99 7.92

12 8.00 7.99 8.08 8.06 8.13 7.96 8.10 8.12

15 8.16 8.20 8.15 8.12 8.13 8.15 8.13 8.13

19 8.06 8.06 8.21 8.01 7.96 7.97 7.98 7.99

21 8.04 8.11 8.00 7.99 8.01 8.01 7.98 7.99

27 8.04 8.07 8.00 7.99 7.97 8.01 7.99 7.96

29 8.07 8.08 8.06 8.04 8.02 8.06 8.03 8.02

32 7.92 7.95 7.91 7.93 7.86 7.92 7.90 7.87

36 7.90 7.90 7.88 7.89 8.46 8.27 8.14 8.07

41 7.88 7.86 7.99 7.98 7.98 7.95 7.88 7.84

43 8.00 7.96 7.97 8.12 7.96 7.98 7.94 7.92

46 8.01 7.91 7.97 8.03 8.02 8.06 8.01 7.96

50 8.13 8.10 8.04 8.12 8.10 8.10 8.12 8.05

53 8.16 8.18 8.16 8.19 8.14 8.21 8.21 8.15

57 8.24 8.20 8.20 8.21 8.21 8.30 7.97 8.17

60 7.78 7.77 7.82 7.81 7.72 7.84 7.73 7.76

64 7.93 7.98 7.93 7.90 7.86 8.05 7.85 7.86

67 7.77 7.75 7.71 7.77 7.68 7.77 7.70 7.71

71 8.09 8.05 8.06 8.17 7.99 8.10 8.03 8.06

75 7.99 7.99 8.00 7.99 7.98 7.99 7.93 7.97

78 7.91 7.94 7.79 7.85 7.83 7.88 7.82 7.84

82 7.98 7.95 7.97 8.01 7.93 7.98 7.87 7.91

85 7.91 7.89 7.85 7.92 7.86 7.91 7.78 7.80

88 7.80 7.83 7.72 7.80 7.33 7.81 7.80 7.84

92 7.97 8.03 7.87 7.85 7.86 7.87 7.91 7.83

97 7.74 7.79 7.76 7.77 7.73 7.74 7.78 7.77

99 7.81 7.91 7.76 7.87 7.53 7.80 7.89 7.82

102 7.69 7.72 7.63 7.67 7.61 7.64 7.68 7.61

106 7.90 7.84 7.71 7.79 7.72 7.77 7.75 7.69

111 8.03 7.86 7.94 7.79 7.70 7.78 7.77 7.75

112 7.81 -- 7.76 -- -- -- 7.75 7.65

113 -- 7.83 -- 7.88 7.75 7.90 -- --

‘--’ indicates measurement not collected. 
ABC Study No. 66086 

Page 234 of 261 

APPENDIX M – PLASMA VITELLOGENIN DATA
ABC Study No. 66086 

Page 235 of 261 

Appendix M-1. Vitellogenin Data for NF Stage 62 Pooled Plasma

Treatment

(μg/L) REP Sample ID

Vtg Conc.

(mn/mL) Plate #

min. 

detection

max. 

detection 

Control A CA-180 0 3 28,442 2,318,572

B CB-180 0 3 28,442 2,318,572

C CC-90 8999.685 11 17,999 1,111,687

D CD-90 8999.685 11 17,999 1,111,687

E CE-90 9156.555 12 18,313 1,166,195

F CF-90 9156.555 12 18,313 1,166,195

G CG-180 16495.74 4 32,991 2,305,985

H CH-180 16495.74 4 32,991 2,305,985

4.97 A L1A-180 16495.74 4 32,991 2,305,985

B L1B-180 16495.74 4 32,991 2,305,985

C L1C-180 0 3 28,442 2,318,572

D L1D-180 0 3 28,442 2,318,572

10.3 A L2A-90 8999.685 11 17,999 1,111,687

B L2B-180 0 3 28,442 2,318,572

C L2C-180 16495.74 4 32,991 2,305,985

D L2D-180 0 3 28,442 2,318,572

19.5 A L3A-180 0 3 28,442 2,318,572

B L3B-180 16495.74 4 32,991 2,305,985

C L3C-180 0 3 28,442 2,318,572

D L3D-180 16495.74 4 32,991 2,305,985

38.0 A L4A-180 16495.74 4 32,991 2,305,985

B L4B-180 0 3 28,442 2,318,572

C L4C-180 16495.74 4 32,991 2,305,985

D L4D-90 9156.555 12 18,313 1,166,195
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Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination, Based on Phenotypic Sex 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

Control A CA1-T 1-90 1 9227.7 1-R1 18455.4 1196909

A CA3-T 3-90 3 9227.7 1-R1 18455.4 1196909

A CA4-T 4-180 4 17144.91 7 34289.82 2215452

A CA9-T 9-180 9 0 3 28442.16 2318572

A CA10-T 10-180 10 0 3 28442.16 2318572

B CB1-T 11-90 11 8999.685 11 17999.37 1111687

B CB4-T 14-90 14 0 2-R1 15544.53 1079139

B CB6-T 16-180 16 50113.582 7 34289.82 2215452

B CB7-T 17-180 17 17144.91 7 34289.82 2215452

B CB8-T 18-180 18 64891.308 7 34289.82 2215452

C CC1-T 21-180 21 16495.74 4 32991.48 2305985

C CC2-T 22-90 22 8999.685 11 17999.37 1111687

C CC3-T 23-90 23 9227.7 1-R1 18455.4 1196909

C CC5-T 25-90 25 9227.7 1-R1 18455.4 1196909

C CC9-T 29-180 29 14221.08 3 28442.16 2318572

D CD3-T 33-180 33 0 8 38758.5 2745362

D CD4-T 34-90 34 9156.555 12 18313.11 1166195

D CD5-T 35-90 35 0 2-R1 15544.53 1079139

D CD6-T 36-90 36 0 2-R1 15544.53 1079139

D CD7-T 37-90 37 0 2-R1 15544.53 1079139

D CD9-T 39-90 39 19905.85 11 17999.37 1111687
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Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

Control E CE2-T 42-180 42 16495.74 4 32991.48 2305984.5

E CE3-T 43-180 43 16495.74 4 32991.48 2305984.5

E CE4-T 44-180 44 16495.74 4 32991.48 2305984.5

E CE5-T 45-180 45 17144.91 7 34289.82 2215452.06

E CE6-T 46-180 46 17144.91 7 34289.82 2215452.06

E CE7-T 47-180 47 17144.91 7 34289.82 2215452.06

E CE8-T 48-180 48 0 8 38758.5 2745361.98

F CF3-T 53-90 53 9227.7 1-R1 18455.4 1196909.01

F CF5-T 55-90 55 9156.555 12 18313.11 1166195.25

F CF8-T 58-180 58 0 3 28442.16 2318571.9

F CF9-T 59-90 59 38540.873 12 18313.11 1166195.25

G CG1-T 61-90 61 8999.685 11 17999.37 1111687.29

G CG3-T 63-90 63 0 2-R1 15544.53 1079138.7

G CG5-T 65-90 65 42828.694 2-R1 15544.53 1079138.7

G CG6-T 66-180 66 19379.25 8 38758.5 2745361.98

G CG8-T 68-180 68 0 8 38758.5 2745361.98

G CG9-T 69-90 69 8999.685 11 17999.37 1111687.29

H CH1-T 71-180 71 16495.74 4 32991.48 2305984.5

H CH2-T 72-180 72 16495.74 4 32991.48 2305984.5

H CH4-T 74-90 74 9156.555 12 18313.11 1166195.25

H CH6-T 76-90 76 8999.685 11 17999.37 1111687.29

H CH8-T 78-90 78 9156.555 12 18313.11 1166195.25

H CH9-T 79-180 79 0 3 28442.16 2318571.9
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Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

4.97 A L1A2-T 82-90 82 8999.685 11 17999.37 1111687.29

A L1A4-T 84-90 84 9227.7 1-R1 18455.4 1196909.01

A L1A6-T 86-180 86 17144.91 7 34289.82 2215452.06

A L1A9-T 89-180 89 0 8 38758.5 2745361.98

B L1B3-T 93-90 93 9227.7 1-R1 18455.4 1196909.01

B L1B4-T 94-90 94 9227.7 1-R1 18455.4 1196909.01

B L1B5-T 95-90 95 8999.685 11 17999.37 1111687.29

B L1B8-T 98-180 98 0 3 28442.16 2318571.9

B L1B10-T 100-180 100 17144.91 7 34289.82 2215452.06

C L1C1-T 101-180 101 36151.126 7 34289.82 2215452.06

C L1C3-T 103-90 103 0 2-R1 15544.53 1079138.7

C L1C6-T 106-180 106 16495.74 4 32991.48 2305984.5

C L1C8-T 108-180 108 16495.74 4 32991.48 2305984.5

D L1D1-T 111-180 111 0 8 38758.5 2745361.98

D L1D3-T 113-90 113 0 2-R1 15544.53 1079138.7

D L1D5-T 115-90 115 9156.555 12 18313.11 1166195.25

D L1D7-T 117-180 117 0 3 28442.16 2318571.9

D L1D9-T 119-180 119 0 3 28442.16 2318571.9
ABC Study No. 66086 

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Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

10.3 A L2A2-T 122-90 122 8999.685 11 17999.37 1111687.29

A L2A3-T 123-90 123 9227.7 1-R1 18455.4 1196909.01

A L2A4-T 124-90 124 9227.7 1-R1 18455.4 1196909.01

A L2A5-T 125-90 125 168474.727 1-R1 18455.4 1196909.01

A L2A6-T 126-90 126 8999.685 11 17999.37 1111687.29

A L2A7-T 127-180 127 16495.74 4 32991.48 2305984.5

A L2A9-T 129-180 129 16495.74 4 32991.48 2305984.5

A L2A10-T 130-90 130 8999.685 11 17999.37 1111687.29

B L2B1-T 131-90 131 9156.555 12 18313.11 1166195.25

B L2B4-T 134-90 134 9227.7 1-R1 18455.4 1196909.01

B L2B5-T 135-90 135 9156.555 12 18313.11 1166195.25

B L2B8-T 138-180 138 0 3 28442.16 2318571.9

B L2B9-T 139-180 139 17144.91 7 34289.82 2215452.06

B L2B10-T 140-180 140 17144.91 7 34289.82 2215452.06

C L2C2-T 142-90 142 9156.555 12 18313.11 1166195.25

C L2C4-T 144-90 144 0 2-R1 15544.53 1079138.7

C L2C5-T 145-90 145 0 2-R1 15544.53 1079138.7

C L2C6-T 146-90 146 0 2-R1 15544.53 1079138.7

C L2C8-T 148-180 148 0 8 38758.5 2745361.98

C L2C9-T 149-180 149 0 8 38758.5 2745361.98

D L2D2-T 152-180 152 0 3 28442.16 2318571.9

D L2D10-T 160-180 160 17144.91 7 34289.82 2215452.06
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Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

19.5 A L3A4-T 164-90 164 9227.7 1-R1 18455.4 1196909.01

A L3A5-T 165-90 165 9156.555 12 18313.11 1166195.25

A L3A6-T 166-90 166 9156.555 12 18313.11 1166195.25

B L3B3-T 173-90 173 0 2-R1 15544.53 1079138.7

B L3B4-T 174-90 174 0 2-R1 15544.53 1079138.7

B L3B5-T 175-180 175 17144.91 7 34289.82 2215452.06

B L3B6-T 176-180 176 17144.91 7 34289.82 2215452.06

B L3B8-T 178-90 178 8999.685 11 17999.37 1111687.29

B L3B10-T 180-90 180 9156.555 12 18313.11 1166195.25

C L3C2-T 182-90 182 9227.7 1-R1 18455.4 1196909.01

C L3C3-T 183-90 183 9227.7 1-R1 18455.4 1196909.01

C L3C4-T 184-180 184 17144.91 7 34289.82 2215452.06

C L3C7-T 187-180 187 0 8 38758.5 2745361.98

C L3C8-T 188-180 188 0 8 38758.5 2745361.98

C L3C9-T 189-180 189 0 8 38758.5 2745361.98

C L3C10-T 190-90 190 8999.685 11 17999.37 1111687.29

D L3D3-T 193-90 193 0 2-R1 15544.53 1079138.7

D L3D4-T 194-90 194 0 2-R1 15544.53 1079138.7

D L3D6-T 196-180 196 0 8 38758.5 2745361.98

D L3D8-T 198-90 198 8999.685 11 17999.37 1111687.29

D L3D9-T 199-90 199 8999.685 11 17999.37 1111687.29
ABC Study No. 66086 

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Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

38.0 A L4A1-T 201-180 201 16495.74 4 32991.48 2305984.5

A L4A4-T 204-180 204 17144.91 7 34289.82 2215452.06

A L4A5-T 205-180 205 17144.91 7 34289.82 2215452.06

A L4A6-T 206-90 206 9156.555 12 18313.11 1166195.25

A L4A7-T 207-90 207 8999.685 11 17999.37 1111687.29

A L4A9-T 209-90 209 0 2-R1 15544.53 1079138.7

B L4B1-T 211-180 211 0 8 38758.5 2745361.98

B L4B2-T 212-180 212 19379.25 8 38758.5 2745361.98

B L4B3-T 213-180 213 19379.25 8 38758.5 2745361.98

B L4B5-T 215-90 215 9156.555 12 18313.11 1166195.25

B L4B9-T 219-180 219 0 8 38758.5 2745361.98

B L4B10-T 220-180 220 19379.25 8 38758.5 2745361.98

C L4C2-T 222-90 222 27180.931 12 18313.11 1166195.25

C L4C3-T 223-90 223 9156.555 12 18313.11 1166195.25

C L4C4-T 224-90 224 9156.555 12 18313.11 1166195.25

C L4C5-T 225-90 225 9227.7 1-R1 18455.4 1196909.01

C L4C7-T 227-90 227 29156.71 1-R1 18455.4 1196909.01

C L4C8-T 228-180 228 0 8 38758.5 2745361.98

D L4D2-T 232-180 232 74942.043 4 32991.48 2305984.5

D L4D6-T 236-180 236 17144.91 7 34289.82 2215452.06

D L4D8-T 238-90 238 9227.7 1-R1 18455.4 1196909.01

D L4D9-T 239-90 239 9227.7 1-R1 18455.4 1196909.01
ABC Study No. 66086 

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Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination, Based on Phenotypic Sex 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

Control A CA2-T 2-90 2 9227.7 1-R1 18455.4 1196909.01

A CA5-T 5-180 5 17144.91 7 34289.82 2215452.06

A CA6-T 6-180 6 17144.91 7 34289.82 2215452.06

A CA8-T 8-180 8 0 3 28442.16 2318571.9

B CB2-T 12-90 12 8999.685 11 17999.37 1111687.29

B CB3-T 13-90 13 7772.265 2-R1 15544.53 1079138.7

B CB5-T 15-90 15 0 2-R1 15544.53 1079138.7

B CB9-T 19-180 19 40189.923 4 32991.48 2305984.5

B CB10-T 20-180 20 16495.74 4 32991.48 2305984.5

C CC4-T 24-90 24 9227.7 1-R1 18455.4 1196909.01

C CC6-T 26-90 26 8999.685 11 17999.37 1111687.29

C CC7-T 27-180 27 0 3 28442.16 2318571.9

C CC8-T 28-180 28 0 3 28442.16 2318571.9

D CD1-T 31-180 31 0 8 38758.5 2745361.98

D CD2-T 32-180 32 0 8 38758.5 2745361.98

D CD8-T 38-90 38 9156.555 12 18313.11 1166195.25
ABC Study No. 66086 

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Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

Control E CE1-T 41-90 41 8999.685 11 17999.37 1111687.29

E CE9-T 49-180 49 0 8 38758.5 2745361.98

E CE10-T 50-180 50 0 8 38758.5 2745361.98

F CF1-T 51-90 51 9227.7 1-R1 18455.4 1196909.01

F CF2-T 52-90 52 9227.7 1-R1 18455.4 1196909.01

F CF4-T 54-90 54 8999.685 11 17999.37 1111687.29

F CF6-T 56-180 56 0 3 28442.16 2318571.9

F CF7-T 57-180 57 0 3 28442.16 2318571.9

F CF10-T 60-90 60 9156.555 12 18313.11 1166195.25

G CG2-T 62-90 62 8999.685 11 17999.37 1111687.29

G CG4-T 64-90 64 0 2-R1 15544.53 1079138.7

G CG7-T 67-180 67 0 8 38758.5 2745361.98

G CG10-T 70-180 70 16495.74 4 32991.48 2305984.5

H CH3-T 73-90 73 9156.555 12 18313.11 1166195.25

H CH5-T 75-90 75 8999.685 11 17999.37 1111687.29

H CH7-T 77-90 77 8999.685 11 17999.37 1111687.29

H CH10-T 80-180 80 14221.08 3 28442.16 2318571.9
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Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

4.97 A L1A1-T 81-180 81 0 3 28442.16 2318571.9

A L1A3-T 83-90 83 9227.7 1-R1 18455.4 1196909.01

A L1A5-T 85-90 85 9227.7 1-R1 18455.4 1196909.01

A L1A7-T 87-180 87 17144.91 7 34289.82 2215452.06

A L1A8-T 88-180 88 17144.91 7 34289.82 2215452.06

A L1A10-T 90-180 90 0 8 38758.5 2745361.98

B L1B1-T 91-180 91 0 8 38758.5 2745361.98

B L1B2-T 92-90 92 9227.7 1-R1 18455.4 1196909.01

B L1B6-T 96-90 96 8999.685 11 17999.37 1111687.29

B L1B7-T 97-180 97 0 3 28442.16 2318571.9

B L1B9-T 99-180 99 0 3 28442.16 2318571.9

C L1C2-T 102-180 102 17144.91 7 34289.82 2215452.06

C L1C4-T 104-90 104 0 2-R1 15544.53 1079138.7

C L1C5-T 105-90 105 0 2-R1 15544.53 1079138.7

C L1C7-T 107-180 107 16495.74 4 32991.48 2305984.5

C L1C9-T 109-180 109 0 8 38758.5 2745361.98

C L1C10-T 110-180 110 0 8 38758.5 2745361.98

D L1D2-T 112-90 112 7772.265 2-R1 15544.53 1079138.7

D L1D4-T 114-90 114 0 2-R1 15544.53 1079138.7

D L1D6-T 116-90 116 9156.555 12 18313.11 1166195.25

D L1D8-T 118-180 118 0 3 28442.16 2318571.9
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Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

10.3 A L2A1-T 121-90 121 9156.555 12 18313.11 1166195.25

A L2A8-T 128-180 128 45364.625 4 32991.48 2305984.5

B L2B2-T 132-90 132 9227.7 1-R1 18455.4 1196909.01

B L2B3-T 133-90 133 9227.7 1-R1 18455.4 1196909.01

B L2B6-T 136-180 136 0 3 28442.16 2318571.9

B L2B7-T 137-180 137 0 3 28442.16 2318571.9

C L2C1-T 141-180 141 17144.91 7 34289.82 2215452.06

C L2C3-T 143-90 143 40739.093 11 17999.37 1111687.29

C L2C7-T 147-180 147 0 8 38758.5 2745361.98

C L2C10-T 150-90 150 8999.685 11 17999.37 1111687.29

D L2D1-T 151-180 151 0 3 28442.16 2318571.9

D L2D3-T 153-180 153 0 3 28442.16 2318571.9

D L2D4-T 154-90 154 8999.685 11 17999.37 1111687.29

D L2D5-T 155-90 155 9156.555 12 18313.11 1166195.25

D L2D6-T 156-90 156 0 2-R1 15544.53 1079138.7

D L2D7-T 157-90 157 0 2-R1 15544.53 1079138.7

D L2D8-T 158-90 158 0 2-R1 15544.53 1079138.7

D L2D9-T 159-180 159 17144.91 7 34289.82 2215452.06
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Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

19.5 A L3A1-T 161-180 161 17144.91 7 34289.82 2215452.06

A L3A2-T 162-90 162 9227.7 1-R1 18455.4 1196909.01

A L3A3-T 163-90 163 9227.7 1-R1 18455.4 1196909.01

A L3A8-T 168-180 168 16495.74 4 32991.48 2305984.5

A L3A9-T 169-180 169 16495.74 4 32991.48 2305984.5

A L3A10-T 170-90 170 8999.685 11 17999.37 1111687.29

B L3B1-T 171-90 171 8999.685 11 17999.37 1111687.29

B L3B2-T 172-90 172 0 2-R1 15544.53 1079138.7

B L3B7-T 177-180 177 17144.91 7 34289.82 2215452.06

B L3B9-T 179-90 179 9156.555 12 18313.11 1166195.25

C L3C1-T 181-90 181 9227.7 1-R1 18455.4 1196909.01

C L3C5-T 185-180 185 17144.91 7 34289.82 2215452.06

C L3C6-T 186-180 186 17144.91 7 34289.82 2215452.06

D L3D1-T 191-90 191 9156.555 12 18313.11 1166195.25

D L3D2-T 192-90 192 0 2-R1 15544.53 1079138.7

D L3D5-T 195-180 195 19379.25 8 38758.5 2745361.98

D L3D7-T 197-180 197 0 8 38758.5 2745361.98

D L3D10-T 200-180 200 77088.478 4 32991.48 2305984.5
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Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID

Vtg Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

38.0 A L4A2-T 202-180 202 16495.74 4 32991.48 2305984.5

A L4A3-T 203-180 203 17144.91 7 34289.82 2215452.06

A L4A8-T 208-90 208 0 2-R1 15544.53 1079138.7

A L4A10-T 210-90 210 0 2-R1 15544.53 1079138.7

B L4B4-T 214-90 214 9156.555 12 18313.11 1166195.25

B L4B6-T 216-90 216 45506.302 2-R1 15544.53 1079138.7

B L4B7-T 217-90 217 0 2-R1 15544.53 1079138.7

B L4B8-T 218-90 218 0 2-R1 15544.53 1079138.7

C L4C1-T 221-180 221 0 8 38758.5 2745361.98

C L4C6-T 226-90 226 9227.7 1-R1 18455.4 1196909.01

C L4C9-T 229-180 229 0 8 38758.5 2745361.98

C L4C10-T 230-180 230 0 8 38758.5 2745361.98

D L4D1-T 231-180 231 16495.74 4 32991.48 2305984.5

D L4D3-T 233-180 233 16495.74 4 32991.48 2305984.5

D L4D4-T 234-180 234 107361.268 7 34289.82 2215452.06

D L4D5-T 235-180 235 17144.91 7 34289.82 2215452.06

D L4D7-T 237-90 237 32859.473 12 18313.11 1166195.25

D L4D10-T 240-90 240 9227.7 1-R1 18455.4 1196909.01
ABC Study No. 66086 

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APPENDIX N – PLASMA T4 DATA
ABC Study No. 66086 

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Appendix N-1. T4 Data for NF Stage 62 Pooled Plasma 

Treatment

(μg/L) REP Sample ID

T4 Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

Control A CA-30 303.46 3 16 7,963

B CB-30 204.6 3 16 7,963

C CC-30 173.95 7-R1 15 7,665

D CD-30 109.91 7-R1 15 7,665

E CE-30 241.35 8-R1 12 6,961

F CF-30 320.03 8-R1 12 6,961

G CG-30 182.48 4 13 7,496

H CH-30 283.01 4 13 7,496

4.97 A L1A-30 149.9 4 13 7,496

B L1B-30 234.93 4 13 7,496

C L1C-30 445.28 3 16 7,963

D L1D-30 303.38 3 16 7,963

10.3 A L2A-30 333.14 7-R1 15 7,665

B L2B-30 208.8 3 16 7,963

C L2C-30 122.9 4 13 7,496

D L2D-30 145.12 3 16 7,963

4

19.5 A L3A-30 314.03 3 16 7,963

B L3B-30 185.12 4 13 7,496

C L3C-30 505.01 3 16 7,963

D L3D-30 117.37 4 13 7,496

38.0 A L4A-30 222.3 4 13 7,496

B L4B-30 410.92 3 16 7,963

C L4C-30 266.77 4 13 7,496

D L4D-30 246.56 8-R1 12 6,961
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Appendix N-2. T4 Data for Juvenile Males at Study Termination, Based on Phenotypic Sex 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

Control A CA1-T 1-30 1 1 65.89 15.3 4228.5

A CA3-T 3-30 3 1 35.39 15.3 4228.5

A CA4-T 4-30 4 5 68.5 17.1 6648.3

A CA7-T 7-30 7 7-R1 169.3 15 7665.3

A CA9-T 9-30 9 3 70.01 15.9 7963.2

A CA10-T 10-30 10 3 242.23 15.9 7963.2

B CB1-T 11-30 11 7-R1 24.79 15 7665.3

B CB4-T 14-30 14 2 25.67 18.6 9006.9

B CB6-T 16-30 16 5 66.38 17.1 6648.3

B CB7-T 17-30 17 5 29.63 17.1 6648.3

B CB8-T 18-30 18 5 26.25 17.1 6648.3

C CC1-T 21-30 21 4 70.45 13.2 7496.1

C CC2-T 22-30 22 7-R1 67.02 15 7665.3

C CC3-T 23-30 23 1 46.53 15.3 4228.5

C CC5-T 25-30 25 1 172.67 15.3 4228.5

C CC9-T 29-30 29 3 58.77 15.9 7963.2

D CD3-T 33-30 33 6 & 6-R1 25.36 13.8 8004

D CD4-T 34-30 34 8-R1 44.84 12 6960.6

D CD5-T 35-30 35 2 49.65 18.6 9006.9

D CD6-T 36-30 36 2 23.88 18.6 9006.9

D CD7-T 37-30 37 2 37.32 18.6 9006.9

D CD9-T 39-30 39 7-R1 99.43 15 7665.3
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Appendix N-2. T4 Data for Juvenile Males at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

Control E CE2-T 42-30 42 4 34.77 13.2 7496.1

E CE3-T 43-30 43 4 76.21 13.2 7496.1

E CE4-T 44-30 44 4 59.93 13.2 7496.1

E CE5-T 45-30 45 5 31.45 17.1 6648.3

E CE6-T 46-30 46 5 38.91 17.1 6648.3

E CE7-T 47-30 47 5 41.36 17.1 6648.3

E CE8-T 48-30 48 6 & 6-R1 16.545 13.8 8004

F CF3-T 53-30 53 1 24.95 15.3 4228.5

F CF5-T 55-30 55 8-R1 78.15 12 6960.6

F CF8-T 58-30 58 3 86.42 15.9 7963.2

F CF9-T 59-30 59 8-R1 57.08 12 6960.6

G CG1-T 61-30 61 7-R1 30.11 15 7665.3

G CG3-T 63-30 63 2 43.32 18.6 9006.9

G CG5-T 65-30 65 2 91.87 18.6 9006.9

G CG6-T 66-15 66 9 264.94 16.65 3474.15

G CG8-T 68-30 68 6 & 6-R1 129.085 13.8 8004

G CG9-T 69-30 69 7-R1 85.45 15 7665.3

H CH1-T 71-30 71 4 101.97 13.2 7496.1

H CH2-T 72-30 72 4 42.32 13.2 7496.1

H CH4-T 74-30 74 8-R1 59.77 12 6960.6

H CH6-T 76-30 76 7-R1 20.35 15 7665.3

H CH8-T 78-30 78 8-R1 62.57 12 6960.6

H CH9-T 79-30 79 3 45.85 15.9 7963.2
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Appendix N-2. T4 Data for Juvenile Males at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

4.97 A L1A2-T 82-30 82 7-R1 185.63 15 7665.3

A L1A4-T 84-30 84 1 82.9 15.3 4228.5

A L1A6-T 86-30 86 5 31.78 17.1 6648.3

A L1A9-T 89-30 89 6 & 6-R1 20.47 13.8 8004

B L1B3-T 93-30 93 1 52.1 15.3 4228.5

B L1B4-T 94-30 94 1 25.57 15.3 4228.5

B L1B5-T 95-30 95 7-R1 47.06 15 7665.3

B L1B8-T 98-30 98 3 58.44 15.9 7963.2

B L1B10-T 100-30 100 5 8.55 17.1 6648.3

C L1C1-T 101-30 101 5 30.29 17.1 6648.3

C L1C3-T 103-30 103 2 26.08 18.6 9006.9

C L1C6-T 106-30 106 4 97.24 13.2 7496.1

C L1C8-T 108-30 108 4 69.73 13.2 7496.1

D L1D1-T 111-15 111 9 56.49 16.65 3474.15

D L1D3-T 113-30 113 2 78.36 18.6 9006.9

D L1D5-T 115-30 115 8-R1 42.31 12 6960.6

D L1D7-T 117-30 117 3 165.72 15.9 7963.2

D L1D9-T 119-30 119 3 28.14 15.9 7963.2
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Appendix N-2. T4 Data for Juvenile Males at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

10.3 A L2A2-T 122-30 122 7-R1 31.76 15 7665.3

A L2A3-T 123-30 123 1 27.29 15.3 4228.5

A L2A4-T 124-30 124 1 58.71 15.3 4228.5

A L2A5-T 125-30 125 1 132.74 15.3 4228.5

A L2A6-T 126-30 126 7-R1 24.79 15 7665.3

A L2A7-T 127-30 127 4 39.29 13.2 7496.1

A L2A9-T 129-30 129 4 50.09 13.2 7496.1

A L2A10-T 130-30 130 7-R1 54.72 15 7665.3

B L2B1-T 131-30 131 8-R1 42.86 12 6960.6

B L2B4-T 134-30 134 1 76.3 15.3 4228.5

B L2B5-T 135-30 135 8-R1 94.1 12 6960.6

B L2B8-T 138-30 138 3 128.22 15.9 7963.2

B L2B9-T 139-30 139 5 19.55 17.1 6648.3

B L2B10-T 140-30 140 5 55.06 17.1 6648.3

C L2C2-T 142-30 142 8-R1 58.3 12 6960.6

C L2C4-T 144-30 144 2 63.12 18.6 9006.9

C L2C5-T 145-30 145 2 48.52 18.6 9006.9

C L2C6-T 146-30 146 2 103.53 18.6 9006.9

C L2C8-T 148-30 148 6 & 6-R1 29.37 13.8 8004

C L2C9-T 149-15 149 9 79.97 16.65 3474.15

D L2D2-T 152-30 152 3 125.32 15.9 7963.2

D L2D10-T 160-30 160 5 29.81 17.1 6648.3
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Appendix N-2. T4 Data for Juvenile Males at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

19.5 A L3A4-T 164-30 164 1 54.03 15.3 4228.5

A L3A5-T 165-30 165 8-R1 37.3 12 6960.6

A L3A6-T 166-30 166 8-R1 48.21 12 6960.6

A L3A7-T 167-30 167 4 56.04 13.2 7496.1

B L3B3-T 173-30 173 2 19.8 18.6 9006.9

B L3B4-T 174-30 174 2 226.14 18.6 9006.9

B L3B5-T 175-30 175 5 20.68 17.1 6648.3

B L3B6-T 176-30 176 5 76.35 17.1 6648.3

B L3B8-T 178-30 178 7-R1 47.88 15 7665.3

B L3B10-T 180-30 180 8-R1 199.9 12 6960.6

C L3C2-T 182-30 182 1 23.94 15.3 4228.5

C L3C3-T 183-30 183 1 94.87 15.3 4228.5

C L3C4-T 184-30 184 5 40.97 17.1 6648.3

C L3C7-T 187-30 187 6 & 6-R1 102.745 13.8 8004

C L3C8-T 188-30 188 6 & 6-R1 30.19 13.8 8004

C L3C9-T 189-30 189 6 & 6-R1 42.23 13.8 8004

C L3C10-T 190-30 190 7-R1 49.9 15 7665.3

D L3D3-T 193-15 193 9 25 16.65 3474.15

D L3D4-T 194-30 194 2 63.87 18.6 9006.9

D L3D6-T 196-15 196 9 157.31 16.65 3474.15

D L3D8-T 198-30 198 7-R1 38.93 15 7665.3

D L3D9-T 199-30 199 7-R1 108.09 15 7665.3
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Appendix N-2. T4 Data for Juvenile Males at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

38.0 A L4A1-T 201-30 201 4 63.43 13.2 7496.1

A L4A4-T 204-15 204 9 8.325 16.65 3474.15

A L4A5-T 205-30 205 5 46.06 17.1 6648.3

A L4A6-T 206-30 206 8-R1 47.57 12 6960.6

A L4A7-T 207-30 207 8-R1 35.87 12 6960.6

A L4A9-T 209-30 209 2 41.76 18.6 9006.9

B L4B1-T 211-30 211 6 & 6-R1 156.54 13.8 8004

B L4B2-T 212-15 212 9 88.12 16.65 3474.15

B L4B3-T 213-30 213 6 & 6-R1 34.295 13.8 8004

B L4B5-T 215-30 215 8-R1 49.92 12 6960.6

B L4B9-T 219-15 219 9 78.38 16.65 3474.15

B L4B10-T 220-15 220 9 8.325 16.65 3474.15

C L4C2-T 222-30 222 8-R1 43.81 12 6960.6

C L4C3-T 223-30 223 8-R1 83.18 12 6960.6

C L4C4-T 224-30 224 8-R1 90.75 12 6960.6

C L4C5-T 225-30 225 1 47.14 15.3 4228.5

C L4C7-T 227-30 227 1 50.88 15.3 4228.5

C L4C8-T 228-30 228 6 & 6-R1 16.56 13.8 8004

D L4D2-T 232-15 232 9 47.7 16.65 3474.15

D L4D6-T 236-30 236 9 132.76 33.3 6948.3

D L4D8-T 238-15 238 9 8.325 16.65 3474.15

D L4D9-T 239-30 239 1 36.36 15.3 4228.5
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Appendix N-3. T4 Data for Juvenile Females at Study Termination, Based on Phenotypic Sex 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

Control A CA2-T 2-30 2 1 58.99 15.3 4228.5

A CA5-T 5-30 5 5 66.81 17.1 6648.3

A CA6-T 6-30 6 5 48.16 17.1 6648.3

A CA8-T 8-30 8 9 147.02 33.3 6948.3

B CB2-T 12-30 12 7-R1 58.42 15 7665.3

B CB3-T 13-30 13 2 126.17 18.6 9006.9

B CB5-T 15-30 15 2 50.23 18.6 9006.9

B CB9-T 19-30 19 9 289.28 33.3 6948.3

B CB10-T 20-30 20 4 99.13 13.2 7496.1

C CC4-T 24-30 24 1 69.26 15.3 4228.5

C CC6-T 26-30 26 7-R1 46.9 15 7665.3

C CC7-T 27-30 27 3 34.39 15.9 7963.2

C CC8-T 28-30 28 3 116.12 15.9 7963.2

D CD1-T 31-30 31 9 93.38 33.3 6948.3

D CD2-T 32-15 32 9 110.57 16.65 3474.15

D CD8-T 38-30 38 8-R1 45.88 12 6960.6
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Appendix N-3. T4 Data for Juvenile Females at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

Control E CE1-T 41-30 41 7-R1 42.2 15 7665.3

E CE9-T 49-15 49 9 38.94 16.65 3474.15

E CE10-T 50-30 50 6 & 6-R1 70.01 13.8 8004

F CF1-T 51-30 51 1 44.18 15.3 4228.5

F CF2-T 52-30 52 1 43.02 15.3 4228.5

F CF4-T 54-30 54 7-R1 140.62 15 7665.3

F CF6-T 56-30 56 3 46.73 15.9 7963.2

F CF7-T 57-30 57 3 80.16 15.9 7963.2

F CF10-T 60-30 60 8-R1 46.6 12 6960.6

G CG2-T 62-30 62 7-R1 35.19 15 7665.3

G CG4-T 64-30 64 2 72.39 18.6 9006.9

G CG7-T 67-15 67 9 90.73 16.65 3474.15

G CG10-T 70-30 70 4 68.47 13.2 7496.1

H CH3-T 73-30 73 8-R1 41.86 12 6960.6

H CH5-T 75-30 75 7-R1 34.85 15 7665.3

H CH7-T 77-30 77 7-R1 25.8 15 7665.3

H CH10-T 80-30 80 3 50.76 15.9 7963.2
ABC Study No. 66086 

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Appendix N-3. T4 Data for Juvenile Females at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

4.97 A L1A1-T 81-30 81 3 111.96 15.9 7963.2

A L1A3-T 83-30 83 1 32.01 15.3 4228.5

A L1A5-T 85-30 85 1 38.04 15.3 4228.5

A L1A7-T 87-30 87 5 40.11 17.1 6648.3

A L1A8-T 88-30 88 5 68.36 17.1 6648.3

A L1A10-T 90-30 90 6 & 6-R1 63.735 13.8 8004

B L1B1-T 91-15 91 9 56.64 16.65 3474.15

B L1B2-T 92-30 92 1 84.12 15.3 4228.5

B L1B6-T 96-30 96 7-R1 129.12 15 7665.3

B L1B7-T 97-30 97 3 143.09 15.9 7963.2

B L1B9-T 99-15 99 9 8.325 16.65 3474.15

C L1C2-T 102-30 102 5 94.54 17.1 6648.3

C L1C4-T 104-30 104 2 50.42 18.6 9006.9

C L1C5-T 105-30 105 2 74.71 18.6 9006.9

C L1C7-T 107-30 107 4 24.87 13.2 7496.1

C L1C9-T 109-30 109 6 & 6-R1 73.185 13.8 8004

C L1C10-T 110-30 110 6 & 6-R1 64.5 13.8 8004

D L1D2-T 112-30 112 2 101.96 18.6 9006.9

D L1D4-T 114-30 114 2 119.87 18.6 9006.9

D L1D6-T 116-30 116 8-R1 22.36 12 6960.6

D L1D8-T 118-30 118 3 99 15.9 7963.2
ABC Study No. 66086 

Page 259 of 261 

Appendix N-3. T4 Data for Juvenile Females at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

10.3 A L2A1-T 121-30 121 8-R1 80.87 12 6960.6

A L2A8-T 128-30 128 4 85.93 13.2 7496.1

B L2B2-T 132-30 132 1 44.44 15.3 4228.5

B L2B3-T 133-15 133 9 30.49 16.65 3474.15

B L2B6-T 136-30 136 3 25.26 15.9 7963.2

B L2B7-T 137-30 137 3 137.9 15.9 7963.2

C L2C1-T 141-30 141 5 93.78 17.1 6648.3

C L2C3-T 143-30 143 7-R1 148.32 15 7665.3

C L2C7-T 147-15 147 9 66.65 16.65 3474.15

C L2C10-T 150-30 150 7-R1 17.09 15 7665.3

D L2D1-T 151-30 151 3 231.11 15.9 7963.2

D L2D3-T 153-30 153 3 88.86 15.9 7963.2

D L2D4-T 154-30 154 7-R1 28.97 15 7665.3

D L2D5-T 155-30 155 8-R1 6 12 6960.6

D L2D6-T 156-30 156 2 22.91 18.6 9006.9

D L2D7-T 157-30 157 2 80.08 18.6 9006.9

D L2D8-T 158-30 158 2 88.16 18.6 9006.9

D L2D9-T 159-30 159 5 57.71 17.1 6648.3
ABC Study No. 66086 

Page 260 of 261 

Appendix N-3. T4 Data for Juvenile Females at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc. 

(ng/mL)

min. 

detection

max. 

detection 

19.5 A L3A1-T 161-30 161 5 62.82 17.1 6648.3

A L3A2-T 162-15 162 9 30.12 16.65 3474.15

A L3A3-T 163-15 163 9 8.325 16.65 3474.15

A L3A8-T 168-30 168 4 57.05 13.2 7496.1

A L3A9-T 169-30 169 4 33.98 13.2 7496.1

A L3A10-T 170-30 170 7-R1 28.04 15 7665.3

B L3B1-T 171-30 171 7-R1 95.73 15 7665.3

B L3B2-T 172-30 172 2 42.59 18.6 9006.9

B L3B7-T 177-30 177 5 18.47 17.1 6648.3

B L3B9-T 179-30 179 8-R1 6 12 6960.6

C L3C1-T 181-15 181 9 8.325 16.65 3474.15

C L3C5-T 185-30 185 5 35.01 17.1 6648.3

C L3C6-T 186-30 186 5 70.7 17.1 6648.3

D L3D1-T 191-30 191 8-R1 45.7 12 6960.6

D L3D2-T 192-30 192 2 69.3 18.6 9006.9

D L3D5-T 195-30 195 6 & 6-R1 45.625 13.8 8004

D L3D7-T 197-15 197 9 97.94 16.65 3474.15

D L3D10-T 200-30 200 4 109 13.2 7496.1
ABC Study No. 66086 

Page 261 of 261 

Appendix N-3. T4 Data for Juvenile Females at Study Termination, Based on Phenotypic Sex (continued) 

Treatment

(μg/L) REP Frog Code

Sample

ID Plasma Vial ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

38.0 A L4A2-T 202-30 202 4 38.24 13.2 7496.1

A L4A3-T 203-30 203 5 183.11 17.1 6648.3

A L4A8-T 208-30 208 2 39.81 18.6 9006.9

A L4A10-T 210-30 210 2 63.67 18.6 9006.9

B L4B4-T 214-30 214 8-R1 73.37 12 6960.6

B L4B6-T 216-30 216 2 134.75 18.6 9006.9

B L4B7-T 217-30 217 2 47.59 18.6 9006.9

B L4B8-T 218-30 218 9 44.81 33.3 6948.3

C L4C1-T 221-15 221 9 42.06 16.65 3474.15

C L4C6-T 226-30 226 1 47.34 15.3 4228.5

C L4C9-T 229-30 229 6 & 6-R1 32.43 13.8 8004

C L4C10-T 230-15 230 9 145.98 16.65 3474.15

D L4D1-T 231-30 231 4 72.78 13.2 7496.1

D L4D3-T 233-30 233 4 34.65 13.2 7496.1

D L4D4-T 234-15 234 9 24.16 16.65 3474.15

D L4D5-T 235-30 235 5 77.26 17.1 6648.3

D L4D7-T 237-30 237 8-R1 41.08 12 6960.6

D L4D10-T 240-30 240 9 100.5 33.3 6948.3