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STUDY TITLE

Prochloraz: Larval Amphibian Growth and Development Assay Using Xenopus laevis 

AUTHOR

Stephen L. Hicks

STUDY INITIATION DATE

20 December 2010 

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. 66085




ABC Study No. 66085 

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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 ..................................................................................................................9

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

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

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

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

2.2 Test Species ...........................................................................................................13

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

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

2.4.1 Stock Solution Preparations .......................................................................14

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

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

2.4.3.1 Test Initiation ..............................................................................16

2.4.3.2 Larval Sub-Sampling ..................................................................16

2.4.3.3 End of the Larval Exposure ........................................................17

2.4.3.4 Juvenile Sampling .......................................................................17

2.4.3.5 Water Chemistry .........................................................................18

2.4.3.6 Feeding ........................................................................................18

2.4.3.7 Vitellogenin Analysis..................................................................19

2.4.3.8 Thyroxine (T4) Analysis .............................................................20

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 ................................................................................21

2.5.4 Calculations for Prochloraz........................................................................22

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

2.7 Statistical Analyses ................................................................................................23

3.0 RESULTS AND DISCUSSION ........................................................................................24

3.1 Method Check ........................................................................................................24

3.2 Analytical Results ..................................................................................................25

3.3 Biological Results ..................................................................................................26
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3.4 Genotypic/Phenotypic Sex Ratios..........................................................................38

3.5 Water Quality .........................................................................................................38

3.6 Vitellogenin Results ...............................................................................................39

3.7 Plasma Thyroxine (T4) Results .............................................................................41

3.8 Validity and Performance Criteria .........................................................................44

4.0 CONCLUSIONS................................................................................................................44

5.0 RECORDS .........................................................................................................................45

LIST OF TABLES

Page

Table 3.1.1. Measured concentrations of prochloraz during the method check in 

freshwater. ..............................................................................................................25

Table 3.2.1. Summary of analytical results for the Xenopus laevis prochloraz in-life 

exposure. ................................................................................................................26

Table 3.3.1. Mean (and standard deviation) time to NF stage 62 during the Xenopus 

laevis prochloraz exposure. ....................................................................................27

Table 3.3.2. Mean (and standard deviation) whole body weight and snout-to-vent length 

for NF stage 62 larvae during the Xenopus laevis prochloraz exposure. ...............29

Table 3.3.3. Mean (and standard deviation) whole body weight and snout-to-vent length 

at the NF stage 66 cull of the Xenopus laevis prochloraz exposure. ......................31

Table 3.3.4. Mean (and standard deviation) whole body weight and snout-to-vent length 

at the termination of the Xenopus laevis prochloraz exposure. .............................32

Table 3.3.5. Mean (standard deviation) proportion of female and male frogs and total 

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

prochloraz exposure. ..............................................................................................34

Table 3.3.6. Proportion of total frogs within each treatment having nuptial pads at the 

termination of the Xenopus laevis prochloraz exposure. .......................................36

Table 3.3.7. Mean (and standard deviation) liver weight:whole body weight for male and 

female frogs at the termination of the Xenopus laevis prochloraz exposure. ........36

Table 3.4.1. Frog phenotypic and genotypic sex ratios at the termination of the Xenopus 

laevis prochloraz exposure. ....................................................................................38

Table 3.5.1. Ranges for Water Quality Measurements During the 110-Day Exposure with 

the African Clawed Frog, Xenopus laevis, Exposed to Prochloraz .......................39

Table 3.6.1. Mean (and standard deviation) vitellogenin concentration for frogs at the 

termination of the Xenopus laevis prochloraz exposure. .......................................40

Table 3.7.1. Mean (and standard deviation) plasma thyroxine (T4) for NF Stage 62 frogs 

during the Xenopus laevis prochloraz exposure. ....................................................42

Table 3.7.2. Mean (and standard deviation) plasma thyroxine (T4) for frogs at the 

termination of the Xenopus laevis prochloraz exposure. .......................................42
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Table 4.0.1. Calculated No-Observable-Effect Concentration and Lowest-ObservableEffect Concentration based on nominal prochloraz concentrations for 

parameter measured for Xenopus laevis exposed to prochloraz. ...........................45

LIST OF FIGURES

Page

Figure 3.3.1. Boxplot of time to NF stage 62 during the Xenopus laevis prochloraz 

exposure based on the replicate analysis. The box represents the 

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

median. ...................................................................................................................28

Figure 3.3.2. Boxplot of whole body weight for NF stage 62 larvae during the Xenopus 

laevis prochloraz exposure. The box represents the interquartile range, the 

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

Figure 3.3.3. Boxplot of snout-to-vent length for NF stage 62 larvae during the Xenopus 

laevis prochloraz exposure. The box represents the interquartile range, the 

circle is the mean, and the horizontal line is the median. ......................................30

Figure 3.3.4. Boxplot of whole body weight for frogs at the NF stage 66 cull of the 

Xenopus laevis prochloraz exposure. The box represents the interquartile 

range, the circle is the mean, and the horizontal line is the median. .....................31

Figure 3.3.5. Boxplot of snout-to-vent length for frogs at the NF stage 66 cull of the 

Xenopus laevis prochloraz exposure. The box represents the interquartile 

range, the circle is the mean, and the horizontal line is the median. .....................32

Figure 3.3.6. Boxplot of whole body weight for frogs at the termination of the Xenopus 

laevis prochloraz exposure. The box represents the interquartile range, the 

circle is the mean, and the horizontal line is the median. ......................................33

Figure 3.3.7. Boxplot of snout-to-vent length for frogs at the termination of the Xenopus 

laevis prochloraz exposure. The box represents the interquartile range, the 

circle is the mean, and the horizontal line is the median. ......................................33

Figure 3.3.8. Boxplot of the proportion of female frogs having nuptial pads at the 

termination of the Xenopus laevis prochloraz exposure. The box represents 

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

median. ...................................................................................................................35

Figure 3.3.9. Boxplot of the proportion of male frogs having nuptial pads at the 

termination of the Xenopus laevis prochloraz exposure. The box represents 

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

median. ...................................................................................................................35

Figure 3.3.10. Boxplot of liver weight:whole body weight for male frogs at the termination 

of the Xenopus laevis prochloraz exposure. The box represents the 

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

median. ...................................................................................................................37
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Figure 3.3.11. Boxplot of liver weight:whole body weight for male frogs at the termination 

of the Xenopus laevis prochloraz exposure. The box represents the 

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

median. ...................................................................................................................37

Figure 3.6.1. Boxplot of plasma vitellogenin concentration for male frogs at the 

termination of the Xenopus laevis prochloraz exposure. The box represents 

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

median. ...................................................................................................................40

Figure 3.6.2. Boxplot of plasma vitellogenin concentration for female frogs at the 

termination of the Xenopus laevis prochloraz exposure. The box represents 

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

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

Figure 3.7.1. Boxplot of plasma thyroxine (T4) concentration for NF Stage 62 larvae 

during the Xenopus laevis prochloraz exposure. The box represents the 

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

median ....................................................................................................................43

Figure 3.7.2. Boxplot of plasma thyroxine concentration for male frogs at the termination 

of the Xenopus laevis prochloraz exposure. The box represents the 

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

horizontal line is the median. .................................................................................43

Figure 3.7.3. Boxplot of plasma thyroxine concentration for female frogs at the 

termination of the Xenopus laevis prochloraz exposure. The box represents 

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

median. ...................................................................................................................44

LIST OF APPENDICES

Page

APPENDIX A - CERTIFICATE OF ANALYSIS ........................................................................50

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

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

APPENDIX D – GENDER REPORT ...........................................................................................99

APPENDIX E – STATISTICAL REPORT .................................................................................145

APPENDIX F – ANALYTICAL DATA SUMMARY ...............................................................174

APPENDIX G - REPLICATE SURVIVAL DATA ....................................................................177

APPENDIX H – NF STAGE 62 DATA ......................................................................................186

APPENDIX I – NF STAGE 66 CULL DATA ............................................................................196

APPENDIX J – TERMINATION DATA – GROWTH AND GENDER ...................................200

APPENDIX K – TERMINATION DATA – NUPTIAL PAD AND LIVER .............................207

APPENDIX L – WATER QUALITY DATA .............................................................................215
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APPENDIX M – PLASMA VITELLOGENIN DATA...............................................................225

APPENDIX N – PLASMA T4 DATA ........................................................................................239
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EXECUTIVE SUMMARY

A 110-day flow-through test with African clawed frog (Xenopus laevis) tadpoles exposed to 

sublethal concentrations (i.e., 6.7, 20, 60, and 180 μg a.i/L nominal concentrations) of prochloraz 

was conducted 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 8 Xenopus laevis tadpoles obtained from an in-house culture of Xenopus 

laevis adults. 

Overall mean measured concentrations of prochloraz in the 6.7, 20, 60, and 180 μg/L nominal 

test substance treatments were 6.27, 15.4, 50.1, and 156 μg/L, respectively, representing 77 to 

94% of the nominal treatment concentrations. All measured concentrations of the test substance 

treatment solutions were 80 to 120% of the overall mean measured concentrations with the 

exception of the following samples: 1) all day 0 samples ranged from 130 to 134%, 2) day 7 

sample from the 20 μg/L nominal treatment was 129%, 3) day 14 sample from the 6.7 μg/L 

nominal treatment was 121%, 4) all day 29 samples ranged from 74 to 76%, 5) day 65 sample 

from the 6.7 μg/L nominal treatment was 77%, and 6) the day 71 samples from the 6.7 and 

20 μg/L nominal treatments were 70 and 71%, respectively. No residues of the prochloraz were 

detected in the controls above the MQL of 3.24 ng/mL. Recoveries from the prochloraz quality 

control fortifications ranged from 81 to 137% of the nominal concentrations. Because the mean 

measured concentrations of prochloraz 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 dissolved oxygen and temperature, remained 

within acceptable levels throughout the course of the 110-day exposure. The dissolved oxygen 

concentrations ranged from 2.7 to 9.2 mg/L, representing 32 to 108% saturation. Dissolved 

oxygen concentration was below acceptable levels (i.e., 40% of saturation) in a single 156 μg/L 

test substance treatment chamber (replicate C) on study days 91 and 100. The continuous 

temperature data collected from a centrally located test chamber indicated the temperature remained 

within the temperature range of 21 ± 1°C specified by the protocol. However, the temperature of all 

test solutions ranged from 20.1 to 22.3°C during the test. Test solution pH ranged from 7.4 to 

8.4 during the test. 

This study satisfied all acceptability and performance criteria stated in the study protocol. The 

No-Observable-Effect Concentration and Lowest-Observable-Effect Concentration were 

calculated as mean measured concentrations of 156 μg/L prochloraz and >156 μg/L prochloraz, 

respectively, for each Xenopus laevis parameter except female liver weight:whole body weight 

ratio. The below table presents the results for each parameter evaluated.

Parameter No-Observable-Effect 

Concentration (μg/L)

Lowest-Observable- Effect Concentration 

(μg/L)

NF Stage 62 

Time to Metamorphosis (Replicate Analysis) 156 >156

Time to Metamorphosis (Kaplan-Meier) 156 >156
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Whole Body Weight 156 >156

Snout-to-Vent Length 156 >156

Plasma Thyroxine Level 156 >156

Plasma Vitellogenin Level 156 >156

NF Stage 66 Cull 

Whole Body Weight 156 >156

Snout-to-Vent Length 156 >156

Frogs at Termination

Whole Body Weight 156 >156

Liver Weight: Whole Body Weight (Male) 156 >156

Liver Weight: Whole Body Weight (Female) 50.1 156

Snout-to-Vent Length 156 >156

Nuptial Pad Development 156 >156

Plasma Thyroxine Level 156 >156

Plasma Vitellogenin Level 156 >156
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1.0 INTRODUCTION

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

Disruptor Screening Program. 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 prochloraz 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 Substance

Two samples of the test substance, prochloraz (CAS No.: 67747-09-5; Lot No.: 450-112B), were

received from Battelle Marine Sciences Laboratory, Sequim, Washington, on 22 September 2010 

and were assigned ABC reference numbers TS9001920 and TS9001921. The samples were 

stored at room temperature. The certificate of analysis reported the purity of the test substance 

as 98.3% (Appendix A). The test substance expiration date was July 2013. The test substance 

was described by the Material Safety Data Sheet as a colorless crystalline solid that is very 

slightly soluble in water with a molecular weight of 376.69 and a vapor pressure of 1.5e-1mPa at 

25oC. A retention sample of the test substance was archived at the U.S. EPA National Pesticide 

Standard Repository Environmental Science Center, Fort Meade, Maryland. A copy of the 

chain-of-custody form accompanied the test substance. 

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) and Sera Micron® (Sera 

North America; Montgomeryville, Pennsylvania).
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Embryos were derived from in-house adults. Eggs were collected from breeding aquaria and 

examined microscopically to determine embryo viability and the developmental stage of the 

embryos. 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 8). 

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 and UV sterilizer, and heated and aerated prior to use. The contaminant screen 

for a representative sample of the base blended water is provided in Appendix B. The dilution 

water was analyzed February 2011 and contained 13 μg iodine/L. 

2.4 Test Methods

Test procedures followed the ABC test protocol entitled “Prochloraz: Larval Amphibian Growth 

and Development Assay using Xenopus laevis,” with amendments and deviations (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 Stock Solution Preparations

Prochloraz was not soluble enough in water to directly prepare a solvent-free stock solution; 

therefore, a saturator column and saturator system was utilized during the definitive test. The 

saturator column consisted of a one-inch stainless steel tube (12-inches in length), packed with 

glass wool, with an aspirator vacuum pump (LEVBOLD Trivac® Model D4A) attached to one 

end. A solution of prochloraz was prepared by dissolving approximately 2 g of prochoraz in 

50 mL of acetone. The solution was then introduced into the saturator column at the end to be 

capped with knotted flexible silicone tubing. The saturator column was then tilted back and forth 

and rotated to distribute the mixture throughout the glass wool. The acetone was then evaporated 

off by using the aspirator vacuum pump, leaving the prochloraz attached to the glass wool. The 

saturator column was then slightly elevated so that the end to be attached to the pump (i.e., end 

opposite of solution addition) was at a higher elevation than the opposite end. The valve on the 

vacuum hose, which was used to control the amount of vacuum applied, was completely closed 

before starting the pump. Once vacuum was established, the valve was opened enough to allow 

flow but not enough to pull the prochloraz solution into the pump. When the condensation had 

dissipated and the saturator column was near room temperature (e.g., ~ 1.5 to 2 hours), the 

saturator column was removed from the vacuum pump and placed on the saturator system. The 

saturator system consisted of an approximately 220-L stainless steel barrel filled with dilution 

water (i.e., approximately 200 L of dilution water). Solution was drawn from the bottom of the 

container through Teflon tubing connected to a Fluid Metering, Inc. (FMI) Micro π–petter pump. 

The FMI pump pumped the solution through the saturator column at a flow rate of approximately 

2.6 mL/minute, collecting the prochloraz. The prochloraz solution passed though the stainless 

steel tubing into a filter (Swagelok 90 micron) at the top of the saturator column prior to entry 

into the top of a separate stainless steel collection vessel through Teflon tubing. Both the 
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dilution water reservoir and collection vessel were covered to reduce evaporation and exposure 

to light. Saturator columns were replaced with a fresh saturator column after at least seven days 

of use. The concentration of the concentrated stock solution was verified analytically prior to 

being diluted with dilution water and placed on the diluter system. The solvent-free concentrated 

stock solutions were prepared January 13, January 21, January 31, February 14, February 21, 

March 1, April 6, April 13, April 27, and May 11, 2011. The approximate concentration of the 

prepared solvent-free concentrated stock solutions ranged from 24.2 to 32.4 mg/L.

2.4.2 Exposure System

The addition of the test substance to the diluter system was started on 17 January 2011. The 

dispenser pump introduced approximately 35-mL volumes of prepared 15.2 mg/L diluter stock 

solution to the diluter system, where the diluter stock solution volume was diluted with 

approximately 3.0 L of dilution water. The usage of the diluter stock solution was monitored and 

recorded daily. 

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

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

solutions and intermittent introduction of the solutions to the test chambers. Four prochloraz 

concentrations selected by the U.S. EPA were 6.7, 20, 60, and 180 μg/L, along with a dilutionwater control (i.e., 0 μg/L) which comprised the test design for the definitive test. The diluter 

system, labeled with the study number, 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. To ensure the test 

substance had reached equilibrium in the test chambers, the test substance was delivered to the 

test chambers for approximately 30 days prior to initiation of the study. 

The diluter system was initially set to a minimum cycle rate of 5.0 cycles per hour. The cycle 

rate was increased on 22 March 2011 (study day 34) to approximately 7.5 cycles per hour to 

increase dissolved oxygen levels after a spot check indicated potentially low levels. The cycle 

rate was increased again on 18 May 2011 (study day 91) to approximately 8.0 cycles per hour. 

The diluter delivered approximately 60 L of dilution water control and test solution to each 

chamber each day at 5.0 cycles per hour, 90 L at 7.5 cycles per hour, and 96 L at 8.0 cycles per 

hour, which was sufficient to provide approximately 6.0, 9.0, and 9.6 volume additions, 

respectively, in a 24-hour period. The minimum flow rates specified above were intended to 

ensure maintenance of test-substance concentrations and acceptable water quality throughout the 

test.

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 
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steel screen to prevent the tadpoles from escaping. 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, measured with a LI-COR Model LI-189 light meter equipped with a photometric 

sensor on day 77 ranged from 600 to 720 lux. 

2.4.3 Biological Methods

The test was initiated on 16 February 2011 when 20 NF stage 8 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 160 embryos for the control and 80 embryos per treatment group. 

Embryos were impartially selected and distributed, two 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) (3).

2.4.3.1 Test Initiation

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

each male with 600 IU hCG and each female with 800 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 and ≥70% viability was 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 8 

were segregated for initiation. Prior to egg placement into test chambers, all impartially selected 

groups of 20 eggs were reexamined to confirm that all eggs were at developmental NF stage 8.

2.4.3.2 Larval Sub-Sampling

When some of the larvae attained NF stage 62, the test chambers were 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. Sub-sampled 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 was 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 MS-222 solution 
ABC Study No. 66085 

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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 carcasses were placed into 

individually labeled containers and placed in Davidson’s fixative within containers large enough 

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 hours, 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 Integrated Laboratory Systems, Inc., Durham, 

North Carolina, 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 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 68 when all animals attained NF 

stage 66. A cull of surviving individuals was performed to reduce animal density to 10 

individuals per test chamber. 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. The glass partition was removed to avoid future 

over-crowding resulting from juvenile growth. 

2.4.3.4 Juvenile Sampling

On study day 110, ten weeks after the median NF stage 62 date (i.e., 28 March 2011; study day 

40) among control larvae, all 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, any gross morphological abnormalities or signs of toxicity were

noted (e.g., scoliosis, petechiae, hemorrhage), and the forelimbs were examined under 10x 

magnification for the presence/absence of nuptial pads prior to dissection. 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 liver was removed and

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

then 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 to Fort Environmental Laboratories who then 

shipped the frozen samples to Battelle for gender genotyping.
ABC Study No. 66085 

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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 Integrated Laboratory Systems, Inc., Durham, North 

Carolina, 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 

and pH were measured with a WTW pH 330i pH meter, or WTW Multi 3500i or 3430 

multimeter. Dissolved oxygen concentration was measured with a WTW OXi 330 dissolved 

oxygen meter or WTW Multi 3500i multimeter. No aeration was provided to any control or test 

substance treatment chamber during the test. A continuous recording of temperature in replicate 

D of the control was made using a data logger and thermistor probe. Alkalinity, hardness, and 

conductivity were measured in the dilution water at initiation and termination, and on study days 50 

and 76. 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 (4).

2.4.3.6 Feeding

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

amount of food offered per animal per day varied depending on the study day as follows: freeswimming 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 on 

weekdays and once daily on weekends using three times the amount of Sera Micron solution and 

the same amount of brine shrimp. The amount of Sera Micron added to each tank was initially 

calculated based on 20 larvae and was adjusted as larval density decreased. Once animals 

completed metamorphosis (NF stage 66), the portion of the test chamber containing juveniles 

received 0.05 mL of crushed sinking frog food (e.g., Frog Brittle) 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 crushed + 0.05 mL whole pellets; weeks 2-3, 0.2 mL whole pellets; weeks 4-5, 

0.3 mL whole pellets; weeks 6-9, 0.4 mL whole pellets. 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. 
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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, pre-adsorbance of the antibody, 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. Fortified quality control samples were created as follows: 

A pool of male Xenopus plasma was divided into two batches and one (1) batch was spiked with 

a known amount of provided VTG standard such that the final fortification concentration would 

fall within the standard curve range. The second batch was used as the unspiked control samples. 

Both batches (fortified and unfortified) were diluted to a final dilution factor of 300 and 

aliquoted into volumes sufficient for one day of analyses as quality control (QC) samples and 

frozen until use. The results of spiked Xenopus samples were to be 70 to 120% 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 120% of the nominal concentration. 

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 mg/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.
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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 1xTris-buffer (0.01M 

Tris, 0.15M NaCl) pH-adjusted to approximately 7.60 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 1 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. 

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 added to each well, and the plates incubated at approximately 

22oC for one hour. The plates were washed (Molecular Devices AquaMax 2000 plate washer) 

five times with water, TMB reagent added to wells, plate contents gently mixed for five seconds, 

and the plates were incubated again at approximately 22oC for approximately 20 minutes in the 

dark. After incubation, the reaction was stopped with 1M HCl and the plate contents gently 

mixed for 30 seconds. Within 15 minutes after stopping the reaction, the absorbance was 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 >0.99. 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 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 T4 value for a particular 

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

measured plasma T4 concentrations were below the standard curve but still calculated by the 

software, the concentration of the sample was reported as the lowest standard concentration 

multiplied by the dilution factor for that sample.
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2.5 Analytical Method

Test solutions were analyzed for the concentrations of prochloraz, 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 prochloraz were prepared in methanol on 03 November 2010 and 

14 April 2011 at concentrations of 3.37 and 0.617 mg/mL, respectively. Subsequent dilutions of 

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

standards for high-performance liquid chromatography (HPLC). 

Primary stock solutions of prochloraz were prepared in methanol on 22 November 2010, 

17 January 2011, and 14 April 2011 at concentrations of 17.5, 1.09, and 1.14 mg/mL, 

respectively. Subsequent dilutions of these stock solutions were 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 prochloraz were measured in test solution samples collected from the 

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

weekly throughout the definitive test. Samples collected on day -9 were from one of the four 

replicates for all treatments. All replicates of all treatments were sampled on day 0 and a single 

alternating replicate (e.g., replicate A on day 7, replicate B on day 14, etc.) sample was collected

from each treatment at the remaining time points. The concentration of prochloraz in the diluter 

stock solution samples was determined on the same days as the test solution samples. The 

concentration of prochloraz in the stock solutions used to prepare the diluter stock solutions was 

determined for each batch. 

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

diluter stock solution. 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., 1.62 to 52.5 ng/mL). The 

samples were analyzed by a HPLC system equipped with a UV detector. Prochloraz-fortified 

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: 

Columns: Zorbax SB-C18, 15 cm × 4.6 mm × 3.5 μm 

Eclipse XDB-C18, 15 cm × 4.6 mm × 5 μm 

Temperature: 25°C 

Mobile Phase: 80% methanol: 20% water (Isocratic)
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Flow Rate: 1.0 mL/minute 

Injection Volume: 150 μL

Wavelength: 220 nm 

Detector: UV (VWD)

Stop Time: 8 minutes

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

the chromatographic conditions.

2.5.4 Calculations for Prochloraz

Calculation of prochloraz concentrations in test samples was performed by the external standard 

analysis function of Empower 2 software. The concentration of prochloraz 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 6.7 μg/L nominal treatment (Level 1 C) on day 0: 

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

where:

y = peak area units 

m = slope of the standard curve 

x = μg/L 

b = y-intercept of the standard curve 

Standard Curve: y = 381.7345x – 51.72735 

Sample Peak Area: = 1,421

μg/L from Standard Curve = 3.857 μg/L 

The concentration of prochloraz in the sample was calculated by the equation: 

( ) .7 71 g a.i./L

10 mL

 g 857.3( a.i./L) 20 mL

μ μ =

Recovery of 6.7 μg/L nominal treatment sample on day 0: 

115% 100x

 6.7 μg/L

 7.71μg a.i./L =
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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: 1.62 μg/L 

Volume for analysis: 20 mL 

Volume of sample: 10 mL 

therefore:

MQL = a.i./L μg 3.24 (10 mL)

 (20 μg/L) (1.62 mL) =

2.6 Genetic Sexing

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

dissection. The hind feet 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 was 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 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 was transformed to the square 

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

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 
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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 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 the 

median time to metamorphosis was determined by a Kaplan-Meier product-limit estimator and 

was analyzed statistically. 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 KW 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. Analyses of all NF stage 62 

parameters, both NF stage 66 cull parameters, and termination whole body weight and snout-tovent length 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 Method Check

Recoveries of prochloraz during the freshwater analytical method check ranged from 73 to 74% 

at the nominal concentration of 4.91 μg/L, from 80 to 89% at the nominal concentration of 

5.40 μg/L, and from 94 to 95% at the nominal concentration of 210 μg/L (Table 3.1.1). Based 

on these recoveries, the analytical method was proven to be valid for recovering prochloraz from 

freshwater over a range of concentrations bracketing the nominal definitive test concentrations. 
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Table 3.1.1. Measured concentrations of prochloraz during the method check in freshwater.

Nominal Measured 

Sample Concentration Concentration % of 

Identification (μg /L) (μg /L) Nominal

Control 1 0 <MQLa NA

Control 2 0 <MQLa NA

Control 3 0 <MQLa NA

Low Spike 1 4.91 3.61 b 74 b

Low Spike 2 4.91 3.57 b 73 b

Low Spike 3 4.91 3.66 b 74 b

Low Spike 1 5.40 4.57 85

Low Spike 2 5.40 4.31 80

Low Spike 3 5.40 4.82 89

High Spike 1 210 199 95

High Spike 2 210 197 94

High Spike 3 210 199 95

a MQL = 3.24 ng/mL

b Average of duplicate re-analyses.

3.2 Analytical Results

Test substance concentrations were measured following an analytical method validated by ABC 

personnel prior to analyzing the samples collected during the definitive test. Measured 

prochloraz concentrations in the 6.7, 20, 60, and 180 μg/L nominal test substance treatments at 

day –9 were 7.24, 16.6, 56.6, and 187 μg/L, respectively, representing 83 to 108% of the 

nominal treatment concentrations. Recoveries from test substance treatments on day -9 indicated 

the diluter had come to equilibrium prior to test initiation. Measured prochloraz concentrations 

in the 6.7, 20, 60, and 180 μg/L nominal test substance treatments on day 0 were 8.17, 20.6, 

67.3, and 206 μg/L, representing 103 to 122% of the nominal treatment concentrations. Overall 

mean measured concentrations of prochloraz in the 6.7, 20, 60, and 180 μg/L nominal test 

substance treatments were 6.27, 15.4, 50.1, and 156 μg/L, respectively, representing 77 to 94% 

of the nominal treatment concentrations (Table 3.2.1). All measured concentrations of the test 

substance treatment solutions were 80 to 120% of the overall mean measured concentrations 

with the exception of the following samples: 1) all day 0 samples ranged from 130 to 134%, 2) 

day 7 sample from the 20 μg/L nominal treatment was 129%, 3) day 14 sample from the 

6.7 μg/L nominal treatment was 121%, 4) all day 29 samples ranged from 74 to 76%, 5) day 65 

sample from the 6.7 μg/L nominal treatment was 77%, and 6) the day 71 samples from the 6.7 

and 20 μg/L nominal treatments were 70 and 71%, respectively. The measured prochloraz 

concentration in diluter stock solution samples ranged from 82 to 111% of the nominal

concentration with the exception of the day 0 sample which was 68%. No residues of the 

prochloraz were detected in the controls above the MQL of 3.24 ng/mL. Recoveries from the 

prochloraz quality control fortifications ranged from 81 to 151% of the nominal concentrations. 
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Because the mean measured concentrations of prochloraz 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. An overall summary of the analyses at each sampling 

event are presented in Appendix F. 

On 17 March 2011 (i.e., study day 29) at approximately 9:30 am the diluter stock delivery pump 

was observed not delivering test substance because of a seized pump head. Based on the stock 

usage at this time, the diluter system had not received test substance for approximately 60 cycles

(i.e., approximately 30 L of water without test substance being delivered over a period of 

11 hours). Test solutions were sampled immediately prior to restoring test substance delivery to 

the diluter system and again approximately five hours later. The recoveries in test solutions 

samples immediately prior to restoring test substance delivery were ≤18% of the nominal 

concentrations and ranged from 60 to 71% of nominal when sampled later that same day.

Table 3.2.1. Summary of analytical results for the Xenopus laevis prochloraz in-life exposure.

Nominal Concentration 

(μg/L)

Mean Measured

Concentration (μg/L) StDev CV Recovery

0 <MQL a NA NA NA

6.7 6.27 1.07 17% 94%

20 15.4 2.40 16% 77%

60 50.1 6.93 14% 84%

180 156 22.0 14% 87%

a MQL = Minimum Quantifiable Limit = 3.24 μg/L

3.3 Biological Results

After 110 days of exposure, mean survival was 92% in the control and 90, 88, 95, and 91% in the 

6.27, 15.4, 50.1, and 156 μg/L test substance treatments, respectively (Appendix G). One 

replicate, replicate A of the 15.4 μg/L treatment, was compromised (i.e., >20 mortality) but was 

not excluded from statistical analyses. Sublethal observations and/or abnormalities observed 

prior to study day 21 included the following: a) a larva on the bottom of the test chamber in the 

control and the 6.27, 15.4, and 156 μg/L test substance treatments, b) a larva swimming 

erratically in the 6.27 and 156 μg/L test substance treatments, c) a larva with loss of equilibrium 

in the 6.27 μg/L test substance treatment, and d) a larva with underdeveloped eye in the 

6.27 μg/L test substance treatment. Spinal curvature began to occur on study day 21 and was no 

longer observed after study day 49. The sublethal observations and/or abnormalities observed on 

study days 21 to 49 included the following: a) spinal curvature in the control (n = 17), the 

6.27 μg/L test substance treatment (n = 17), the 15.4 μg/L test substance treatment (n = 15), the 

50.1 μg/L test substance treatment (n = 8), and the 156 μg/L test substance treatment (n = 10); b) 

a larva with an underdeveloped eye in the 6.27 μg/L test substance treatment; and c) a larva 

missing an eye in the 15.4 and 50.1 μg/L test substance treatments. Beginning on study day 50 

and continuing to test termination (study day 110) no spinal curvature was observed in any of the 

treatments but two juveniles (one in each of the 6.27 and 15.4 μg/L test substance treatments) 

each had a missing eye. No other sublethal effects were observed after study day 50. 
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NF Stage 62 Survival:

Survival through NF stage 62 was 92% for the control treatment. Twelve of 159 controltreatment larvae died before reaching NF stage 62; one larvae was inadvertently lost and not 

counted in the total number of control-treatment larvae. Total survival in the prochloraz 

treatments ranged from 88 to 95%. All of the control- and prochloraz-treatment mortalities 

occurred before NF stage 58. 

Time to NF Stage 62: 

The average number of days until NF stage 62 was reached was 41 in the control, and 39, 40, 41, 

and 40 in the 6.27, 15.4, 50.1, and 156 μg/L test substance treatments, respectively ( Figure 

3.3.1, Figure_3_3_1). The median time to NF stage 62 based on the replicate analysis was 

40 days in the control treatment and 40 days in the prochloraz treatments. No significant 

differences among treatments were detected at p = 0.05. The median time to metamorphosis 

based on the Kaplan-Meier product-limit estimator analysis was 39 days in the control treatment 

and ranged from 38 to 40 days in the prochloraz treatments. The number of days until NF stage 

62 was reached for each NF stage 62 larva sampled are presented in Appendix H. 

Table 3.3.1. Mean (and standard deviation) time to NF stage 62 during the Xenopus laevis prochloraz 

exposure.

Mean Measured

Concentration (μg/L)

Time to NF Stage 62 (d)

Mean (StDev) Median a Median b

<MQL 41 (2.5) 40 39

6.27 40 (1.1) 40 38

15.4 40 (1.8) 40 39

50.1 41 (2.1) 40 40

156 40 (1.3) 40 39

p-value 0.660 0.502

Test: K-W 1-Way ANOVA a Median of treatment replicates 

b Median of replicate medians based on the Kaplan-Meier product-limit estimator analysis
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<MQL 6.27 15.4 50.1 156

46

45

44

43

42

41

40

39

38

Mean Measured Dose (μg/L)

Time to NF Stage 62 (d)

Figure 3.3.1. Boxplot of time to NF stage 62 during the Xenopus laevis prochloraz exposure based on the 

replicate analysis. The box represents the interquartile range, the circle is the mean, 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 1.08 g in 

the control treatment and ranged from 0.92 to 1.21 g in the prochloraz treatments (Table 3.3.2; 

Figure 3.3.2). No significant differences in whole body weight among treatments were detected 

at p = 0.050. However, the calculated p-value was nearly significant (p = 0.055), which was 

likely because of relatively high median weight and relatively low median weight for frogs in the 

6.27 μg/L and 156 μg/L exposures, respectively. Mean snout-to-vent length of NF stage 62 

larvae in the control treatment was 21 mm and ranged from 20 to 21 mm in the prochloraz 

treatments (Table 3.3.2; Figure 3.3.3). No significant differences in snout-to-vent length among 

treatments were detected at p = 0.050. However, the calculated p-value was nearly significant 

(p = 0.054), which was likely because of a relatively low median value for larvae in the 156 μg/L 

exposure. 
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Table 3.3.2. Mean (and standard deviation) whole body weight and snout-to-vent length for NF stage 62 

larvae during the Xenopus laevis prochloraz exposure.

Mean Measured

Concentration (μg/L)

Whole Body Weight 

(g)

Snout-to-Vent Length 

(mm)

<MQL 1.08 (0.110) 21 (0.6) 

6.27 1.21 (0.112) 21 (0.6)

15.4 1.08 (0.197) 21 (1.1)

50.1 1.13 (0.067) 21 (0.6) 

156 0.92 (0.074) 20 (0.5)

p-value 0.055 0.054

Test: K-W K-W

<MQL 6.27 15.4 50.1 156

1.3

1.2

1.1

1.0

0.9

0.8

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 3.3.2. Boxplot of whole body weight for NF stage 62 larvae during the Xenopus laevis prochloraz 

exposure. The box represents the interquartile range, the circle is the mean, and the 

horizontal line is the median.
ABC Study No. 66085 

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<MQL 6.27 15.4 50.1 156

22.0

21.5

21.0

20.5

20.0

19.5

19.0

Mean Measured Dose (μg/L)

Snout-to-Vent Length (mm)

Figure 3.3.3. Boxplot of snout-to-vent length for NF stage 62 larvae during the Xenopus laevis prochloraz 

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

prochloraz treatments. Replicate A of the 15.4 μg/L treatment only had nine juveniles at the 

time of the cull; therefore, no juveniles were removed from this replicate at the cull.

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 measured are presented in Appendix I. Mean whole body (wet) weight of frogs measured 

at the NF stage 66 cull was 3.18 g in the control treatment and ranged from 2.10 to 3.17 g in the 

prochloraz treatments (Table 3.3.3; Figure 3.3.4). No significant differences in whole body 

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

measured at the NF stage 66 cull was 29 mm in the control treatment and ranged from 27 to 

31 mm in the prochloraz treatments (Table 3.3.3; Figure 3.3.5). No significant differences in 

snout-to-vent length among treatments were detected for these frogs.
ABC Study No. 66085 

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Table 3.3.3. Mean (and standard deviation) whole body weight and snout-to-vent length at the NF stage 

66 cull of the Xenopus laevis prochloraz exposure.

Mean Measured

Concentration (μg/L)

Whole Body Weight 

(g)

Snout-to-Vent Length 

(mm)

<MQL 3.18 (0.832) 29 (2.3)

6.27 3.17 (0.624) 31 (1.4) 

15.4 2.37 (0.752) 28 (2.5)

50.1 2.36 (0.449) 28 (1.6)

156 2.10 (0.467) 27 (1.3) 

p-value 0.068 0.079

Test: 1-Way ANOVA K-W 

<MQL 6.27 15.4 50.1 156

4.5

4.0

3.5

3.0

2.5

2.0

1.5

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 3.3.4. Boxplot of whole body weight for frogs at the NF stage 66 cull of the Xenopus laevis

prochloraz exposure. The box represents the interquartile range, the circle is the mean, and 

the horizontal line is the median.
ABC Study No. 66085 

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<MQL 6.27 15.4 50.1 156

33

32

31

30

29

28

27

26

25

24

Mean Measured Dose (μg/L)

Snout-to-Vent Length (mm)

Figure 3.3.5. Boxplot of snout-to-vent length for frogs at the NF stage 66 cull of the Xenopus laevis

prochloraz exposure. The box represents the interquartile range, the circle is the mean, 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 measured at termination are presented in Appendix J. Mean whole body (wet) weight of 

frogs measured at the termination of the prochloraz exposure was 12.6 g in the control treatment 

and ranged from 11.4 to 12.8 g in the prochloraz treatments (Table 3.3.4; Figure 3.3.6). No 

significant differences among treatments were detected in whole body weight for frogs at the 

termination of the exposure. Mean snout-to-vent length of frogs measured at the termination of 

the prochloraz exposure was 45 cm in the control treatment and ranged from 42 to 44 cm in the 

prochloraz treatments (Table 3.3.4; Figure 3.3.7). No significant differences in snout-to-vent 

length among treatments were detected for these frogs.

Table 3.3.4. Mean (and standard deviation) whole body weight and snout-to-vent length at the 

termination of the Xenopus laevis prochloraz exposure.

Mean Measured

Concentration (μg/L)

Whole Body Weight 

(g)

Snout-to-Vent Length 

(mm)

<MQL 12.6 (1.75) 45 (1.9)

6.27 12.8 (0.80) 44 (1.1) 

15.4 12.6 (2.67) 44 (3.2)

50.1 12.2 (1.64) 43 (2.0)

156 11.4 (2.20) 42 (3.3)

p-value 0.782 0.842

Test: K-W K-W 
ABC Study No. 66085 

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<MQL 6.27 15.4 50.1 156

17

16

15

14

13

12

11

10

9

8

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 3.3.6. Boxplot of whole body weight for frogs at the termination of the Xenopus laevis prochloraz 

exposure. The box represents the interquartile range, the circle is the mean, and the 

horizontal line is the median.

<MQL 6.27 15.4 50.1 156

48

46

44

42

40

38

Mean Measured Dose (μg/L)

Snout-to-Vent Length (mm)

Figure 3.3.7. Boxplot of snout-to-vent length for frogs at the termination of the Xenopus laevis prochloraz 

exposure. The box represents the interquartile range, the circle is the mean, and the 

horizontal line is the median.

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 female and male frogs in the control treatment that had 

nuptial pads at the termination of the exposure were 0.37 and 0.37, respectively. The mean 

proportions of female and male frogs in the prochloraz treatments that had nuptial pads at the 

termination of the exposure ranged from 0.47 to 0.70 and from 0.51 to 0.71, respectively. 
ABC Study No. 66085 

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(Table 3.3.5; Figure 3.3.8; Figure 3.3.9). No significant differences among treatment medians 

were detected for females or males. 

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 3.3.6). The expected proportion of nuptial pad occurrence was 

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

(probability of nuptial pads occurring by chance = 0.41 for females, 0.35 for males; n = 8). The 

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

treatment) from chance alone was calculated independently for each exposure 

concentration. The proportions of females in the 50.1- and 156-μg/L prochloraz concentrations 

having nuptial pads were 0.63 and 0.64, respectively, which were considered significantly 

greater than chance (p = 0.016 and 0.015). Similarly, the proportions of males in the 50.1- and 

156-μg/L prochloraz concentrations having nuptial pads were 0.69 and 0.73, respectively, which 

were considered significantly greater than chance (p = 0.019 and 0.005). 

Table 3.3.5. Mean (standard deviation) proportion of female and male frogs and total number of frogs 

having nuptial pads at the termination of the Xenopus laevis prochloraz exposure.

Mean Measured

Concentration (μg/L)

Nuptial Pads 

(proportion of females)

Nuptial Pads 

(proportion of males)

Total Number of 

Frogs with Nuptial 

Pads 1 

<MQL 0.37 (0.312) 0.37 (0.285) 31

6.27 0.52 (0.137) 0.56 (0.086) 21

15.4 0.47 (0.452) 0.51 (0.156) 19

50.1 0.62 (0.109) 0.71 (0.276) 26

156 0.70 (0.281) 0.71 (0.288) 27

p-value 0.436 0.175 – 

Test: K-W K-W –

1 Total number of frogs = 80 for control and 40 for each prochloraz dose (39 frogs in the 15.4 μg/L dose). 
ABC Study No. 66085 

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<MQL 6.27 15.4 50.1 156

1.0

0.8

0.6

0.4

0.2

0.0

Mean Measured Dose (μg/L)

Proportion with Nuptial Pads

Figure 3.3.8. Boxplot of the proportion of female frogs having nuptial pads at the termination of the 

Xenopus laevis prochloraz exposure. The box represents the interquartile range, the circle is 

the mean, and the horizontal line is the median.

<MQL 6.27 15.4 50.1 156

1.0

0.8

0.6

0.4

0.2

0.0

Mean Measured Dose (μg/L)

Proportion with Nuptial Pads

Figure 3.3.9. Boxplot of the proportion of male frogs having nuptial pads at the termination of the 

Xenopus laevis prochloraz exposure. The box represents the interquartile range, the circle is 

the mean, and the horizontal line is the median.
ABC Study No. 66085 

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Table 3.3.6. Proportion of total frogs within each treatment having nuptial pads at the termination of the 

Xenopus laevis prochloraz exposure. 

Mean Measured

Concentration (μg/L)

Proportion of Total 

Females with 

Nuptial Pads

Binomial 

Probability

Proportion of 

Total Males with 

Nuptial Pads

Binomial 

Probability

<MQL 0.41 – 0.37 –

6.27 0.50 0.279 0.55 0.074

15.4 0.44 0.491 0.52 0.104

50.1 0.63 0.016 0.69 0.019

156 0.64 0.015 0.73 0.005

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 prochloraz exposure were 0.057 for males and 0.067 for females. The ratio for males ranged 

from 0.047 to 0.064 in the prochloraz treatments (Table 3.3.7; Figure 3.3.10). Significant 

differences among treatments in liver weight:whole body weight for male frogs were detected. 

However, no prochloraz treatment differed significantly from the control treatment. The ratio for 

females ranged from 0.049 to 0.059 in the prochloraz treatments (Table 3.3.7; Figure 3.3.11). 

Significant differences among treatments were detected in liver weight:whole body weight for 

female frogs at the termination of the exposure. The median liver weight:whole body weight 

ratio for the 156-μg/L prochloraz treatment (0.049) was significantly less than the median value 

for the control treatments (p < 0.001). 

Table 3.3.7. Mean (and standard deviation) liver weight:whole body weight for male and female 

frogs at the termination of the Xenopus laevis prochloraz exposure.

Mean Measured

Concentration (μg/L)

Liver 

Weight:Whole Body 

Weight (Males)

Liver 

Weight:Whole Body 

Weight (Females)

<MQL 0.057 (0.004) 0.067 (0.010)

6.27 0.064 (0.005) 0.059 (0.005)

15.4 0.052 (0.009) 0.056 (0.005)

50.1 0.053 (0.004) 0.055 (0.002)

156 0.047 (0.005) 0.049 (0.006)

p-value 0.013 0.006

Test: K-W K-W
ABC Study No. 66085 

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<MQL 6.27 15.4 50.1 156

0.075

0.070

0.065

0.060

0.055

0.050

0.045

0.040

Mean Measured Dose (μg/L)

Liver Weight:Body Weight

Figure 3.3.10. Boxplot of liver weight:whole body weight for male frogs at the termination of the 

Xenopus laevis prochloraz exposure. The box represents the interquartile range, the 

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

<MQL 6.27 15.4 50.1 156

0.09

0.08

0.07

0.06

0.05

0.04

Mean Measured Dose (μg/L)

Liver Weight:Body Weight

Figure 3.3.11. Boxplot of liver weight:whole body weight for male frogs at the termination of the Xenopus 

laevis prochloraz exposure. The box represents the interquartile range, the circle is the 

mean, and the horizontal line is the median.
ABC Study No. 66085 

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3.4 Genotypic/Phenotypic Sex Ratios

The genetic sexing of the juvenile frogs using PCR followed by gel separation did confirm in all 

cases the phenotypic sex at study termination was correct (Appendix D and Appendix J). The 

phenotypic and genotypic sex ratios at the termination were essentially the same within each 

dose (Table 3.4.1). Genotypic sex determinations did not differ from the phenotypic 

determinations. 

Table 3.4.1. Frog phenotypic and genotypic sex ratios at the termination of the Xenopus laevis prochloraz 

exposure.

Mean Measured

Concentration (μg/L) N

Phenotype Genotype

Males Females Males Females

<MQL 80 5.4 4.6 5.4 4.6

6.27 40 5.5 4.5 5.5 4.5

15.4 39 5.8 4.0 5.8 4.0

50.1 40 3.3 6.8 3.3 6.8

156 40 3.8 6.3 3.8 6.3

3.5 Water Quality

All water quality parameters, with the exception of dissolved oxygen and temperature, remained 

within acceptable levels throughout the course of the 110-day exposure (Table 3.5.1). The 

dissolved oxygen concentrations ranged from 2.7 to 9.2 mg/L, representing 32 to 108% 

saturation. Dissolved oxygen concentration was below acceptable levels (i.e., 40% of saturation) 

in a single 156 μg/L test substance treatment chamber (replicate C) on study days 91 and 100. 

The continuous temperature data collected from a centrally located test chamber indicated the 

temperature remained within the temperature range of 21 ± 1°C specified by the protocol. 

However, the temperature of all test solutions ranged from 20.1 to 22.3°C during the test. Test 

solution temperature deviated from the acceptable range (i.e., 21 ± 1oC) on study day 91 when the 

temperature in replicate B of the 50.1 μg/L test substance treatment was 22.1°C and on study day 

107 when the temperature was 22.3°C in replicate A of the 6.27 μg/L test substance treatment and 

replicate C of the 15.4 μg/L test substance treatment. Test solution pH ranged from 7.4 to 8.4 

during the test. Hardness, alkalinity, and conductivity in the dilution water ranged from 138 to

148 mg CaCO3/L, from 152 to 164 mg CaCO3/L, and from 315 to 356 μS, respectively. All 

individual water parameter measurements during the 110-day exposure are presented in 

Appendix L. 
ABC Study No. 66085 

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Table 3.5.1. Ranges for Water Quality Measurements During the 110-Day Exposure with the African 

Clawed Frog, Xenopus laevis, Exposed to Prochloraz

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 – 22.0 3.41 – 9.16 7.35 – 8.35 316 – 352 152 – 164 138 – 148

6.27 20.3 – 22.3 3.82 – 9.19 7.54 – 8.35 315 – 350 152 – 164 138 – 142

15.4 20.3 – 22.3 4.18 – 9.15 7.56 – 8.39 -- -- --

50.1 20.4 – 22.1 3.48 – 9.10 7.54 – 8.32 -- -- --

156 20.4 – 22.0 2.71 – 9.21 7.47 – 8.34 356 164 148

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.

3.6 Vitellogenin Results

The measured VTG concentrations in the plasma of NF stage 62 tadpoles ranged from 0.000 

(i.e., not detected) to 0.017 mg/mL (i.e., 1⁄2 the lowest standard) in all treatments and all detected 

VTG was below the concentration of the lowest standard (i.e., 0.034 mg/mL) (Appendix M). No 

statistical analyses were conducted for this parameter.

The measured VTG concentrations in plasma of the juveniles at study day 110 are presented in 

Appendix M. The measured VTG concentrations in the plasma of juvenile males at study day 

110 ranged from 0.000 (i.e., not detected) to 0.057 mg/mL in all treatments. There was a greater 

number of males (e.g., >90%) with no detectable VTG or below the lowest standard (i.e., 0.033 

to 0.037 mg/mL) than those males with a quantifiable concentration of VTG. The measured 

VTG concentrations in the plasma of juvenile females at study day 110 ranged from 0.000 (i.e., 

not detected) to 0.171 mg/mL in all treatments. There was a greater number of females (e.g., 

>90%) with no detectable VTG or below the lowest standard (i.e., 0.034 or 0.036 mg/mL) than 

those females with a quantifiable concentration of VTG. The mean plasma VTG concentrations 

for frogs in the control treatment were 0.0065 and 0.0095 mg/mL for males and females, 

respectively (Table 3.6.1; Figure 3.6.1; Figure 3.6.2). The mean plasma VTG concentrations for 

frogs in the prochloraz treatments ranged from 0.0056 to 0.0166 mg/mL for males and from 

0.0047 to 0.0310 mg/mL for females (Table 3.6.1; Figure 3.6.1; Figure 3.6.2). No significant 

differences among treatments were detected for male or female frogs. 
ABC Study No. 66085 

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Table 3.6.1. Mean (and standard deviation) vitellogenin concentration for frogs at the termination of the 

Xenopus laevis prochloraz exposure.

Mean Measured

Concentration (μg/L)

Male Plasma 

Vitellogenin 

(mg/mL)

Female Plasma 

Vitellogenin 

(mg/mL)

<MQL 0.0065 (0.0073) 0.0095 (0.0104)

6.27 0.0166 (0.0169) 0.0310 (0.0115)

15.4 0.0056 (0.0067) 0.0047 (0.0055)

50.1 0.0066 (0.0082) 0.0069 (0.0074)

156 0.0066 (0.0093) 0.0058 (0.0082)

p-value 0.789 0.051

Test: K-W K-W

<MQL 6.27 15.4 50.1 156

0.04

0.03

0.02

0.01

0.00

Mean Measured Dose (μg/L)

VTG Concentration (mg/mL)

Figure 3.6.1. Boxplot of plasma vitellogenin concentration for male frogs at the termination of the 

Xenopus laevis prochloraz exposure. The box represents the interquartile range, the circle is 

the mean, and the horizontal line is the median.
ABC Study No. 66085 

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<MQL 6.27 15.4 50.1 156

0.05

0.04

0.03

0.02

0.01

0.00

Mean Measured Dose (μg/L)

VTG Concentration (mg/mL)

Figure 3.6.2. Boxplot of plasma vitellogenin concentration for female frogs at the termination of the 

Xenopus laevis prochloraz exposure. The box represents the interquartile range, the circle is 

the mean, and the horizontal line is the median.

3.7 Plasma Thyroxine (T4) Results

The measured T4 concentrations in the plasma of NF stage 62 larvae in all treatments ranged 

from 35.23 to 315.0 ng/mL (Appendix N). The mean T4 concentration was 140 ng/mL in the 

control and ranged from 92.0 to 204 ng/mL in the test substance treatments (Table 3.7.1; Figure 

3.7.1). No significant differences in plasma T4 concentration among treatments were detected.

The measured T4 concentrations in the plasma of juvenile males in all treatments at study day 

110 ranged from 12.19 to 216.1 ng/mL (Appendix N). The mean T4 concentration was 

41.8 ng/mL in the control males and ranged from 41.3 to 64.1 ng/mL in the test substance 

treatment males (Table 3.7.2; Figure 3.7.2). No significant differences in plasma T4 

concentrations among treatments were detected for males.

The measured T4 concentrations in the plasma of juvenile females in all treatments at study day 

110 ranged from 3.730 to 318.4 ng/mL (Appendix N). The mean T4 concentration was 

57.3 ng/mL in the control females and ranged from 37.8 to 84.5 ng/mL in the test substance 

treatment females (Table 3.7.2; Figure 3.7.3). No significant differences in plasma T4 

concentrations among treatments were detected for females.
ABC Study No. 66085 

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Table 3.7.1. Mean (and standard deviation) plasma thyroxine (T4) for NF Stage 62 frogs during the 

Xenopus laevis prochloraz exposure. 

Mean Measured

Concentration (μg/L)

Plasma Thyroxine (T4) 

(ng/mL)

<MQL 140 (84.3)

6.27 142 (87.9)

15.4 92.0 (59.4)

50.1 204 (48.5)

156 122 (72.0)

p-value 0.339

Test: 1-Way ANOVA

Table 3.7.2. Mean (and standard deviation) plasma thyroxine (T4) for frogs at the termination of the Xenopus 

laevis prochloraz exposure.

Mean Measured

Concentration (μg/L)

Male Plasma 

Thyroxine (T4) 

(ng/mL)

Female Plasma 

Thyroxine (T4) 

(ng/mL)

<MQL 41.8 (16.92) 57.3 (18.99)

6.27 58.2 (11.41) 37.8 (11.63)

15.4 64.1 (6.70) 57.8 (38.06)

50.1 41.3 (7.00) 62.5 (22.88)

156 58.7 (35.29) 84.5 (56.95)

p-value 0.056 0.401

Test: K-W K-W
ABC Study No. 66085 

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<MQL 6.27 15.4 50.1 156

350

300

250

200

150

100

50

0

Mean Measured Dose (μg/L)

Thyroxine (T4) Concentration (ng/mL) 

Figure 3.7.1. Boxplot of plasma thyroxine (T4) concentration for NF Stage 62 larvae during the Xenopus 

laevis prochloraz exposure. The box represents the interquartile range, the circle is the 

mean, and the horizontal line is the median

<MQL 6.27 15.4 50.1 156

100

90

80

70

60

50

40

30

20

Mean Measured Dose (μg/L)

Thyroxine Concentration (ng/mL)

Figure 3.7.2. Boxplot of plasma thyroxine concentration for male frogs at the termination of the Xenopus 

laevis prochloraz exposure. The box represents the interquartile range, the circle is the 

mean, the asterisk is an outlier, and the horizontal line is the median.
ABC Study No. 66085 

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<MQL 6.27 15.4 50.1 156

180

160

140

120

100

80

60

40

20

0

Mean Measured Dose (μg/L)

Thyroxine Concentration (ng/mL)

Figure 3.7.3. Boxplot of plasma thyroxine concentration for female frogs at the termination of the 

Xenopus laevis prochloraz exposure. The box represents the interquartile range, the circle is 

the mean, and the horizontal line is the median.

3.8 Validity and Performance Criteria

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 dissolved oxygen and temperature. The dissolved oxygen concentration was below 

acceptable levels (i.e., 40% of saturation) in a single 156 μg/L test substance treatment chamber 

(replicate C) on study days 91 and 100. The test solution temperature deviated from the 

acceptable range (i.e., 21 ± 1 oC) on study day 91 when the temperature in replicate B of the 

50.1 μg/L test substance treatment was 22.1°C and on study day 107 when the temperature was 

22.3°C in replicate A of the 6.27 μg/L test substance treatment and replicate C of the 15.4 μg/L test 

substance treatment. Delivery of chemical to the test organisms (e.g., concentrations of the 

chemical in test water) was maintained at ≤20% CV over the entire test. Survival of test 

organisms was ≥90% in all control replicates and mean survival was ≥88% in all test substance 

treatments. Time to NF stage 62 among control larvae ranged from 41 to 46 days. The mean 

weight of culled NF stage 66 control larvae was 3.181 g. 

4.0 CONCLUSIONS

This study satisfied all acceptability and performance criteria stated in the study protocol. The 

No-Observable-Effect Concentration and Lowest-Observable-Effect Concentration were 

calculated as mean measured concentrations of 156 μg/L prochloraz and >156 μg/L prochloraz, 

respectively, for each Xenopus laevis parameter, except female liver weight:whole body weight 

ratio (Table 4.0.1).
ABC Study No. 66085 

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Table 4.0.1. Calculated No-Observable-Effect Concentration and Lowest-Observable-Effect 

Concentration based on nominal prochloraz concentrations for parameter measured for 

Xenopus laevis exposed to prochloraz.

Parameter No-Observable-Effect 

Concentration (μg/L)

Lowest-ObservableEffect Concentration 

(μg/L)

NF stage 62

Time to Metamorphosis (Replicate Analysis) 156 >156

Time to Metamorphosis (Kaplan-Meier) 156 >156

Whole Body Weight 156 >156

Snout-to-Vent Length 156 >156

Plasma Thyroxine Level 156 >156

Plasma Vitellogenin Level 156 >156

NF stage 66 Cull

Whole Body Weight 156 >156

Snout-to-Vent Length 156 >156

Frogs at Termination

Whole Body Weight 156 >156

Liver Weight:Whole Body Weight (Male) 156 >156

Liver Weight:Whole Body Weight (Female) 50.1 156

Snout-to-Vent Length 156 >156

Nuptial Pad Development 156 >156

Plasma Thyroxine Level 156 >156

Plasma Vitellogenin Level 156 >156

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. 
ABC Study No. 66085 

Page 46 of 252 

PROTOCOL DEVIATIONS 

Item 1 Section 10.0 Test Substance Delivery/Dosing System

Deviation: On 17 March 2011 (i.e., study day 29) at approximately 9:30 am the diluter 

stock delivery pump was observed not delivering test substance to the 

diluter system. Based on the stock usage at this time, the diluter system had 

not received test substance for approximately 60 cycles (i.e., approximately 

30 L of water without test substance being delivered to each chamber over a 

period of 11 hours).

Reason for 

Deviation:

The pump head on the diluter stock delivery pump had seized and this stopped 

delivery of the test substance to the diluter system.

Effect on Study: No adverse effect on study integrity. The duration the diluter system had 

operated without test substance was <2% of the first 29 days of the exposure 

and <1% of the entire 110 day exposure. Test solutions were sampled 

immediately prior to restoring test substance delivery to the diluter system 

and again approximately five hours later. The recoveries in test solutions 

samples immediately prior to restoring test substance delivery were ≤18% of 

the nominal concentrations and ranged from 60 to 71% of the nominal 

concentrations when sampled later that same day.

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: study day 91 when the temperature 

in replicate B of the 60 μg/L nominal treatment was 22.1°C and on study day 

107 when the temperature was 22.3 °C in replicate A of the 6.7 μg/L nominal 

treatment and replicate C of the 20 μg/L nominal treatment.

Reason for 

Deviation:

Efforts made to maintain test solution temperature at 21 ± 1oC during the test 

(e.g., regulating water bath temperature with a chiller and/or heaters and recirculation of water within the water bath) were not sufficient to achieve 

maintain all test solutions at this temperature. 

Effect on Study: No adverse effect on study integrity. With the above noted exceptions, test 

solution temperatures were 21 ± 1 oC and the continuous temperature record 

maintained from a centrally located test chamber indicated the temperature 

was 21 ± 1oC throughout the test.

Item 3 Section 13.0 Temperature

Deviation: Test solution temperature differences among treatments on a given 

measurement day exceeded 0.5 oC on the following study days: 8, 20, 29, 36, 

48, 50, 62, 65, 69, 72, 76, 79, 83, 91, 107, and 110. Temperature differences 

exceeding 0.5oC among treatments ranged from 0.6 to 1.0oC. The temperature 
ABC Study No. 66085 

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differences among treatment replicate solutions on a given measurement day 

exceeded 0.5oC in the following treatments: the control on days 48, 50, 62, 69, 

and 76; the 6.7 μg/L nominal treatment on day 8; the 20 μg/L nominal 

treatment on days 29 and 107; the 60 μg/L nominal treatment on day 91; and 

the 180 μg/L nominal treatment on days 50, 62, 69, 72, and 79. Temperature 

differences exceeding 0.5 oC among treatment replicate solutions ranged from 

0.6 to 0.9 oC.

Reason for 

Deviation:

Efforts made to limit test solution temperature variation to ≤0.5oC across 

treatments and treatment replicates within a given day (e.g., regulating water 

bath temperature with a chiller and/or heaters and re-circulation of water 

within the water bath) were not sufficient to achieve this criterion 

throughout the course of the study.

Effect on Study: No adverse effect on study integrity. With the above noted exceptions, test 

solution temperatures across treatments and replicates on a given day were 

consistent (i.e., ≤0.5oC variation) throughout the test. 

Item 4 Section 15.0 Physical Parameters Monitored

Deviation: Dissolved oxygen concentration was below acceptable levels (i.e., 40% of 

saturation) in a single test substance treatment chamber (replicate C of the 

180 μg/L nominal treatment) on study days 91 (34% of saturation) and 100

(32% of saturation).

Reason for 

Deviation:

The biological oxygen demand in this single chamber may have been greater 

than all other chambers on these days.

Effect on Study: No adverse effect on study integrity. With the above noted exception, the 

dissolved oxygen concentration remained ≥40% of saturation on all 

measurement days.

Item 5 Section 16.0 Analytical Measurements

Deviation: At the study termination, Day 110, samples were not collected from all 

replicates of the control and each test substance treatment. Samples were 

collected from a single replicate of the control and each test substance as was 

done on all previous sample points with the exception of test initiation when 

all replicates were sampled.

Reason for 

Deviation: Technical oversight. 

Effect on Study: None. Analytical samples were collected at least weekly throughout the 

110 day exposure.
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Item 6 Section 16.1 Analytical Method

Deviation Column: Zorbax SB-C18, 15 cm x 4.6 mm x 3.5 μm, was replaced on Day 35 

sample analysis to Column: Eclipse XDB-C18, 15 cm x 4.6 mm x 5 μm.

Reason for 

Deviation: The Zorbax column stopped functioning, so a replacement column was 

found.

Effect on Study: None. The results were consistent from one column to the other.
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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. Mount, D.I. and W.A. Brungs. 1967. A Simplified Dosing Apparatus for Fish 

Toxicological Studies. Water Res. 1: 21-29. 

3. Nieuwkoop,P.D. & Faber,J. (1994) Normal Table of Xenopus laevis. Garland Publishing, 

New York

4. 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 - CERTIFICATE 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 2011 ABC Well Water Screen (non-GLP)

Organophosphate 

(μg/L) 2011

Historical 

Range 

1998-2011 1 Elements (mg/L) 2011

Historical Range 

1998-2011 1

Azinphos ethyl5, 7 --- <1.0 Aluminum4 <0.0500 <0.0500

Azinphos-methyl3 <1.00 <0.200-<1.00 Antimony4 <0.0500 <0.0500

Bolstar4 <1.00 <0.200-<1.00 Arsenic <0.0030 <0.0030-<0.050

Chloropyrifos4 <1.00 <0.200-<1.00 Barium4 0.0206 0.0189-0.0206

Coumaphos4 <1.00 <0.400-<1.00 Beryllium4 <0.0010 <0.0010

Demeton, Total <1.00 <0.200-<1.0 Boron 0.432 0.37-0.432

Diazinon <1.00 <0.200-<1.0 Cadmium <0.0020 <0.0020-<0.0050

Dichlorvos4 <1.00 <0.200-<1.00 Calcium2 78.4 52-83.1

Dimethoate4 <1.00 <1.00 Chromium <0.0100 <0.010

Disulfoton <1.00 <0.200-<1.0 Cobalt4 <0.0100 <0.0100

EPN4 <1.00 <0.200-<1.00 Copper <0.0100 <0.010

Ethion5, 7 --- <1.0 Iron 0.0294 <0.0059-0.16

Ethoprop4 <1.00 <0.200-<1.00 Lead <0.0020 <0.0020-0.0400

Famphur6 <1.00 <1.00 Magnesium2 33.3 27-33.3

Fensulfothion4 <1.00 <1.00 Manganese4 <0.0050 <0.0050

Fenthion4 <1.00 <0.200-<1.00 Molybdenum4 <0.0100 <0.0100

Malathion <1.00 <0.200-<1.0 Mercury5 <0.00020 <0.00020-

<0.00060

Merphos4, 7 --- <0.200 Nickel <0.0100 <0.0100-<0.020

Mevinphos4 <1.00 <1.00 Potassium2 8.42 6.6-8.42

Monocrotophos4, 7 --- <1.00 Selenium <0.0500 <0.050

Naled4, 7 --- <2.00 Silver <0.0100 <0.010

O,O,O-Triethyl 

phosphorothioate6 

<1.00 <1.00 Sodium2 30.0 27-32.2

Thallium4 <0.0500 <0.0500

Parathion5, 7 --- <1.0 Tin4 <0.0200 <0.0200

Parathion, ethyl3 <1.00 <0.200-<1.00 Vanadium4 <0.0100 <0.0100

Parathion, methyl3 <1.00 <0.200-<1.00 Zinc 0.0198 0.0118-0.078

Phorate4 <1.00 <0.200-<1.00

Ronnel4 <1.00 <0.200-<1.00

Stirophos4 <1.00 <0.200-<1.00 Polychlorinated 

Sulfotepp Biphenyls (μg/L) 4 <1.00 <0.200-<1.00

TEPP4, 7 --- <0.200 Aroclor 1016 <1.00 <1.00

Thionazin6 <1.00 1.00 Aroclor 1221 <1.00 <1.00

Tokuthion4 <1.00 <0.200-<1.00 Aroclor 1232 <1.00 <1.00

Trichloronate4 <1.00 <0.200-<1.00 Aroclor 1242 <1.00 <1.00

Aroclor 1248 <1.00 <1.00

Aroclor 1254 <1.00 <1.00

Aroclor 1260 <1.00 <1.00
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Chemical Characteristics of ABC Well Water Used by

ABC Laboratories' Chemical Services Group (continued)

February 2011 ABC Well Water Screen (non-GLP)

Chlorinated 

Herbicides (μg/L) 2011

Historical 

Range 

1998-2011 1

Chlorinated 

Hydrocarbons (μg/L) 2011

Historical 

Range 

1998-2011 1

2,4,5-TP (Silvex) <0.200 <0.200-<50 4,4’-DDD <0.04 <0.040-<0.04

2,4-D <0.200 <0.200-<250 4,4’-DDE <0.04 <0.040-<0.04

4,4’-DDT <0.04 <0.040-<0.04

Aldrin <0.04 <0.040-<0.04

Miscellaneous (mg/L)

α-BHC <0.04 <0.040-<0.04

β-BHC <0.04 <0.040-<0.04

∆-BHC <0.04 <0.040-<0.04

Nitrite N <0.05 <0.01-≤0.050 Chlordane <0.05 <0.05-<0.48

Nitrate N 0.329 <0.11-0.329 Dieldrin <0.04 <0.040-<0.04

Total Phosphorus as P <0.050 <0.050-0.64 Endosulfan I <0.04 <0.040-<0.04

Endosulfan II <0.04 <0.040-<0.04

Endosulfan sulfate <0.04 <0.040-<0.04

Endrin <0.04 <0.040-<0.04

Endrin aldehyde <0.04 <0.040-<0.04

Endrin ketone <0.04 <0.040-<0.04

γ-BHC <0.04 <0.040-<0.04

Heptachlor <0.04 <0.040-<0.04

Heptachlor epoxide <0.04 <0.040-<0.04

Methoxychlor <0.04 <0.04-<0.095

Toxaphene <0.50 <0.50-<3.8

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.

Note: ABC well water is the base water for ABC Aged-Blended Water.
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APPENDIX C - PROTOCOL, AMENDMENTS, AND DEVIATIONS
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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 Prochloraz During the Larval Growth and Development Assay with the African Clawed 

Frog, Xenopus laevis 

Study 

Day

Measured Concentration as μg/L

Control 

0

6.7 20 60 180 Stock

15,200 

Meas. % Nom % Day 0 % Mean Meas. % Nom % Day 0 % Mean Meas. % Nom % Day 0 % Mean Meas. % Nom % Day 0 % Mean (% nominal)

-N a <MQL 7.24 108 NA NA 16.6 83 NA NA 56.6 94 NA NA 187 104 NA NA 16,000 (105)

0 <MQL 8.17 122 NA 130 20.6 103 NA 134 67.3 112 NA 134 206 114 NA 132 10,400 (68)

7 <MQL 7.32 109 90 117 19.9 100 97 129 58.7 98 87 117 186 103 90 119 15,500 (102)

14 <MQL 7.58 113 93 121 17.7 89 86 115 56.9 95 85 114 173 96 84 111 14,400 (95)

21 <MQL 5.78 86 71 92 16.1 81 78 105 53.1 89 79 106 178 99 86 114 14,000 (92)

28 <MQL 6.38 95 78 102 15.8 79 77 103 52.8 88 78 105 174 97 84 112 14,400 (95)

29 (pm) <MQL 4.78 71 59 76 12.0 60 58 78 37.8 63 56 75 115 64 56 74 14,100 (93)

30 <MQL 6.59 98 81 105 15.4 77 75 100 49.2 82 73 98 146 81 71 94 13,900 (91)

35 <MQL 7.24 108 89 115 16.5 83 80 107 47.5 79 71 95 150 83 73 96 13,500 (89)

42 <MQL 6.66 99 82 106 16.5 83 80 107 54.4 91 81 109 162 90 79 104 13,500 (89)

49 <MQL 6.37 95 78 102 16.4 82 80 106 49.4 82 73 99 174 97 84 112 13,700 (90)

56 <MQL 6.83 102 84 109 16.5 83 80 107 46.0 77 68 92 156 87 76 100 13,400 (88)

65 <MQL 4.81 72 59 77 13.9 70 67 90 57.8 96 86 115 175 97 85 112 13,700 (90)

71 <MQL 4.37 65 53 70 10.9 55 53 71 41.6 69 62 83 142 79 69 91 13,200 (87)

76 <MQL 6.55 98 80 104 14.0 70 68 91 48.9 82 73 98 144 80 70 92 12,500 (82)

84 <MQL 5.78 86 71 92 15.1 76 73 98 46.3 77 69 92 145 81 70 93 13,000 (86)

91 <MQL 7.27 109 89 116 15.1 76 73 98 40.7 68 60 81 132 73 64 85 14,800 (97)

98 <MQL 5.02 75 61 80 15.9 80 77 103 53.6 89 80 107 132 73 64 85 16,800 (111)

99 <MQL 5.63 84 69 90 12.5 63 61 81 48.2 80 72 96 141 78 68 90 13,300 (88)

105 <MQL 5.04 75 62 80 13.2 66 64 86 46.4 77 69 93 145 81 70 93 14,000 (92)

110 <MQL 7.17 107 88 114 14.0 70 68 91 45.7 76 68 91 143 79 69 92 12,800 (84)

Overall 

Mean c <MQL 6.27 94 77 -- 15.4 77 75 -- 50.1 84 74 -- 156 87 76 -- -- 

MQL = 3.24 ng/mL

a Day –N sampled 9 days prior to initiation. Day –N values not included in overall mean calculation.

b Average of duplicate re-analyses.
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Table F-2. Measured Concentrations of Prochloraz 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

(5.40)a 

High Spike

(210)a 

0 7.24 (134) b 233 (111) b

7 5.95 (110) 232 (110)

14 5.78 (107) 229 (109)

21 6.13 (114) 250 (119)

28 6.32 (117) 247 (118)

29 6.73 (125) 242 (115)

30 6.37 (118) 241 (115)

35 6.10 (113) 233 (111)

42 7.40 (137) c 224 (107) b

49 6.77 (125) 246 (117)

56 8.15 (151) 234 (111)

65 4.80 (89) 253 (117)

71 4.35 (81) 223 (106)

76 6.00 (111) 210 (100)

84 5.82 (108) 200 (95)

91 5.85 (108) 254 (121)

98 6.73 (125) b 223 (106) b

99 6.36 (118) 222 (106)

105 6.45 (119) b 220 (105) b

110 6.54 (121) 243 (116)

a Nominal concentration as μg/L. 

b Average of duplicate re-analyses. c Value confirmed by reanalysis. 
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APPENDIX G - REPLICATE SURVIVAL DATA
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Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to Prochloraz for 110-Days

Mean 

Measured 

Concentration

(μg/L) Replicate

Day 0 

Initial 

Number

Day 4 Day 7 Day 14

Number 

Hatched

Cumulative

Number 

Dead 

% 

Survival

Number 

Surviving

Cumulative

Number 

Dead

% 

Survival

Number 

Surviving

Cumulative

Number 

Dead

% 

Survival

0 (Control) A 20 19 1 95 19 1 95 19 1 95

B 20 19 1 95 19 1 95 19 1 95

C 20 19 1 95 19 1 95 19 1 95

D 20 17 3 85 17 3 85 17 3 85

E 20 18 2 90 18 2 90 18 2 90

F 20 18 2 90 18 2 90 18 2 90

G 20 20 0 100 20 0 100 20 0 100

H 20 19 1 95 19 1 95 18 a 1 95

6.27 A 20 19 1 95 18 2 90 18 2 90

B 20 19 1 95 18 2 90 18 2 90

C 20 18 2 90 18 2 90 18 2 90

D 20 18 2 90 18 2 90 18 2 90

15.4 A 21 14 7 67 14 7 67 14 7 67

B 20 19 1 95 19 1 95 19 1 95

C 20 20 0 100 20 0 100 20 0 100

D 20 19 1 95 19 1 95 19 1 95

50.1 A 20 19 1 95 19 1 95 19 1 95

B 20 20 0 100 20 0 100 20 0 100

C 20 19 1 95 19 1 95 19 1 95

D 20 18 2 90 18 2 90 18 2 90

156 A 20 18 2 90 18 2 90 18 2 90

B 20 19 1 95 19 1 95 19 1 95

C 20 19 1 95 19 1 95 19 1 95

D 20 19 1 95 18 2 90 18 2 90 a One organism could not be accounted for on study day 13 after cleaning and siphoning the test chamber. This unaccounted for organism was likely removed 

during the cleaning procedure; therefore, was not treated as an exposure-related mortality.
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Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to Prochloraz for 110-Days (continued) 

Mean 

Measured 

Concentration

(μg/L) Replicate

Day 0 

Initial 

Number

Day 21 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 20 19 1 95 19 1 95 19 1 95

B 20 19 1 95 19 1 95 19 1 95

C 20 19 1 95 19 1 95 19 1 95

D 20 17 3 85 17 3 85 17 3 85

E 20 18 2 90 18 2 90 18 2 90

F 20 18 2 90 18 2 90 18 2 90

G 20 20 0 100 20 0 100 20 0 100

H 20 18 1 95 18 1 95 18 1 95

6.27 A 20 18 2 90 18 2 90 18 2 90

B 20 18 2 90 18 2 90 18 2 90

C 20 18 2 90 18 2 90 18 2 90

D 20 18 2 90 18 2 90 18 2 90

15.4 A 21 14 7 67 14 7 67 14 7 67

B 20 19 1 95 19 1 95 19 1 95

C 20 20 0 100 20 0 100 20 0 100

D 20 19 1 95 19 1 95 18 2 90

50.1 A 20 19 1 95 19 1 95 19 1 95

B 20 20 0 100 20 0 100 20 0 100

C 20 19 1 95 19 1 95 19 1 95

D 20 18 2 90 18 2 90 18 2 90

156 A 20 18 2 90 18 2 90 18 2 90

B 20 19 1 95 19 1 95 19 1 95

C 20 18 2 90 18 2 90 18 2 90

D 20 18 2 90 18 2 90 18 2 90
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Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to Prochloraz for 110-Days (continued) 

Mean 

Measured 

Concentration

(μg/L) Replicate

Day 0 

Initial 

Number

Number 

Removed for 

Sampling

Day 42 Number 

Removed for 

Sampling

Day 49

Number 

Alive

Cumulative 

Number Dead

% 

Survival

Number 

Alive

Cumulative 

Number Dead

% 

Survival

0 (Control) A 20 3 16 1 95 5 14 1 95

B 20 1 18 1 95 5 14 1 95

C 20 4 15 1 95 5 14 1 95

D 20 1 16 3 85 4 13 3 85

E 20 5 13 2 90 5 13 2 90

F 20 5 13 2 90 5 13 2 90

G 20 0 20 0 100 5 15 0 100

H 20 4 14 1 95 5 13 1 95

6.27 A 20 5 13 2 90 5 13 2 90

B 20 4 14 2 90 5 13 2 90

C 20 3 15 2 90 5 13 2 90

D 20 3 15 2 90 5 13 2 90

15.4 A 21 3 11 7 67 5 9 7 67

B 20 1 18 1 95 5 14 1 95

C 20 5 15 0 100 5 15 0 100

D 20 4 14 2 90 5 13 2 90

50.1 A 20 3 16 1 95 5 14 1 95

B 20 5 15 0 100 5 15 0 100

C 20 0 19 1 95 5 14 1 95

D 20 3 15 2 90 5 13 2 90

156 A 20 4 14 2 90 5 13 2 90

B 20 4 15 1 95 5 14 1 95

C 20 1 17 2 90 5 13 2 90

D 20 4 14 2 90 5 13 2 90
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Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to Prochloraz for 110-Days (continued) 

Mean 

Measured 

Concentration

(μg/L) Replicate

Day 0 

Initial 

Number

Number 

Removed for 

Sampling

Day 56 Day 63

Number 

Alive

Cumulative 

Number Dead

% 

Survival

Number 

Alive

Cumulative 

Number Dead

% 

Survival

0 (Control) A 20 5 14 1 95 14 1 95

B 20 5 14 1 95 14 1 95

C 20 5 14 1 95 14 1 95

D 20 5 12 3 85 12 3 85

E 20 5 13 2 90 13 2 90

F 20 5 12 a 3 85 12 3 85

G 20 5 15 0 100 15 0 100

H 20 5 13 1 95 13 1 95

6.27 A 20 5 13 2 90 13 2 90

B 20 5 13 2 90 13 2 90

C 20 5 13 2 90 13 2 90

D 20 5 13 2 90 13 2 90

15.4 A 21 5 9 7 67 9 7 67

B 20 5 14 1 95 14 1 95

C 20 5 15 0 100 15 0 100

D 20 5 13 2 90 13 2 90

50.1 A 20 5 14 1 95 14 1 95

B 20 5 15 0 100 15 0 100

C 20 5 14 1 95 14 1 95

D 20 5 13 2 90 13 2 90

156 A 20 5 13 2 90 13 2 90

B 20 5 14 1 95 14 1 95

C 20 5 13 2 90 13 2 90

D 20 5 13 2 90 13 2 90 a A thirteenth individual was not found starting on this day. This individual had not completed metamorphosis prior to this day and was approximately 

NF stage 44. The loss of this organism was considered to be an exposure-related mortality and was added to the cumulative number dead.
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Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to Prochloraz for 110-Days (continued) 

Mean 

Measured 

Concentration

(μg/L) Replicate

Day 0 

Initial 

Number

Number 

Removed for 

Juvenile Cull

Day 70 Day 77

Number 

Alive

Cumulative 

Number Dead

% 

Survival

Number 

Alive

Cumulative 

Number Dead

% 

Survival

0 (Control) A 20 4 10 1 95 10 1 95

B 20 4 10 1 95 10 1 95

C 20 4 10 1 95 10 1 95

D 20 2 10 3 85 10 3 85

E 20 3 10 2 90 10 2 90

F 20 2 10 3 85 10 3 85

G 20 5 10 0 100 10 0 100

H 20 3 10 1 95 10 1 95

6.27 A 20 3 10 2 90 10 2 90

B 20 3 10 2 90 10 2 90

C 20 3 10 2 90 10 2 90

D 20 3 10 2 90 10 2 90

15.4 A 21 0 9 7 67 9 7 67

B 20 4 10 1 95 10 1 95

C 20 5 10 0 100 10 0 100

D 20 3 10 2 90 10 2 90

50.1 A 20 4 10 1 95 10 1 95

B 20 5 10 0 100 10 0 100

C 20 4 10 1 95 10 1 95

D 20 3 10 2 90 10 2 90

156 A 20 3 10 2 90 10 2 90

B 20 4 10 1 95 10 1 95

C 20 3 10 2 90 10 2 90

D 20 3 10 2 90 10 2 90
ABC Study No. 66085 

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Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to Prochloraz for 110-Days (continued) 

Mean 

Measured 

Concentration

(μg/L) Replicate

Day 0 

Initial 

Number

Day 84 Day 91 Day 98

Number 

Surviving

Cumulative

Number 

Dead

% 

Survival

Number 

Surviving

Cumulative

Number 

Dead

% 

Survival

Number 

Surviving

Cumulative

Number 

Dead

% 

Survival

0 (Control) A 20 10 1 95 10 1 95 10 1 95

B 20 10 1 95 10 1 95 10 1 95

C 20 10 1 95 10 1 95 10 1 95

D 20 10 3 85 10 3 85 10 3 85

E 20 10 2 90 10 2 90 10 2 90

F 20 10 3 85 10 3 85 10 3 85

G 20 10 0 100 10 0 100 10 0 100

H 20 10 1 95 10 1 95 10 1 95

6.27 A 20 10 2 90 10 2 90 10 2 90

B 20 10 2 90 10 2 90 10 2 90

C 20 10 2 90 10 2 90 10 2 90

D 20 10 2 90 10 2 90 10 2 90

15.4 A 21 9 7 67 9 7 67 9 7 67

B 20 10 1 95 10 1 95 10 1 95

C 20 10 0 100 10 0 100 10 0 100

D 20 10 2 90 10 2 90 10 2 90

50.1 A 20 10 1 95 10 1 95 10 1 95

B 20 10 0 100 10 0 100 10 0 100

C 20 10 1 95 10 1 95 10 1 95

D 20 10 2 90 10 2 90 10 2 90

156 A 20 10 2 90 10 2 90 10 2 90

B 20 10 1 95 10 1 95 10 1 95

C 20 10 2 90 10 2 90 10 2 90

D 20 10 2 90 10 2 90 10 2 90
ABC Study No. 66085 

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Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis, Exposed to Prochloraz for 110-Days (continued) 

Mean 

Measured 

Concentration

(μg/L) Replicate

Day 0 

Initial 

Number

Day 105 Day 110

Number 

Hatched

Cumulative 

Number 

Dead

% 

Survival

Number 

Surviving

Cumulative 

Number 

Dead

% 

Survival

Treatment 

Total % 

Survival

0 (Control) A 20 10 1 95 10 1 95 92

B 20 10 1 95 10 1 95

C 20 10 1 95 10 1 95

D 20 10 3 85 10 3 85

E 20 10 2 90 10 2 90

F 20 10 3 85 10 23 85

G 20 10 0 100 10 0 100

H 20 10 1 95 10 1 95

6.27 A 20 10 2 90 10 2 90 90

B 20 10 2 90 10 2 90

C 20 10 2 90 10 2 90

D 20 10 2 90 10 2 90

15.4 A 21 9 7 67 9 7 67 88

B 20 10 1 95 10 1 95

C 20 10 0 100 10 0 100

D 20 10 2 90 10 2 90

50.1 A 20 10 1 95 10 1 95 95

B 20 10 0 100 10 0 100

C 20 10 1 95 10 1 95

D 20 10 2 90 10 2 90

156 A 20 10 2 90 10 2 90 91

B 20 10 1 95 10 1 95

C 20 10 2 90 10 2 90

D 20 10 2 90 10 2 90
ABC Study No. 66085 

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Table G-2. Survival of the African Clawed Frog, Xenopus laevis, Exposed to Prochloraz for 110 Days 

Mean 

Measured 

Concentration

(μg/L) Rep

Day 0 

Initial 

Number

NF Stage 62 

Survivors

NF Stage 62 

% Survival

Number

Removed a

Day 68 Initial 

Number

Day 110 

Survivors

Overall

% Survival

Overall

Treatment

% Survival

0 (Control) A 20 19 95 9 10 10 95 92

B 20 19 95 9 10 10 95

C 20 19 95 9 10 10 95

D 20 17 85 7 10 10 85

E 20 18 90 8 10 10 90

F 20 17 85 7 10 10 90

G 20 20 100 10 10 10 100

H 20 18 b 95 8 10 10 95

6.27 A 20 18 90 8 10 10 90 90

B 20 18 90 8 10 10 90

C 20 18 90 8 10 10 90

D 20 18 90 8 10 10 90

15.4 A 21 14 67 5 9 9 67 88

B 20 19 95 9 10 10 95

C 20 20 100 10 10 10 100

D 20 18 90 8 10 10 90

50.1 A 20 19 95 9 10 10 95 95

B 20 20 100 10 10 10 100

C 20 19 95 9 10 10 95

D 20 18 90 8 10 10 90

156 A 20 18 90 8 10 10 90 91

B 20 19 95 9 10 10 95

C 20 18 90 8 10 10 90

D 20 18 90 8 10 10 90 a Five NF stage 62 animals were sampled and the remaining were removed at cull to 10 animals on study day 68.

b One organism could not be accounted for on study day 13 after cleaning and siphoning the test chamber and was not considered an exposure-related mortality.
ABC Study No. 66085 

Page 186 of 252 

APPENDIX H – NF STAGE 62 DATA
ABC Study No. 66085 

Page 187 of 252 

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 38 0.814 19

0 (Control) A 2 39 1.137 21

0 (Control) A 3 40 0.832 19

0 (Control) A 4 42 1.066 21

0 (Control) A 5 42 1.255 21

0 (Control) B 1 38 0.945 21

0 (Control) B 2 42 0.854 20

0 (Control) B 3 43 1.141 21

0 (Control) B 4 44 1.188 22

0 (Control) B 5 44 0.921 20

0 (Control) C 1 36 0.950 20

0 (Control) C 2 38 1.225 21

0 (Control) C 3 39 1.239 22

0 (Control) C 4 39 1.298 21

0 (Control) C 5 42 1.382 23

0 (Control) D 1 39 1.098 21

0 (Control) D 2 42 1.010 20

0 (Control) D 3 43 1.237 22

0 (Control) D 4 44 1.051 21

0 (Control) D 5 50 1.189 22

0 (Control) E 1 37 0.887 19

0 (Control) E 2 38 1.174 21

0 (Control) E 3 38 1.004 21

0 (Control) E 4 39 1.303 23

0 (Control) E 5 41 1.305 21

0 (Control) F 1 37 1.111 20

0 (Control) F 2 38 1.150 21

0 (Control) F 3 39 1.488 22

0 (Control) F 4 40 1.212 22

0 (Control) F 5 41 1.204 21

0 (Control) G 1 42 1.156 22

0 (Control) G 2 43 1.017 21

0 (Control) G 3 43 1.113 21

0 (Control) G 4 44 0.713 19

0 (Control) G 5 48 0.979 21

0 (Control) H 1 38 0.914 20

0 (Control) H 2 39 0.849 20

0 (Control) H 3 39 1.110 20

0 (Control) H 4 41 0.912 20

0 (Control) H 5 43 0.862 19
ABC Study No. 66085 

Page 188 of 252 

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)

6.27 A 1 36 0.907 20

6.27 A 2 37 1.044 21

6.27 A 3 37 1.192 21

6.27 A 4 39 1.353 23

6.27 A 5 40 1.512 22

6.27 B 1 36 1.227 22

6.27 B 2 36 1.168 21

6.27 B 3 38 1.503 22

6.27 B 4 39 1.002 20

6.27 B 5 42 1.681 24

6.27 C 1 37 1.186 21

6.27 C 2 39 1.179 21

6.27 C 3 40 1.140 21

6.27 C 4 42 1.422 22

6.27 C 5 43 1.398 22

6.27 D 1 38 1.045 20

6.27 D 2 39 1.123 20

6.27 D 3 40 1.227 22

6.27 D 4 42 1.150 21

6.27 D 5 43 0.742 19

15.4 A 1 39 1.152 22

15.4 A 2 40 1.050 21

15.4 A 3 40 1.082 21

15.4 A 4 43 1.203 22

15.4 A 5 44 1.347 22

15.4 B 1 40 1.184 21

15.4 B 2 42 1.454 22

15.4 B 3 43 1.473 22

15.4 B 4 44 1.268 21

15.4 B 5 44 0.928 20

15.4 C 1 36 1.212 22

15.4 C 2 37 1.091 20

15.4 C 3 38 1.208 22

15.4 C 4 39 0.846 19

15.4 C 5 41 0.982 20

15.4 D 1 36 0.799 19

15.4 D 2 37 0.816 19

15.4 D 3 39 0.723 19

15.4 D 4 40 0.805 19

15.4 D 5 42 0.879 20
ABC Study No. 66085 

Page 189 of 252 

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)

50.1 A 1 37 1.107 21

50.1 A 2 39 1.100 20

50.1 A 3 40 0.870 20

50.1 A 4 42 1.175 21

50.1 A 5 44 1.197 21

50.1 B 1 37 1.203 21

50.1 B 2 38 1.155 22

50.1 B 3 38 1.257 21

50.1 B 4 40 1.142 21

50.1 B 5 41 1.188 22

50.1 C 1 42 1.020 21

50.1 C 2 43 1.294 22

50.1 C 3 43 1.173 22

50.1 C 4 44 1.044 21

50.1 C 5 48 1.417 22

50.1 D 1 36 0.843 19

50.1 D 2 38 0.983 20

50.1 D 3 40 1.027 20

50.1 D 4 42 1.185 21

50.1 D 5 48 1.265 22

156 A 1 36 0.689 19

156 A 2 38 0.548 17

156 A 3 39 0.828 19

156 A 4 39 0.832 19

156 A 5 42 1.268 21

156 B 1 37 0.877 19

156 B 2 39 0.718 18

156 B 3 40 0.982 20

156 B 4 40 1.029 20

156 B 5 42 0.886 20

156 C 1 38 0.942 19

156 C 2 42 0.955 21

156 C 3 43 0.955 19

156 C 4 43 1.014 21

156 C 5 44 1.131 21

156 D 1 37 0.760 18

156 D 2 39 0.930 19

156 D 3 39 0.945 19

156 D 4 40 0.935 20

156 D 5 42 1.257 22
ABC Study No. 66085 

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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 38 11 40

0 (Control) A 2 39 12 41

0 (Control) A 3 39 13 41

0 (Control) A 4 39 14 41

0 (Control) A 5 39 15 42

0 (Control) A 6 39 16 42

0 (Control) A 7 39 17 43

0 (Control) A 8 39 18 43

0 (Control) A 9 40 19 46

0 (Control) A 10 40 20 --

0 (Control) B 1 38 11 43

0 (Control) B 2 39 12 44

0 (Control) B 3 39 13 44

0 (Control) B 4 40 14 44

0 (Control) B 5 41 15 44

0 (Control) B 6 41 16 45

0 (Control) B 7 42 17 46

0 (Control) B 8 42 18 46

0 (Control) B 9 43 19 51

0 (Control) B 10 43 20 --

0 (Control) C 1 36 11 39

0 (Control) C 2 37 12 39

0 (Control) C 3 37 13 39

0 (Control) C 4 37 14 39

0 (Control) C 5 38 15 40

0 (Control) C 6 38 16 40

0 (Control) C 7 39 17 42

0 (Control) C 8 39 18 43

0 (Control) C 9 39 19 45

0 (Control) C 10 39 20 --

0 (Control) D 1 39 11 44

0 (Control) D 2 40 12 44

0 (Control) D 3 40 13 45

0 (Control) D 4 41 14 48

0 (Control) D 5 41 15 49

0 (Control) D 6 41 16 50

0 (Control) D 7 41 17 50

0 (Control) D 8 42 18 --

0 (Control) D 9 42 19 --

0 (Control) D 10 43 20 --

‘---’ denotes a mortality that occurred before NF stage 62
ABC Study No. 66085 

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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

0 (Control) E 1 37 11 39

0 (Control) E 2 37 12 39

0 (Control) E 3 38 13 39

0 (Control) E 4 38 14 39

0 (Control) E 5 38 15 39

0 (Control) E 6 38 16 41

0 (Control) E 7 38 17 41

0 (Control) E 8 38 18 42

0 (Control) E 9 38 19 --

0 (Control) E 10 39 20 --

0 (Control) F 1 37 11 40

0 (Control) F 2 38 12 40

0 (Control) F 3 38 13 40

0 (Control) F 4 38 14 40

0 (Control) F 5 38 15 41

0 (Control) F 6 38 16 41

0 (Control) F 7 38 17 41

0 (Control) F 8 38 18 --

0 (Control) F 9 39 19 --

0 (Control) F 10 40 20 --

0 (Control) G 1 41 11 44

0 (Control) G 2 42 12 45

0 (Control) G 3 42 13 45

0 (Control) G 4 42 14 45

0 (Control) G 5 43 15 46

0 (Control) G 6 43 16 47

0 (Control) G 7 43 17 48

0 (Control) G 8 43 18 50

0 (Control) G 9 43 19 54

0 (Control) G 10 44 20 58

0 (Control) H 1 38 11 40

0 (Control) H 2 38 12 40

0 (Control) H 3 38 13 41

0 (Control) H 4 39 14 41

0 (Control) H 5 39 15 41

0 (Control) H 6 39 16 41

0 (Control) H 7 39 17 41

0 (Control) H 8 39 18 43

0 (Control) H 9 39 19 --

0 (Control) H 10 40 20 IL

‘---’ denotes a mortality that occurred before NF stage 62; IL = inadvertently lost before NF stage 62
ABC Study No. 66085 

Page 192 of 252 

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

6.27 A 1 36 11 38

6.27 A 2 36 12 38

6.27 A 3 36 13 39

6.27 A 4 37 14 39

6.27 A 5 37 15 39

6.27 A 6 37 16 39

6.27 A 7 37 17 40

6.27 A 8 37 18 56

6.27 A 9 37 19 --

6.27 A 10 38 20 --

6.27 B 1 36 11 38

6.27 B 2 36 12 39

6.27 B 3 36 13 39

6.27 B 4 36 14 40

6.27 B 5 36 15 40

6.27 B 6 37 16 41

6.27 B 7 37 17 42

6.27 B 8 37 18 47

6.27 B 9 38 19 --

6.27 B 10 38 20 --

6.27 C 1 37 11 40

6.27 C 2 38 12 41

6.27 C 3 38 13 41

6.27 C 4 38 14 41

6.27 C 5 39 15 41

6.27 C 6 39 16 42

6.27 C 7 39 17 43

6.27 C 8 39 18 54

6.27 C 9 40 19 --

6.27 C 10 40 20 --

6.27 D 1 38 11 41

6.27 D 2 38 12 41

6.27 D 3 39 13 41

6.27 D 4 39 14 41

6.27 D 5 39 15 42

6.27 D 6 40 16 42

6.27 D 7 40 17 43

6.27 D 8 40 18 44

6.27 D 9 40 19 --

6.27 D 10 41 20 --

‘---’ denotes a mortality that occurred before NF stage 62
ABC Study No. 66085 

Page 193 of 252 

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

15.4 A 1 39 11 41

15.4 A 2 39 12 41

15.4 A 3 39 13 43

15.4 A 4 39 14 44

15.4 A 5 40 15 --

15.4 A 6 40 16 --

15.4 A 7 40 17 --

15.4 A 8 40 18 --

15.4 A 9 40 19 --

15.4 A 10 40 20 --

15.4 B 1 40 11 42

15.4 B 2 40 12 43

15.4 B 3 40 13 44

15.4 B 4 41 14 44

15.4 B 5 41 15 45

15.4 B 6 41 16 46

15.4 B 7 41 17 48

15.4 B 8 41 18 48

15.4 B 9 41 19 48

15.4 B 10 42 20 --

15.4 C 1 36 11 39

15.4 C 2 37 12 39

15.4 C 3 37 13 39

15.4 C 4 37 14 40

15.4 C 5 37 15 40

15.4 C 6 38 16 40

15.4 C 7 38 17 41

15.4 C 8 38 18 42

15.4 C 9 38 19 43

15.4 C 10 38 20 44

15.4 D 1 36 11 39

15.4 D 2 37 12 40

15.4 D 3 37 13 40

15.4 D 4 37 14 40

15.4 D 5 37 15 40

15.4 D 6 38 16 42

15.4 D 7 39 17 42

15.4 D 8 39 18 43

15.4 D 9 39 19 --

15.4 D 10 39 20 --

‘---’ denotes a mortality that occurred before NF stage 62
ABC Study No. 66085 

Page 194 of 252 

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

50.1 A 1 37 11 41

50.1 A 2 38 12 41

50.1 A 3 38 13 41

50.1 A 4 39 14 41

50.1 A 5 39 15 42

50.1 A 6 39 16 42

50.1 A 7 39 17 42

50.1 A 8 39 18 44

50.1 A 9 40 19 45

50.1 A 10 40 20 --

50.1 B 1 37 11 40

50.1 B 2 37 12 40

50.1 B 3 37 13 40

50.1 B 4 37 14 40

50.1 B 5 38 15 40

50.1 B 6 38 16 41

50.1 B 7 38 17 41

50.1 B 8 38 18 42

50.1 B 9 38 19 43

50.1 B 10 39 20 43

50.1 C 1 41 11 44

50.1 C 2 41 12 44

50.1 C 3 41 13 44

50.1 C 4 41 14 45

50.1 C 5 42 15 46

50.1 C 6 42 16 46

50.1 C 7 43 17 47

50.1 C 8 43 18 48

50.1 C 9 43 19 52

50.1 C 10 44 20 --

50.1 D 1 36 11 41

50.1 D 2 37 12 41

50.1 D 3 38 13 41

50.1 D 4 38 14 41

50.1 D 5 38 15 42

50.1 D 6 38 16 43

50.1 D 7 39 17 48

50.1 D 8 39 18 50

50.1 D 9 40 19 --

50.1 D 10 40 20 --

‘---’ denotes a mortality that occurred before NF stage 62
ABC Study No. 66085 

Page 195 of 252 

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

156 A 1 36 11 39

156 A 2 37 12 39

156 A 3 37 13 39

156 A 4 38 14 40

156 A 5 38 15 41

156 A 6 38 16 41

156 A 7 38 17 42

156 A 8 39 18 47

156 A 9 39 19 --

156 A 10 39 20 --

156 B 1 37 11 40

156 B 2 37 12 40

156 B 3 38 13 40

156 B 4 39 14 41

156 B 5 39 15 41

156 B 6 39 16 42

156 B 7 39 17 43

156 B 8 40 18 44

156 B 9 40 19 44

156 B 10 40 20 --

156 C 1 38 11 43

156 C 2 39 12 43

156 C 3 41 13 43

156 C 4 41 14 44

156 C 5 41 15 44

156 C 6 41 16 44

156 C 7 42 17 44

156 C 8 42 18 47

156 C 9 42 19 --

156 C 10 42 20 --

156 D 1 37 11 40

156 D 2 37 12 40

156 D 3 39 13 40

156 D 4 39 14 40

156 D 5 39 15 40

156 D 6 39 16 42

156 D 7 39 17 42

156 D 8 39 18 44

156 D 9 39 19 --

156 D 10 40 20 --

‘---’ denotes a mortality that occurred before NF stage 62
ABC Study No. 66085 

Page 196 of 252 

APPENDIX I – NF STAGE 66 CULL DATA 
ABC Study No. 66085 

Page 197 of 252 

Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull

Treatment

(μg/L) Replicate Juvenile Weight (g)

Snout-to-Vent 

Length (mm) Phenotypic Sex

0 (Control) A 1 3.607 30 INDETERMINATE

0 (Control) A 2 3.395 30 FEMALE

0 (Control) A 3 2.874 28 FEMALE

0 (Control) A 4 2.243 25 MALE

0 (Control) A 5 --- --- ---

0 (Control) B 1 3.430 29 FEMALE

0 (Control) B 2 3.079 30 FEMALE

0 (Control) B 3 2.531 27 MALE

0 (Control) B 4 1.694 25 FEMALE

0 (Control) B 5 --- --- ---

0 (Control) C 1 2.726 27 FEMALE

0 (Control) C 2 5.648 35 FEMALE

0 (Control) C 3 2.772 28 MALE

0 (Control) C 4 3.821 30 FEMALE

0 (Control) C 5 --- --- ---

0 (Control) D 1 3.946 30 MALE

0 (Control) D 2 2.818 29 FEMALE

0 (Control) D 3 --- --- ---

0 (Control) D 4 --- --- ---

0 (Control) D 5 --- --- ---

0 (Control) E 1 3.349 30 FEMALE

0 (Control) E 2 3.888 31 MALE

0 (Control) E 3 3.474 29 FEMALE

0 (Control) E 4 --- --- ---

0 (Control) E 5 --- --- ---

0 (Control) F 1 4.814 33 FEMALE

0 (Control) F 2 4.297 31 MALE

0 (Control) F 3 --- --- ---

0 (Control) F 4 --- --- ---

0 (Control) F 5 --- --- ---

0 (Control) G 1 1.608 24 FEMALE

0 (Control) G 2 2.354 26 MALE

0 (Control) G 3 1.723 24 MALE

0 (Control) G 4 1.652 24 MALE

0 (Control) G 5 1.599 24 FEMALE

0 (Control) H 1 3.078 29 FEMALE

0 (Control) H 2 2.159 26 FEMALE

0 (Control) H 3 2.851 28 MALE

0 (Control) H 4 --- --- ---

0 (Control) H 5 --- --- ---

‘---’ denotes a juvenile was not available for this sample
ABC Study No. 66085 

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Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull (cont.)

Treatment

(μg/L) Replicate Juvenile

Weight 

(g)

Snout-to-Vent 

Length (mm) Phenotypic Sex

6.27 A 1 3.897 32 FEMALE

6.27 A 2 3.195 31 MALE

6.27 A 3 5.153 35 FEMALE

6.27 A 4 --- . .

6.27 A 5 --- . .

6.27 B 1 2.938 31 FEMALE

6.27 B 2 3.114 31 MALE

6.27 B 3 2.469 29 MALE

6.27 B 4 --- --- ---

6.27 B 5 --- --- ---

6.27 C 1 2.456 29 INDETERMINATE

6.27 C 2 3.006 30 MALE

6.27 C 3 2.665 30 MALE

6.27 C 4 --- --- ---

6.27 C 5 --- --- ---

6.27 D 1 2.252 29 FEMALE

6.27 D 2 4.025 31 FEMALE

6.27 D 3 2.867 30 MALE

6.27 D 4 --- --- ---

6.27 D 5 --- --- ---

15.4 A 1 --- --- ---

15.4 A 2 --- --- ---

15.4 A 3 --- --- ---

15.4 A 4 --- --- ---

15.4 A 5 --- --- ---

15.4 B 1 1.690 25 FEMALE

15.4 B 2 4.208 37 FEMALE

15.4 B 3 2.229 28 FEMALE

15.4 B 4 2.213 26 FEMALE

15.4 B 5 --- --- ---

15.4 C 1 1.962 27 FEMALE

15.4 C 2 3.360 31 FEMALE

15.4 C 3 4.879 33 FEMALE

15.4 C 4 2.826 27 FEMALE

15.4 C 5 1.948 27 MALE

15.4 D 1 2.108 28 MALE

15.4 D 2 1.519 25 FEMALE

15.4 D 3 0.985 21 MALE

15.4 D 4 --- --- ---

15.4 D 5 --- --- ---

‘---’ denotes a juvenile was not available for this sample
ABC Study No. 66085 

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Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull (cont.)

Treatment

(μg/L) Replicate Juvenile

Weight 

(g)

Snout-to-Vent 

Length (mm) Phenotypic Sex

50.1 A 1 2.256 28 FEMALE

50.1 A 2 3.186 29 MALE

50.1 A 3 2.120 27 MALE

50.1 A 4 1.684 25 MALE

50.1 A 5 --- --- ---

50.1 B 1 2.901 30 MALE

50.1 B 2 2.056 28 FEMALE

50.1 B 3 3.574 31 FEMALE

50.1 B 4 3.154 30 FEMALE

50.1 B 5 2.884 31 FEMALE

50.1 C 1 2.389 30 MALE

50.1 C 2 2.048 26 FEMALE

50.1 C 3 1.708 25 FEMALE

50.1 C 4 1.131 24 MALE

50.1 C 5 --- --- ---

50.1 D 1 2.627 29 MALE

50.1 D 2 2.220 28 MALE

50.1 D 3 2.396 27 MALE

50.1 D 4 --- --- ---

50.1 D 5 --- --- ---

156 A 1 2.649 29 MALE

156 A 2 2.026 27 MALE

156 A 3 1.402 25 MALE

156 A 4 --- --- ---

156 A 5 --- --- ---

156 B 1 2.183 28 MALE

156 B 2 1.868 31 MALE

156 B 3 1.730 26 MALE

156 B 4 1.682 25 FEMALE

156 B 5 --- --- ---

156 C 1 1.809 26 FEMALE

156 C 2 1.959 27 MALE

156 C 3 1.393 25 FEMALE

156 C 4 --- --- ---

156 C 5 --- --- ---

156 D 1 3.143 31 MALE

156 D 2 3.187 30 FEMALE

156 D 3 1.979 26 MALE

156 D 4 --- --- ---

156 D 5 --- --- ---

‘---’ denotes a juvenile was not available for this sample
ABC Study No. 66085 

Page 200 of 252 

APPENDIX J – TERMINATION DATA – GROWTH AND GENDER
ABC Study No. 66085 

Page 201 of 252 

Appendix J-1. Replicate Weight, Length, and Sex Data at Termination 

Treatment

(μg/L) Replicate Juvenile Weight (g) Snout-to-Vent Length (mm)

Phenotypic/Genotypic

Sex a

0 (Control) A CA1-T 10.449 49 FEMALE

0 (Control) A CA2-T 7.216 39 FEMALE 

0 (Control) A CA3-T 9.902 41 MALE

0 (Control) A CA4-T 11.884 43 MALE

0 (Control) A CA5-T 13.842 46 MALE

0 (Control) A CA6-T 10.944 41 MALE

0 (Control) A CA7-T 7.150 37 MALE

0 (Control) A CA8-T 8.444 41 MALE

0 (Control) A CA9-T 10.631 44 MALE

0 (Control) A CA10-T 7.017 40 MALE

0 (Control) B CB1-T 10.912 43 FEMALE

0 (Control) B CB2-T 9.964 40 MALE

0 (Control) B CB3-T 11.187 44 FEMALE

0 (Control) B CB4-T 5.193 33 MALE

0 (Control) B CB5-T 18.508 50 MALE

0 (Control) B CB6-T 5.437 35 MALE

0 (Control) B CB7-T 9.404 42 FEMALE 

0 (Control) B CB8-T 15.856 49 MALE

0 (Control) B CB9-T 12.980 45 FEMALE

0 (Control) B CB10-T 6.995 37 FEMALE 

0 (Control) C CC1-T 15.379 48 MALE

0 (Control) C CC2-T 9.779 41 MALE

0 (Control) C CC3-T 7.476 38 MALE

0 (Control) C CC4-T 12.542 46 FEMALE

0 (Control) C CC5-T 9.587 42 MALE

0 (Control) C CC6-T 20.856 53 FEMALE

0 (Control) C CC7-T 11.908 44 MALE

0 (Control) C CC8-T 15.526 49 MALE

0 (Control) C CC9-T 14.765 47 MALE

0 (Control) C CC10-T 11.780 43 FEMALE 

0 (Control) D CD1-T 12.513 45 MALE

0 (Control) D CD2-T 21.852 56 FEMALE 

0 (Control) D CD3-T 12.397 45 FEMALE

0 (Control) D CD4-T 3.764 31 FEMALE

0 (Control) D CD5-T 10.268 43 FEMALE 

0 (Control) D CD6-T 12.275 46 MALE

0 (Control) D CD7-T 8.713 42 FEMALE 

0 (Control) D CD8-T 14.511 48 FEMALE

0 (Control) D CD9-T 17.036 49 FEMALE

0 (Control) D CD10-T 9.601 42 FEMALE a Genotypic sex confirmed the phenotypic sex
ABC Study No. 66085 

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Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued) 

Treatment

(μg/L) Replicate Juvenile Weight (g) Snout-to-Vent Length (mm)

Phenotypic/Genotypic

Sex a

0 (Control) E CE1-T 8.191 39 MALE

0 (Control) E CE2-T 20.472 53 FEMALE

0 (Control) E CE3-T 15.332 49 FEMALE

0 (Control) E CE4-T 13.235 44 MALE

0 (Control) E CE5-T 14.549 43 MALE

0 (Control) E CE6-T 14.091 48 MALE

0 (Control) E CE7-T 13.835 47 FEMALE

0 (Control) E CE8-T 22.758 55 FEMALE

0 (Control) E CE9-T 8.918 40 MALE

0 (Control) E CE10-T 6.084 36 MALE

0 (Control) F CF1-T 10.263 43 FEMALE

0 (Control) F CF2-T 16.313 49 FEMALE

0 (Control) F CF3-T 16.985 50 MALE

0 (Control) F CF4-T 15.825 50 FEMALE

0 (Control) F CF5-T 11.289 43 MALE

0 (Control) F CF6-T 7.865 38 MALE

0 (Control) F CF7-T 11.826 45 FEMALE

0 (Control) F CF8-T 14.935 48 FEMALE

0 (Control) F CF9-T 11.660 44 MALE

0 (Control) F CF10-T 4.231 32 MALE

0 (Control) G CG1-T 10.075 43 MALE

0 (Control) G CG2-T 27.468 58 FEMALE

0 (Control) G CG3-T 14.389 48 FEMALE

0 (Control) G CG4-T 11.008 43 MALE

0 (Control) G CG5-T 19.297 50 MALE

0 (Control) G CG6-T 14.503 48 MALE

0 (Control) G CG7-T 9.523 42 FEMALE

0 (Control) G CG8-T 11.407 43 MALE

0 (Control) G CG9-T 7.915 40 FEMALE

0 (Control) G CG10-T 15.605 48 MALE

0 (Control) H CH1-T 16.728 50 FEMALE

0 (Control) H CH2-T 20.414 53 FEMALE

0 (Control) H CH3-T 12.040 43 MALE

0 (Control) H CH4-T 15.063 47 FEMALE

0 (Control) H CH5-T 13.938 46 MALE

0 (Control) H CH6-T 14.074 47 MALE

0 (Control) H CH7-T 16.774 49 FEMALE

0 (Control) H CH8-T 14.737 47 FEMALE

0 (Control) H CH9-T 11.557 43 MALE

0 (Control) H CH10-T 13.939 46 FEMALE

a Genotypic sex confirmed the phenotypic sex
ABC Study No. 66085 

Page 203 of 252 

Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued) 

Treatment

(μg/L) Replicate Juvenile Weight (g) Snout-to-Vent Length (mm)

Phenotypic/Genotypic

Sex a

6.27 A L1A1-T 8.949 42 MALE

6.27 A L1A2-T 2.218 25 FEMALE

6.27 A L1A3-T 15.207 48 MALE

6.27 A L1A4-T 16.864 49 MALE

6.27 A L1A5-T 16.797 50 FEMALE

6.27 A L1A6-T 19.396 52 MALE

6.27 A L1A7-T 15.038 48 MALE

6.27 A L1A8-T 17.784 51 FEMALE

6.27 A L1A9-T 10.706 42 FEMALE

6.27 A L1A10-T 12.572 45 FEMALE

6.27 B L1B1-T 11.034 43 MALE

6.27 B L1B2-T 6.241 34 MALE

6.27 B L1B3-T 5.405 35 FEMALE

6.27 B L1B4-T 10.840 50 MALE

6.27 B L1B5-T 12.055 43 MALE

6.27 B L1B6-T 18.299 50 FEMALE

6.27 B L1B7-T 11.455 43 FEMALE

6.27 B L1B8-T 20.692 51 MALE

6.27 B L1B9-T 19.467 52 FEMALE

6.27 B L1B10-T 13.277 45 FEMALE

6.27 C L1C1-T 9.439 41 MALE

6.27 C L1C2-T 19.113 52 MALE

6.27 C L1C3-T 18.422 50 MALE

6.27 C L1C4-T 16.890 49 MALE

6.27 C L1C5-T 10.360 42 MALE

6.27 C L1C6-T 13.348 47 FEMALE

6.27 C L1C7-T 10.904 42 FEMALE

6.27 C L1C8-T 5.836 36 FEMALE

6.27 C L1C9-T 9.370 39 MALE

6.27 C L1C10-T 3.005 30 MALE

6.27 D L1D1-T 8.011 40 FEMALE

6.27 D L1D2-T 12.745 46 FEMALE

6.27 D L1D3-T 15.974 47 MALE

6.27 D L1D4-T 19.955 51 FEMALE

6.27 D L1D5-T 12.143 45 FEMALE

6.27 D L1D6-T 24.688 58 MALE

6.27 D L1D7-T 11.137 42 MALE

6.27 D L1D8-T 9.334 41 MALE

6.27 D L1D9-T 10.428 43 FEMALE

6.27 D L1D10-T 5.949 35 MALE

a Genotypic sex confirmed the phenotypic sex
ABC Study No. 66085 

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Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued) 

Treatment

(μg/L) Replicate Juvenile Weight (g) Snout-to-Vent Length (mm)

Phenotypic/Genotypic

Sex a

15.4 A L2A1-T 6.851 37 FEMALE

15.4 A L2A2-T 9.714 42 FEMALE

15.4 A L2A3-T 9.396 42 FEMALE

15.4 A L2A4-T 18.293 50 MALE

15.4 A L2A5-T 11.069 45 MALE

15.4 A L2A6-T 11.608 43 FEMALE

15.4 A L2A7-T 9.644 40 FEMALE

15.4 A L2A8-T 5.843 35 MALE

15.4 A L2A9-T 13.373 46 MALE

15.4 A L210-T --- --- ---

15.4 B L2B1-T 10.355 44 MALE

15.4 B L2B2-T 22.894 53 FEMALE

15.4 B L2B3-T 12.179 45 MALE

15.4 B L2B4-T 8.809 41 FEMALE

15.4 B L2B5-T 12.935 45 MALE

15.4 B L2B6-T 6.926 37 MALE

15.4 B L2B7-T 17.136 49 FEMALE

15.4 B L2B8-T 17.262 49 FEMALE

15.4 B L2B9-T 13.281 45 MALE

15.4 B L2B10-T 6.432 47 MALE

15.4 C L2C1-T 15.050 46 FEMALE

15.4 C L2C2-T 14.374 47 MALE

15.4 C L2C3-T 9.429 41 MALE

15.4 C L2C4-T 22.699 51 MALE

15.4 C L2C5-T 17.515 49 MALE

15.4 C L2C6-T 17.385 50 MALE

15.4 C L2C7-T 14.242 46 FEMALE

15.4 C L2C8-T 17.681 49 MALE

15.4 C L2C9-T 16.135 49 FEMALE

15.4 C L2C10-T 18.225 50 MALE

15.4 D L2D1-T 13.231 45 MALE

15.4 D L2D2-T 11.563 44 MALE

15.4 D L2D3-T 5.817 34 MALE

15.4 D L2D4-T 4.539 33 MALE

15.4 D L2D5-T 21.190 51 MALE

15.4 D L2D6-T 7.780 39 FEMALE

15.4 D L2D7-T 13.488 45 MALE

15.4 D L2D8-T 10.128 41 FEMALE

15.4 D L2D9-T 14.279 44 FEMALE

15.4 D L2D10-T 3.797 31 FEMALE

a Genotypic sex confirmed the phenotypic sex; ‘---’ denotes a juvenile was not available for sample
ABC Study No. 66085 

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Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued) 

Treatment

(μg/L) Replicate Juvenile Weight (g) Snout-to-Vent Length (mm)

Phenotypic/Genotypic

Sex a

50.1 A L3A1-T 14.350 47 FEMALE

50.1 A L3A2-T 10.970 42 MALE

50.1 A L3A3-T 19.999 49 FEMALE

50.1 A L3A4-T 8.800 41 FEMALE

50.1 A L3A5-T 10.492 41 MALE

50.1 A L3A6-T 13.770 45 MALE

50.1 A L3A7-T 6.560 37 FEMALE

50.1 A L3A8-T 7.182 36 FEMALE

50.1 A L3A9-T 7.739 38 MALE

50.1 A L3A10-T 4.708 33 FEMALE

50.1 B L3B1-T 18.686 50 MALE

50.1 B L3B2-T 20.590 54 FEMALE

50.1 B L3B3-T 11.173 43 MALE

50.1 B L3B4-T 12.986 44 FEMALE

50.1 B L3B5-T 9.348 40 MALE

50.1 B L3B6-T 14.883 46 FEMALE

50.1 B L3B7-T 4.466 33 FEMALE

50.1 B L3B8-T 15.332 46 FEMALE

50.1 B L3B9-T 7.311 38 FEMALE

50.1 B L3B10-T 6.687 36 FEMALE

50.1 C L3C1-T 11.332 44 MALE

50.1 C L3C2-T 8.683 43 FEMALE

50.1 C L3C3-T 11.659 45 MALE

50.1 C L3C4-T 12.731 44 FEMALE

50.1 C L3C5-T 9.879 42 FEMALE

50.1 C L3C6-T 16.359 50 MALE

50.1 C L3C7-T 12.243 44 FEMALE

50.1 C L3C8-T 10.164 41 MALE

50.1 C L3C9-T 12.169 42 FEMALE

50.1 C L3C10-T 11.350 43 FEMALE

50.1 D L3D1-T 18.874 53 FEMALE

50.1 D L3D2-T 15.681 48 FEMALE

50.1 D L3D3-T 12.232 45 FEMALE

50.1 D L3D4-T 24.565 56 FEMALE

50.1 D L3D5-T 1.688 25 FEMALE

50.1 D L3D6-T 9.475 41 FEMALE

50.1 D L3D7-T 12.879 45 MALE

50.1 D L3D8-T 15.870 48 FEMALE

50.1 D L3D9-T 14.641 47 FEMALE

50.1 D L3D10-T 17.928 49 MALE

a Genotypic sex confirmed the phenotypic sex
ABC Study No. 66085 

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Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued) 

Treatment

(μg/L) Replicate Juvenile Weight (g) Snout-to-Vent Length (mm)

Phenotypic/Genotypic

Sex a

156 A L4A1-T 7.319 31 FEMALE

156 A L4A2-T 19.734 52 FEMALE

156 A L4A3-T 5.159 33 MALE

156 A L4A4-T 13.556 46 MALE

156 A L4A5-T 10.536 42 FEMALE

156 A L4A6-T 15.749 47 FEMALE

156 A L4A7-T 11.899 43 FEMALE

156 A L4A8-T 12.292 44 FEMALE

156 A L4A9-T 12.382 45 FEMALE

156 A L4A10-T 14.098 47 MALE

156 B L4B1-T 12.305 42 FEMALE

156 B L4B2-T 15.595 46 FEMALE

156 B L4B3-T 11.846 44 FEMALE

156 B L4B4-T 18.316 50 FEMALE

156 B L4B5-T 7.853 39 MALE

156 B L4B6-T 14.598 46 FEMALE

156 B L4B7-T 9.879 42 FEMALE

156 B L4B8-T 9.685 40 MALE

156 B L4B9-T 9.609 41 FEMALE

156 B L4B10-T 6.054 35 MALE

156 C L4C1-T 5.896 35 FEMALE

156 C L4C2-T 17.163 50 MALE

156 C L4C3-T 1.188 24 FEMALE

156 C L4C4-T 17.022 51 FEMALE

156 C L4C5-T 14.261 47 FEMALE

156 C L4C6-T 2.465 29 MALE

156 C L4C7-T 8.866 39 FEMALE

156 C L4C8-T 4.396 32 FEMALE

156 C L4C9-T 4.261 33 FEMALE

156 C L4C10-T 8.171 40 FEMALE

156 D L4D1-T 14.774 48 FEMALE

156 D L4D2-T 11.300 45 FEMALE

156 D L4D3-T 15.035 45 MALE

156 D L4D4-T 14.093 46 MALE

156 D L4D5-T 15.489 48 MALE

156 D L4D6-T 13.400 46 FEMALE

156 D L4D7-T 14.151 48 MALE

156 D L4D8-T 13.480 46 MALE

156 D L4D9-T 10.205 43 MALE

156 D L4D10-T 13.259 45 MALE

a

 Genotypic sex confirmed the phenotypic sex
ABC Study No. 66085 

Page 207 of 252 

APPENDIX K – TERMINATION DATA – NUPTIAL PAD AND LIVER 
ABC Study No. 66085 

Page 208 of 252 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data 

Frog Code Phenotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight (g)

Cassette + 

Liver Weight 

(g)

Calculated Liver 

Weight (g)

CA1-T F absent 3.4461 4.7622 1.3161

CA2-T F absent 3.4558 3.8147 0.3589

CA3-T M absent 3.4633 4.0498 0.5865

CA4-T M absent 3.4579 4.2314 0.7735

CA5-T M absent 3.4878 4.5147 1.0269

CA6-T M absent 3.4402 3.9820 0.5418

CA7-T M absent 3.4632 3.9076 0.4444

CA8-T M absent 3.4688 3.9110 0.4422

CA9-T M absent 3.4577 4.0151 0.5574

CA10-T M absent 3.4472 3.7950 0.3478

CB1-T F absent 3.4764 4.1307 0.6543

CB2-T M present 3.4643 3.9740 0.5097

CB3-T F absent 3.4743 4.1005 0.6262

CB4-T M absent 3.4572 3.6216 0.1644

CB5-T M absent 3.4795 4.7351 1.2556

CB6-T M absent 3.4471 3.6431 0.1960

CB7-T F absent 3.4547 4.0420 0.5873

CB8-T M present 3.4931 4.4928 0.9997

CB9-T F absent 3.4611 4.2404 0.7793

CB10-T F absent 3.4650 3.8268 0.3618

CC1-T M present 3.4894 4.6104 1.1210

CC2-T M absent 3.4625 4.0291 0.5666

CC3-T M absent 3.4666 3.8660 0.3994

CC4-T F absent 3.4502 4.3806 0.9304

CC5-T M absent 3.4683 4.0072 0.5389

CC6-T F present 3.4772 5.0792 1.6020

CC7-T M absent 3.4685 4.3022 0.8337

CC8-T M absent 3.4559 4.5462 1.0903

CC9-T M present 3.4813 4.4322 0.9509

CC10-T F absent 3.4922 4.3472 0.8550

CD1-T M absent 3.4807 4.2120 0.7313

CD2-T F present 3.4662 5.0810 1.6148

CD3-T F absent 3.4775 4.2146 0.7371

CD4-T F absent 3.4499 3.6245 0.1746

CD5-T F absent 3.4472 3.9874 0.5402

CD6-T M absent 3.4400 4.0798 0.6398

CD7-T F absent 3.4794 4.0357 0.5563

CD8-T F present 3.4692 4.3794 0.9102

CD9-T F present 3.4603 4.5880 1.1277
ABC Study No. 66085 

Page 209 of 252 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Frog Code Phenotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight (g)

Cassette + 

Liver Weight 

(g)

Calculated Liver 

Weight (g)

CD10-T F absent 3.4915 4.0024 0.5109

CE1-T M absent 3.4755 3.9638 0.4883

CE2-T F present 3.4582 4.8251 1.3669

CE3-T F absent 3.4631 4.3235 0.8604

CE4-T M present 3.4504 4.2671 0.8167

CE5-T M present 3.4890 4.2535 0.7645

CE6-T M absent 3.4882 4.2669 0.7787

CE7-T F absent 3.4917 4.1904 0.6987

CE8-T F present 3.4871 5.2029 1.7158

CE9-T M absent 3.4586 3.9009 0.4423

CE10-T M absent 3.4480 3.7734 0.3254

CF1-T F absent 3.4584 4.0348 0.5764

CF2-T F absent 3.4848 4.6778 1.1930

CF3-T M absent 3.4458 4.5113 1.0655

CF4-T F present 3.4565 4.4401 0.9836

CF5-T M present 3.4413 4.1001 0.6588

CF6-T M absent 3.4604 3.9195 0.4591

CF7-T F absent 3.4493 4.1692 0.7199

CF8-T F absent 3.4610 4.5172 1.0562

CF9-T M present 3.4640 4.0922 0.6282

CF10-T M present 3.4633 3.6571 0.1938

CG1-T M present 3.4589 4.0081 0.5492

CG2-T F present 3.4910 5.6254 2.1344

CG3-T F absent 3.4913 4.3375 0.8462

CG4-T M absent 3.4602 4.0852 0.6250

CG5-T M present 3.4839 4.9029 1.4190

CG6-T M present 3.4505 4.1834 0.7329

CG7-T F present 3.4918 4.0459 0.5541

CG8-T M present 3.4579 4.1258 0.6679

CG9-T F present 3.4869 3.8515 0.3646

CG10-T M present 3.4776 4.6473 1.1697

CH1-T F present 3.4508 4.5458 1.0950

CH2-T F present 3.4575 5.0023 1.5448

CH3-T M present 3.4611 4.0490 0.5879

CH4-T F absent 3.4811 4.4628 0.9817

CH5-T M absent 3.4503 4.2681 0.8178

CH6-T M present 3.4497 4.3053 0.8556

CH7-T F present 3.4905 4.5611 1.0706

CH8-T F present 3.4494 4.3775 0.9281
ABC Study No. 66085 

Page 210 of 252 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Frog Code Phenotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight (g)

Cassette + 

Liver Weight 

(g)

Calculated Liver 

Weight (g)

CH9-T M absent 3.4510 4.0114 0.5604

CH10-T F present 3.4452 4.4157 0.9705

L1A1-T M present 3.4926 3.9672 0.4746

L1A2-T F absent 3.4576 3.4947 0.0371

L1A3-T M present 3.4779 4.5367 1.0588

L1A4-T M absent 3.4592 4.5602 1.1010

L1A5-T F absent 3.4862 4.7132 1.2270

L1A6-T M present 3.4474 4.8023 1.3549

L1A7-T M absent 3.4394 4.3249 0.8855

L1A8-T F present 3.4544 4.5959 1.1415

L1A9-T F absent 3.4846 4.0206 0.5360

L1A10-T F present 3.4662 4.3634 0.8972

L1B1-T M absent 3.4805 4.0973 0.6168

L1B2-T M absent 3.4394 3.7354 0.2960

L1B3-T F absent 3.4493 3.6738 0.2245

L1B4-T M present 3.4913 4.7331 1.2418

L1B5-T M present 3.4462 4.3144 0.8682

L1B6-T F present 3.4758 4.7182 1.2424

L1B7-T F absent 3.4921 4.2516 0.7595

L1B8-T M present 3.4497 4.8375 1.3878

L1B9-T F present 3.4504 4.7532 1.3028

L1B10-T F present 3.4570 4.2632 0.8062

L1C1-T M absent 3.4413 4.0886 0.6473

L1C2-T M present 3.4488 4.6138 1.1650

L1C3-T M present 3.4736 4.6482 1.1746

L1C4-T M present 3.4862 4.6274 1.1412

L1C5-T M absent 3.4451 4.0503 0.6052

L1C6-T F present 3.4792 4.1576 0.6784

L1C7-T F present 3.4498 4.1528 0.7030

L1C8-T F absent 3.4568 3.7466 0.2898

L1C9-T M absent 3.4550 4.0251 0.5701

L1C10-T M absent 3.4858 3.6164 0.1306

L1D1-T F present 3.4601 3.8944 0.4343

L1D2-T F absent 3.4548 4.2535 0.7987

L1D3-T M present 3.4634 4.4685 1.0051

L1D4-T F present 3.4856 4.8708 1.3852

L1D5-T F absent 3.4570 4.2876 0.8306

L1D6-T M present 3.4461 5.5044 2.0583

L1D7-T M absent 3.4568 4.1930 0.7362
ABC Study No. 66085 

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Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Frog Code Phenotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight (g)

Cassette + 

Liver Weight 

(g)

Calculated Liver 

Weight (g)

L1D8-T M absent 3.4502 3.9200 0.4698

L1D9-T F absent 3.4913 4.2218 0.7305

L1D10-T M present 3.4413 3.6993 0.2580

L2A1-T F absent 3.4497 3.7982 0.3485

L2A2-T F absent 3.4905 3.9697 0.4792

L2A3-T F present 3.4798 4.0077 0.5279

L2A4-T M present 3.4461 3.4491 0.0030

L2A5-T M absent 3.4643 4.0691 0.6048

L2A6-T F absent 3.4511 4.1035 0.6524

L2A7-T F absent 3.4653 4.0164 0.5511

L2A8-T M absent 3.4448 3.6961 0.2513

L2A9-T M present 3.6399 4.4755 0.8356

L2A10-T --- --- --- --- ---

L2B1-T M absent 3.6440 4.2923 0.6483

L2B2-T F present 3.6626 5.1816 1.5190

L2B3-T M absent 3.6464 4.3373 0.6909

L2B4-T F present 3.6432 4.0771 0.4339

L2B5-T M absent 3.6299 4.3615 0.7316

L2B6-T M present 3.6393 3.9477 0.3084

L2B7-T F present 3.6340 4.7855 1.1515

L2B8-T F present 3.6340 4.7037 1.0697

L2B9-T M present 3.6520 4.3969 0.7449

L2B10-T M absent 3.6479 3.9814 0.3335

L2C1-T F present 3.6519 4.5970 0.9451

L2C2-T M absent 3.6507 4.4515 0.8008

L2C3-T M absent 3.6605 4.2274 0.5669

L2C4-T M present 3.6448 4.9475 1.3027

L2C5-T M present 3.6227 4.7572 1.1345

L2C6-T M present 3.6424 4.6722 1.0298

L2C7-T F absent 3.6321 4.4743 0.8422

L2C8-T M present 3.6423 4.6441 1.0018

L2C9-T F present 3.6434 4.4818 0.8384

L2C10-T M present 3.6375 4.9387 1.3012

L2D1-T M absent 3.6365 4.3709 0.7344

L2D2-T M present 3.6378 4.3344 0.6966

L2D3-T M absent 3.6458 3.8933 0.2475

L2D4-T M absent 3.6533 3.8148 0.1615

L2D5-T M present 3.6426 4.9660 1.3234

L2D6-T F absent 3.6411 3.9704 0.3293

‘---’ denotes a frog was not available for sample
ABC Study No. 66085 

Page 212 of 252 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Frog Code Phenotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight (g)

Cassette + 

Liver Weight 

(g)

Calculated Liver 

Weight (g)

L2D7-T M present 3.6275 4.3597 0.7322

L2D8-T F absent 3.6465 4.2179 0.5714

L2D9-T F absent 3.6573 4.6325 0.9752

L2D10-T F absent 3.6379 3.7565 0.1186

L3A1-T F present 3.6414 4.3918 0.7504

L3A2-T M present 3.6375 4.1548 0.5173

L3A3-T F present 3.6600 4.9568 1.2968

L3A4-T F absent 3.6407 4.1764 0.5357

L3A5-T M present 3.6391 4.0966 0.4575

L3A6-T M present 3.6403 4.3661 0.7258

L3A7-T F absent 3.6294 3.9957 0.3663

L3A8-T F absent 3.6367 3.9996 0.3629

L3A9-T M absent 3.6644 3.9850 0.3206

L3A10-T F present 3.6467 3.8512 0.2045

L3B1-T M present 3.6414 4.7794 1.1380

L3B2-T F present 3.6310 5.1098 1.4788

L3B3-T M absent 3.6399 4.2507 0.6108

L3B4-T F absent 3.6394 4.4262 0.7868

L3B5-T M absent 3.6385 4.0762 0.4377

L3B6-T F present 3.6381 4.5244 0.8863

L3B7-T F absent 3.6450 3.8946 0.2496

L3B8-T F present 3.6279 4.5138 0.8859

L3B9-T F present 3.6458 4.0446 0.3988

L3B10-T F absent 3.6645 3.9915 0.3270

L3C1-T M present 3.6675 4.2402 0.5727

L3C2-T F present 3.6635 4.0041 0.3406

L3C3-T M absent 3.6368 4.2122 0.5754

L3C4-T F present 3.6246 4.3144 0.6898

L3C5-T F absent 3.6542 4.1549 0.5007

L3C6-T M present 3.6393 4.4648 0.8255

L3C7-T F present 3.6634 4.3281 0.6647

L3C8-T M present 3.6360 4.3200 0.6840

L3C9-T F absent 3.6434 4.2896 0.6462

L3C10-T F present 3.6385 4.3625 0.7240

L3D1-T F present 3.6320 4.6189 0.9869

L3D2-T F present 3.6391 4.6184 0.9793

L3D3-T F present 3.6419 4.2910 0.6491

L3D4-T F present 3.6618 5.2665 1.6047

L3D5-T F absent 3.6637 3.7021 0.0384
ABC Study No. 66085 

Page 213 of 252 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Frog Code Phenotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight (g)

Cassette + 

Liver Weight 

(g)

Calculated Liver 

Weight (g)

L3D6-T F absent 3.6416 4.2215 0.5799

L3D7-T M present 3.6384 4.3993 0.7609

L3D8-T F present 3.6424 4.7240 1.0816

L3D9-T F present 3.6324 4.5338 0.9014

L3D10-T M present 3.6463 4.5898 0.9435

L4A1-T F absent 3.4046 3.7684 0.3638

L4A2-T F present 3.3864 4.5291 1.1427

L4A3-T M absent 3.4096 3.6176 0.2080

L4A4-T M present 3.4047 4.1141 0.7094

L4A5-T F absent 3.4079 3.9405 0.5326

L4A6-T F present 3.3993 4.4155 1.0162

L4A7-T F absent 3.3937 3.9539 0.5602

L4A8-T F present 3.4054 3.9693 0.5639

L4A9-T F present 3.4109 4.0803 0.6694

L4A10-T M present 3.3993 4.2664 0.8671

L4B1-T F absent 3.4006 4.1124 0.7118

L4B2-T F present 3.3947 4.2265 0.8318

L4B3-T F present 3.4053 3.9975 0.5922

L4B4-T F present 3.3991 4.3875 0.9884

L4B5-T M absent 3.3939 3.7789 0.3850

L4B6-T F present 3.3975 4.1466 0.7491

L4B7-T F present 3.3966 3.8527 0.4561

L4B8-T M absent 3.4091 3.8489 0.4398

L4B9-T F present 3.3986 3.8519 0.4533

L4B10-T M present 3.4046 3.6427 0.2381

L4C1-T F absent 3.6455 3.8549 0.2094

L4C2-T M present 3.6540 4.6001 0.9461

L4C3-T F absent 3.6363 3.6483 0.0120

L4C4-T F present 3.6300 4.5125 0.8825

L4C5-T F present 3.6593 4.4001 0.7408

L4C6-T M present 3.6385 3.7011 0.0626

L4C7-T F present 3.6381 4.1021 0.4640

L4C8-T F absent 3.6583 3.8002 0.1419

L4C9-T F absent 3.6535 3.8057 0.1522

L4C10-T F absent 3.6470 4.0779 0.4309

L4D1-T F present 3.6462 4.4290 0.7828

L4D2-T F present 3.6334 4.1408 0.5074

L4D3-T M present 3.6379 4.4453 0.8074

L4D4-T M present 3.6355 4.2228 0.5873
ABC Study No. 66085 

Page 214 of 252 

Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Frog Code Phenotypic

Sex

Nuptial Pads 

(Present/Absent)

Liver Cassette 

Tare Weight (g)

Cassette + 

Liver Weight 

(g)

Calculated Liver 

Weight (g)

L4D5-T M present 3.6392 4.3826 0.7434

L4D6-T F present 3.6412 4.4643 0.8231

L4D7-T M present 3.6431 4.3703 0.7272

L4D8-T M present 3.6632 4.3966 0.7334

L4D9-T M absent 3.6509 4.0947 0.4438

L4D10-T M present 3.6630 4.4552 0.7922
ABC Study No. 66085 

Page 215 of 252 

APPENDIX L – WATER QUALITY DATA
ABC Study No. 66085 

Page 216 of 252 

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 21.6 21.4 21.5 21.7 21.5 21.4 21.6 21.5

6 21.0 20.8 20.9 21.0 21.0 21.0 20.7 20.8

8 20.8 20.5 20.7 20.8 20.8 20.9 20.6 20.7

13 21.0 20.7 21.0 21.0 21.1 21.0 20.7 20.9

15 21.0 20.7 21.0 21.0 21.0 21.0 20.7 20.9

20 20.9 20.6 20.9 21.0 20.9 20.9 20.5 20.7

22 21.0 20.8 21.0 21.0 21.0 21.1 20.7 20.9

27 21.1 20.8 21.1 21.1 21.1 21.0 20.6 20.8

29 21.2 20.9 21.1 21.1 21.2 21.1 21.1 21.4

34 21.6 21.2 21.4 21.5 21.5 21.3 21.2 21.3

36 20.6 20.4 20.5 20.7 20.7 20.6 20.4 20.5

41 20.8 20.3 20.6 20.6 20.8 20.8 20.6 20.5

43 20.6 20.1 20.3 20.3 20.4 20.6 20.6 20.3

48 21.1 20.6 21.0 21.0 20.8 20.7 20.3 20.5

50 21.1 20.6 20.8 20.9 21.0 21.0 20.5 20.6

55 21.3 20.9 21.2 21.2 21.2 21.2 20.9 20.9

57 21.3 21.0 21.3 21.2 21.4 21.2 21.1 21.3

62 21.7 21.0 21.3 21.1 21.4 21.4 21.3 21.6

65 21.0 20.5 20.9 20.7 20.9 20.9 21.0 20.9

69 21.2 20.8 21.1 21.2 21.3 21.2 21.2 21.5

72 21.4 21.1 21.4 21.2 21.5 21.4 21.3 21.6

76 21.3 21.2 21.2 20.9 21.3 21.2 21.3 21.7

79 21.1 20.9 21.1 20.9 21.2 21.0 21.0 21.4

83 21.4 21.1 21.4 21.3 21.5 21.4 21.2 21.6

85 21.6 21.2 21.5 21.4 21.6 21.5 21.4 21.6

91 21.5 21.2 21.5 21.4 21.5 21.4 21.3 21.3

93 21.5 21.4 21.5 21.5 21.6 21.5 21.4 21.4

98 21.7 21.6 21.7 21.7 21.7 21.6 21.6 21.6

100 21.5 21.3 21.4 21.6 21.5 21.4 21.4 21.4

105 21.7 21.4 21.6 21.6 21.6 21.4 21.5 21.5

107 21.6 21.8 21.9 21.6 21.9 21.8 21.9 22.0

110 21.6 21.5 21.9 21.4 21.8 21.8 21.5 21.6
ABC Study No. 66085 

Page 217 of 252 

Table L-1. Test Solution Temperature Measured in Each Replicate Test Chamber (continued)

Temperature °C

Level 1 (6.27 μg/L) Level 2 (15.4 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 21.7 21.4 21.5 21.4 21.3 21.4 21.6 21.6

6 21.0 20.9 21.0 20.9 20.7 20.7 20.9 20.9

8 20.9 20.3 20.8 20.8 20.6 20.6 20.6 20.8

13 21.1 20.9 21.0 20.8 20.6 20.8 20.8 20.8

15 21.1 20.9 21.0 20.8 20.6 20.6 20.9 20.9

20 21.0 20.9 20.9 20.8 20.6 20.6 20.8 20.9

22 21.1 21.0 21.0 20.9 20.9 20.7 20.9 20.9

27 21.1 20.9 21.0 20.9 20.6 20.7 20.9 21.0

29 21.1 21.2 21.0 20.8 20.8 21.4 21.1 21.2

34 21.5 21.3 21.4 21.2 21.1 21.1 21.4 21.5

36 20.7 20.6 20.7 20.5 20.4 20.3 20.6 20.6

41 20.8 20.7 20.7 20.6 20.5 20.3 20.5 20.7

43 20.4 20.4 20.6 20.5 20.6 20.5 20.5 20.5

48 21.0 20.6 20.7 20.5 20.4 20.3 20.7 20.7

50 21.1 20.9 20.9 20.8 20.6 20.5 21.0 21.0

55 21.3 21.1 21.2 21.1 20.9 20.8 21.2 21.2

57 21.4 21.2 21.3 21.2 21.0 20.9 21.2 21.2

62 21.5 21.1 21.4 21.3 21.0 21.1 21.4 21.3

65 21.0 20.7 20.8 20.7 20.5 20.6 20.9 20.8

69 21.4 21.1 21.3 21.1 20.9 21.0 21.4 21.2

72 21.5 21.2 21.3 21.3 21.0 21.2 21.5 21.2

76 21.4 21.3 21.1 21.1 21.4 21.1 21.1 21.0

79 21.3 20.9 21.1 21.0 20.7 20.7 21.2 20.8

83 21.5 21.3 21.4 21.3 21.1 21.2 21.5 21.3

85 21.6 21.4 21.5 21.4 21.2 21.3 21.6 21.4

91 21.5 21.4 21.4 21.3 21.1 21.2 21.5 21.4

93 21.6 21.5 21.5 21.5 21.3 21.4 21.6 21.5

98 21.7 21.6 21.6 21.6 21.5 21.6 21.7 21.7

100 21.5 21.4 21.4 21.4 21.2 21.3 21.5 21.5

105 21.6 21.5 21.5 21.4 21.3 21.4 21.6 21.5

107 22.3 21.8 21.8 21.8 21.7 22.0 22.3 21.6

110 21.7 21.5 21.7 21.8 21.3 21.4 21.5 21.3
ABC Study No. 66085 

Page 218 of 252 

Table L-1. Test Solution Temperature Measured in Each Replicate Test Chamber (continued)

Temperature °C

Level 3 (50.1 μg/L) Level 4 (156 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 21.5 21.4 21.6 21.7 21.6 21.4 21.4 21.6

6 21.0 20.9 20.8 20.9 20.9 20.9 21.0 20.9

8 20.9 20.7 20.6 20.8 20.8 20.7 20.8 20.8

13 20.9 20.8 20.7 20.9 20.9 20.9 21.0 21.0

15 21.0 20.7 20.6 20.9 21.0 20.8 21.0 20.9

20 21.0 20.8 20.6 21.0 21.1 20.9 21.0 20.8

22 21.0 20.9 20.7 20.9 21.0 20.9 21.0 20.9

27 20.9 20.8 20.7 20.9 21.0 20.8 20.9 20.9

29 21.2 21.0 20.8 21.1 21.2 21.1 21.2 21.1

34 21.4 21.2 21.1 21.4 21.4 21.2 21.4 21.4

36 20.8 20.7 20.6 20.9 20.9 20.5 20.6 20.7

41 20.7 20.5 20.4 20.6 20.5 20.5 20.7 20.8

43 20.5 20.4 20.5 20.5 20.5 20.6 20.5 20.4

48 20.7 20.5 20.6 20.7 20.7 20.9 20.6 20.9

50 20.9 20.7 20.4 20.9 20.7 20.6 20.7 21.2

55 21.1 21.0 20.8 21.1 21.1 21.1 21.1 21.3

57 21.2 21.0 20.9 21.2 21.3 21.0 21.1 21.4

62 21.4 21.1 21.0 21.2 21.7 21.1 21.3 21.5

65 20.9 20.6 20.4 20.8 21.0 20.5 20.7 20.9

69 21.2 21.0 20.8 21.2 21.6 20.8 21.0 21.4

72 21.3 21.0 20.9 21.2 21.6 21.0 21.2 21.4

76 21.1 21.3 21.4 21.2 21.1 21.3 21.1 21.2

79 21.0 20.8 20.5 20.9 21.3 20.5 20.7 21.2

83 21.4 21.2 21.0 21.3 21.6 21.1 21.2 21.5

85 21.5 21.3 21.2 21.4 21.6 21.2 21.3 21.5

91 21.4 22.1 21.2 21.4 21.4 21.3 21.4 21.5

93 21.5 21.4 21.5 21.5 21.5 21.4 21.5 21.6

98 21.6 21.5 21.5 21.4 21.6 21.5 21.6 21.7

100 21.4 21.3 21.3 21.5 21.4 21.4 21.4 21.5

105 21.5 21.4 21.4 21.5 21.5 21.4 21.5 21.7

107 21.8 21.8 21.9 21.8 22.0 21.9 21.7 22.0

110 21.7 21.4 21.4 21.5 21.6 21.6 21.5 21.5
ABC Study No. 66085 

Page 219 of 252 

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 8.13 8.18 8.12 8.22 8.23 8.28 8.21 8.25

6 7.63 7.74 7.80 8.02 7.85 7.56 8.19 7.84

8 6.48 6.48 6.25 6.37 6.34 5.76 6.82 6.44

13 7.76 8.06 7.53 7.70 7.03 7.36 8.22 7.07

15 7.19 7.75 7.06 7.10 6.40 6.68 7.86 7.47

20 6.95 6.78 6.89 6.93 6.25 6.61 6.89 6.68

22 6.98 7.14 6.54 7.32 6.33 6.75 7.82 6.97

27 6.47 7.37 6.19 7.21 7.20 7.18 7.47 7.34

29 7.03 7.32 7.85 8.40 7.55 7.39 7.87 7.90

34 7.03 7.87 7.54 7.54 7.27 7.38 7.26 6.62

36 5.97 5.27 5.46 6.43 5.98 5.76 5.90 5.83

41 7.83 7.62 7.80 7.98 8.19 7.88 6.77 8.12

43 8.51 8.65 8.38 8.20 8.25 8.03 8.45 8.61

48 7.71 7.58 7.54 7.96 7.82 7.39 7.01 7.28

50 8.10 8.32 8.55 9.09 8.97 7.67 9.16 8.55

55 7.24 7.65 6.97 7.70 7.10 7.17 7.81 6.68

57 6.63 6.70 5.94 6.92 6.51 6.71 7.35 6.37

62 4.94 7.16 6.39 7.26 6.35 6.99 6.84 5.53

65 7.07 7.49 6.91 7.50 7.63 7.39 5.49 7.51

69 6.74 7.01 7.15 6.16 6.89 7.01 6.84 6.57

72 6.49 6.45 6.51 6.76 6.61 5.65 6.62 6.25

76 7.31 7.38 7.29 7.41 7.19 7.21 7.51 7.25

79 6.90 6.90 7.07 6.98 6.24 6.75 6.78 6.20

83 4.57 4.89 6.14 5.30 4.70 5.10 6.08 4.91

85 5.05 5.93 6.28 4.96 4.58 6.17 6.18 5.47

91 3.41 4.27 4.71 3.91 3.57 3.82 5.86 4.75

92 6.79 7.18 7.48 7.02 6.80 6.93 7.72 6.67

93 5.99 6.41 6.64 5.90 6.42 5.61 6.90 6.47

98 6.52 6.10 6.95 6.43 6.78 5.76 7.13 6.65

100 4.78 5.62 5.89 4.48 5.81 5.08 5.74 6.38

105 5.64 5.66 6.48 6.02 6.76 6.19 5.90 6.52

107 5.86 5.64 5.82 5.77 6.85 6.27 6.19 6.47

110 6.96 6.53 6.62 6.68 7.34 6.45 6.50 6.49

Note: 100% dissolved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21, and 22°C, respectively.
ABC Study No. 66085 

Page 220 of 252 

Table L-2. Test Solution Dissolved Oxygen Concentrations Measured in Each Replicate Test 

Chamber (continued) 

Dissolved Oxygen (mg/L)

Level 1 (6.27 μg/L) Level 2 (15.4 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 8.22 8.24 8.17 8.19 8.21 8.18 8.21 8.19

6 7.53 7.70 8.05 7.73 7.87 7.79 7.91 7.45

8 7.16 6.61 6.33 6.72 6.48 6.10 6.64 5.97

13 7.25 7.74 7.12 7.58 7.92 6.95 7.74 6.66

15 6.38 7.32 7.04 6.86 7.39 7.00 7.50 6.91

20 6.31 6.75 6.50 6.46 6.86 6.71 6.95 6.76

22 6.25 6.73 7.27 6.76 7.00 6.17 6.70 6.39

27 7.44 7.31 7.44 7.07 7.76 7.24 7.67 6.86

29 7.86 7.80 7.77 8.24 7.81 7.51 7.98 7.84

34 6.99 7.72 7.47 7.81 7.79 7.49 7.43 7.20

36 6.33 6.30 6.10 6.19 6.19 5.71 6.20 6.17

41 8.48 8.27 8.05 8.34 8.53 7.91 8.69 8.84

43 8.38 8.29 8.30 8.65 8.54 8.37 8.21 8.02

48 7.68 7.56 7.83 7.47 8.21 7.38 7.86 7.37

50 9.19 8.95 8.92 8.66 9.15 8.36 8.99 8.42

55 7.39 7.62 7.66 7.41 8.07 7.38 7.63 7.22

57 6.66 6.87 7.02 6.77 7.61 6.63 6.74 6.86

62 6.83 6.97 6.81 6.94 7.47 6.48 6.55 6.66

65 7.70 7.49 7.40 7.56 8.04 7.24 7.28 7.58

69 6.64 6.81 6.89 6.75 7.34 6.41 7.05 6.43

72 6.72 6.89 6.62 6.99 6.40 6.63 6.48 6.83

76 7.31 7.27 7.19 7.18 7.24 7.32 7.28 6.99

79 6.74 6.70 6.50 7.01 7.09 6.71 6.81 6.03

83 5.28 5.72 5.02 5.84 5.84 5.32 5.69 4.24

85 5.96 5.78 5.09 6.05 6.37 5.83 6.04 5.35

91 4.60 3.82 4.01 4.54 4.63 4.64 5.92 4.18

92 7.14 6.49 7.08 7.02 7.22 6.47 7.24 7.37

93 6.40 6.23 6.23 6.37 6.61 6.04 6.72 6.28

98 6.75 6.10 6.64 6.46 6.86 5.94 6.96 6.27

100 6.10 5.61 5.30 5.63 5.97 5.36 6.44 5.42

105 5.90 6.08 6.14 6.06 6.23 6.06 6.62 5.72

107 6.47 5.60 6.31 5.72 6.14 5.94 6.65 6.07

110 6.18 5.83 5.18 5.19 5.11 5.62 6.55 5.20

Note: 100% dissolved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21, and 22°C, respectively.
ABC Study No. 66085 

Page 221 of 252 

Table L-2. Test Solution Dissolved Oxygen Concentrations Measured in Each Replicate Test 

Chamber (continued) 

Dissolved Oxygen (mg/L)

Level 3 (50.1 μg/L) Level 4 (156 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 8.20 8.20 8.16 8.10 8.18 8.17 8.21 8.19

6 7.65 7.43 7.86 7.89 7.49 7.47 7.69 8.02

8 6.60 6.45 6.25 7.13 6.16 6.88 7.15 7.51

13 7.44 6.57 7.06 7.34 7.83 7.64 7.63 7.92

15 6.78 7.02 7.03 7.13 7.47 7.30 7.12 7.23

20 6.32 6.43 6.51 6.43 6.59 6.57 6.23 6.55

22 6.98 6.26 7.29 6.88 7.26 7.21 7.07 6.72

27 7.76 7.15 7.01 7.96 7.67 7.34 7.27 7.92

29 7.84 7.35 8.13 8.02 8.08 7.64 7.31 8.07

34 7.44 6.25 7.55 7.21 7.76 7.04 6.46 7.41

36 5.66 5.82 5.21 6.39 6.13 5.86 5.59 6.41

41 8.04 8.64 7.53 8.09 8.47 8.53 8.35 8.73

43 8.29 8.11 8.19 8.59 8.36 8.47 8.52 8.11

48 7.56 7.82 7.13 8.04 8.02 6.87 8.26 8.28

50 8.39 8.72 8.76 9.10 8.98 9.00 9.21 8.22

55 7.31 7.04 7.33 7.48 7.93 4.98 7.82 8.04

57 6.74 6.31 6.87 6.81 6.73 6.73 6.92 7.11

62 6.61 5.67 6.72 6.91 6.59 5.57 6.69 6.87

65 7.26 7.02 7.22 7.66 7.35 7.13 7.29 7.54

69 6.59 6.25 6.25 6.84 3.50 6.97 7.13 6.60

72 6.31 6.44 6.66 6.68 6.60 6.54 6.45 6.49

76 7.24 7.38 7.27 7.32 7.21 7.19 7.01 6.84

79 6.29 6.11 5.96 6.70 6.47 6.38 5.68 5.04

83 4.94 4.62 4.77 5.92 5.22 4.93 4.41 3.81

85 5.55 5.66 5.54 6.25 5.11 5.12 4.06 5.48

91 3.81 3.48 4.32 5.86 4.07 4.58 2.88 5.09

92 6.96 6.99 7.43 7.44 6.89 6.87 6.77 7.29

93 5.15 6.12 6.37 6.96 5.95 5.98 5.45 6.63

98 6.39 6.35 6.47 7.11 6.82 6.58 4.18 6.90

100 5.44 5.65 5.97 6.02 6.29 6.17 2.71 6.38

105 6.06 5.90 6.21 6.66 6.12 6.39 5.13 6.69

107 6.19 6.03 6.31 6.44 5.91 5.94 5.42 6.60

110 5.08 6.10 6.24 6.24 6.23 6.44 5.96 5.37

Note: 100% dissolved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21, and 22°C, respectively.
ABC Study No. 66085 

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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 8.30 8.29 8.31 8.30 8.34 8.35 8.32 8.33

6 8.13 8.13 8.14 8.18 8.19 8.15 8.20 8.16

8 8.03 8.01 8.02 8.03 8.07 7.98 8.07 8.03

13 8.12 8.17 8.16 8.18 8.12 8.15 8.24 8.09

15 8.07 8.12 8.07 8.07 8.04 8.06 8.18 8.11

20 7.96 7.96 7.94 8.01 7.88 7.94 8.01 7.94

22 7.90 7.88 7.81 7.90 7.77 7.83 7.93 7.85

27 8.06 8.05 7.87 8.06 8.04 8.00 8.07 8.06

29 7.91 7.93 7.86 7.84 7.92 7.86 7.91 7.90

34 7.83 7.85 7.84 7.86 7.89 7.88 7.87 7.88

36 8.01 7.96 7.99 8.09 8.03 8.04 8.05 8.01

41 8.17 8.16 8.21 8.21 8.27 8.23 8.06 8.18

43 8.21 8.25 8.27 8.30 8.29 8.29 8.25 8.25

48 7.95 8.03 8.03 8.02 8.10 8.00 8.08 8.04

50 7.99 8.06 8.12 8.16 8.16 8.00 8.16 8.05

55 7.94 8.06 8.00 8.06 8.02 7.98 8.11 7.99

57 7.92 7.95 7.90 7.99 7.95 7.95 8.07 7.96

62 7.71 8.02 7.95 8.05 7.97 8.05 8.02 7.86

65 7.92 7.98 7.94 7.99 8.04 8.00 7.80 8.04

69 7.96 7.98 8.00 7.89 7.95 8.01 8.01 7.92

72 7.90 7.93 7.94 7.97 8.03 7.98 7.85 7.91

76 7.91 7.84 7.91 7.93 7.89 7.85 7.91 7.87

79 7.86 7.60 7.90 7.92 7.86 7.86 7.86 7.82

83 7.55 7.56 7.72 7.62 7.64 7.65 7.70 7.61

85 7.60 7.66 7.68 7.61 7.59 7.66 7.68 7.60

91 7.35 7.45 7.59 7.58 7.55 7.55 7.64 7.53

93 7.78 7.78 7.83 7.76 7.81 7.79 7.85 7.68

98 7.78 7.69 7.79 7.70 7.69 7.66 7.70 7.65

100 7.57 7.60 7.64 7.50 7.62 7.65 7.56 7.59

105 7.74 7.71 7.75 7.74 7.77 7.76 7.63 7.70

107 7.63 7.63 7.69 7.66 7.71 7.78 7.62 7.62

110 8.12 8.03 8.04 8.03 8.10 7.98 7.86 7.81
ABC Study No. 66085 

Page 223 of 252 

Table L-3. Test Solution pH Values Measured in Each Replicate Test Chamber (continued)

pH

Level 1 (6.27 μg/L) Level 2 (15.4 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 8.35 8.34 8.32 8.32 8.31 8.30 8.32 8.31

6 8.12 8.13 8.16 8.15 8.15 8.13 8.15 8.10

8 8.11 8.04 8.03 8.05 8.03 7.98 8.03 8.00

13 8.12 8.16 8.12 8.16 8.16 8.05 8.19 8.07

15 8.01 8.05 8.10 8.05 8.10 8.05 8.12 8.04

20 7.92 7.93 7.99 7.93 7.90 7.94 7.93 7.92

22 7.77 7.80 7.88 7.80 7.83 7.72 7.81 7.79

27 8.06 8.01 8.04 8.03 8.06 7.98 8.03 8.01

29 7.88 7.90 7.90 7.97 7.91 7.82 7.85 7.89

34 7.92 7.92 7.90 7.90 7.93 7.90 7.89 7.88

36 8.05 8.06 8.06 8.08 8.10 8.03 8.07 8.06

41 8.28 8.25 8.25 8.25 8.24 8.17 8.22 8.26

43 8.26 8.24 8.26 8.25 8.34 8.26 8.36 8.39

48 8.10 8.07 8.09 8.09 8.10 8.00 8.08 8.07

50 8.15 8.10 8.08 8.11 8.13 8.01 8.12 8.05

55 8.08 8.01 8.06 8.04 8.12 8.01 8.05 8.04

57 7.99 7.95 7.99 7.96 8.06 7.98 7.98 7.99

62 8.03 7.99 7.98 7.99 8.09 7.97 7.96 8.01

65 8.07 7.99 8.00 8.00 8.11 7.98 7.98 8.06

69 8.02 7.95 7.94 7.94 8.02 7.89 7.98 7.96

72 7.95 7.95 7.97 7.97 7.99 7.94 7.95 7.97

76 7.95 7.93 7.90 7.90 7.91 7.96 7.91 7.92

79 7.83 7.85 7.86 7.89 7.94 7.91 7.92 7.92

83 7.64 7.66 7.65 7.67 7.71 7.63 7.66 7.56

85 7.66 7.60 7.58 7.62 7.66 7.63 7.62 7.60

91 7.57 7.55 7.56 7.54 7.58 7.57 7.64 7.58

93 7.83 7.77 7.83 7.78 7.90 7.70 7.76 7.85

98 7.69 7.66 7.74 7.68 7.82 7.63 7.65 7.73

100 7.61 7.57 7.59 7.56 7.68 7.58 7.61 7.63

105 7.67 7.69 7.71 7.69 7.74 7.65 7.67 7.69

107 7.71 7.68 7.74 7.65 7.76 7.68 7.68 7.74

110 7.79 7.70 7.62 7.61 7.57 7.60 7.66 7.61
ABC Study No. 66085 

Page 224 of 252 

Table L-3. Test Solution pH Values Measured in Each Replicate Test Chamber (continued) 

pH

Level 3 (50.1 μg/L) Level 4 (156 μg/L)

Study Day Rep A Rep B Rep C Rep D Rep E Rep F Rep G Rep H

0 8.31 8.30 8.32 8.31 8.34 8.34 8.33 8.34

6 8.12 8.09 8.13 8.15 8.13 8.11 8.14 8.20

8 8.04 8.05 8.01 8.10 8.03 8.10 8.12 8.19

13 8.13 8.06 8.05 8.14 8.19 8.16 8.15 8.22

15 8.05 8.05 8.04 8.06 8.12 8.12 8.10 8.13

20 7.95 7.91 7.95 7.87 7.95 7.97 7.99 7.99

22 7.82 7.76 7.83 7.84 7.86 7.87 7.88 7.90

27 8.03 7.97 7.98 8.03 8.07 8.04 8.06 8.13

29 7.89 7.87 7.87 7.88 7.91 7.91 7.91 7.95

34 7.87 7.83 7.83 7.84 7.91 7.91 7.82 7.86

36 8.03 8.00 7.93 8.04 8.03 8.04 8.09 8.10

41 8.12 8.24 8.13 8.21 8.23 8.25 8.25 8.32

43 8.29 8.27 8.26 8.26 8.27 8.29 8.29 8.32

48 8.04 8.05 7.98 8.05 8.06 7.95 8.08 8.10

50 8.03 8.04 8.08 8.09 8.08 8.10 8.15 8.12

55 8.05 8.00 8.04 8.07 8.10 7.79 8.10 8.19

57 7.98 7.93 7.95 7.98 7.99 7.97 8.03 8.07

62 7.97 7.83 7.97 7.99 7.99 7.82 8.02 8.09

65 7.95 7.91 8.00 8.03 8.01 7.99 8.05 8.10

69 7.92 7.88 7.93 7.98 7.55 8.01 8.07 8.03

72 7.99 7.98 7.97 7.98 7.98 7.99 8.01 8.02

76 7.91 7.90 7.88 7.92 7.88 7.94 7.85 7.94

79 7.86 7.86 7.86 7.89 7.93 7.92 7.89 7.78

83 7.60 7.59 7.57 7.63 7.65 7.63 7.61 7.54

85 7.61 7.61 7.60 7.66 7.59 7.61 7.58 7.66

91 7.54 7.55 7.56 7.67 7.59 7.51 7.51 7.59

93 7.74 7.78 7.83 7.86 7.78 7.83 7.84 7.86

98 7.70 7.69 7.65 7.68 7.69 7.70 7.59 7.70

100 7.63 7.59 7.62 7.60 7.67 7.68 7.47 7.64

105 7.70 7.63 7.68 7.68 7.63 7.66 7.64 7.65

107 7.76 7.67 7.70 7.69 7.70 7.71 7.75 7.76

110 7.61 7.66 7.63 7.62 7.69 7.76 7.79 7.50
ABC Study No. 66085 

Page 225 of 252 

APPENDIX M – PLASMA VITELLOGENIN DATA
ABC Study No. 66085 

Page 226 of 252 

Appendix M-1. Vitellogenin Data for NF Stage 62 Pooled Plasma 

Treatment

(μg/L) REP Frog Code Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

Control A CA-NF62 CA-180 0.017 66085-VTG-Plate 3 0.033 2.451

B CB-NF62 CB-180 0.017 66085-VTG-Plate 3 0.033 2.451

C CC-NF62 CC-300 0.000 66085-VTG-Plate 1 0.062 4.042

D CD-NF62 CD-300 0.000 66085-VTG-Plate 1 0.062 4.042

E CE-NF62 CE-180 0.000 66085-VTG-Plate 4 0.037 2.505

F CF-NF62 CF-180 0.000 66085-VTG-Plate 4 0.037 2.505

G CG-NF62 CG-300 0.000 66085-VTG-Plate 2 0.056 3.699

H CH-NF62 CH-300 0.000 66085-VTG-Plate 2 0.056 3.699

6.27 A L1A-NF62 L1A-300 0.000 66085-VTG-Plate 2 0.056 3.699

B L1B-NF62 L1B-180 0.017 66085-VTG-Plate 3 0.033 2.451

C L1C-NF62 L1C-300 0.000 66085-VTG-Plate 1 0.062 4.042

D L1D-NF62 L1D-180 0.000 66085-VTG-Plate 4 0.037 2.505

15.4 A L2A-NF62 L2A-300 0.000 66085-VTG-Plate 2 0.056 3.699

B L2B-NF62 L2B-180 0.017 66085-VTG-Plate 3 0.033 2.451

C L2C-NF62 L2C-300 0.000 66085-VTG-Plate 1 0.062 4.042

D L2D-NF62 L2D-180 0.000 66085-VTG-Plate 4 0.037 2.505

4

50.1 A L3A-NF62 L3A-300 0.000 66085-VTG-Plate 1 0.062 4.042

B L3B-NF62 L3B-180 0.000 66085-VTG-Plate 4 0.037 2.505

C L3C-NF62 L3C-180 0.017 66085-VTG-Plate 3 0.033 2.451

D L3D-NF62 L3D-300 0.000 66085-VTG-Plate 2 0.056 3.699

156 A L4A-NF62 L4A-300 0.000 66085-VTG-Plate 1 0.062 4.042

B L4B-NF62 L4B-180 0.000 66085-VTG-Plate 4 0.037 2.505

C L4C-NF62 L4C-180 0.017 66085-VTG-Plate 3 0.033 2.451

D L4D-NF62 L4D-300 0.000 66085-VTG-Plate 2 0.056 3.699
ABC Study No. 66085 

Page 227 of 252 

Appendix M-2. Vitellogenin Data for Juvenile Males on Study Day 110 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

Control A CA3-T 3 3-300 0.000 66085-VTG-Plate 1 0.062 4.042

A CA4-T 4 4-300 0.000 66085-VTG-Plate 1 0.062 4.042

A CA5-T 5 5-180 0.000 66085-VTG-Plate 6 0.036 2.405

A CA6-T 6 6-180 0.000 66085-VTG-Plate 6 0.036 2.405

A CA7-T 7 7-180 0.000 66085-VTG-Plate 6 0.036 2.405

A CA8-T 8 8-180 0.000 66085-VTG-Plate 6 0.036 2.405

A CA9-T 9 9-180 0.000 66085-VTG-Plate 6 0.036 2.405

A CA10-T 10 10-180 0.000 66085-VTG-Plate 6 0.036 2.405

B CB2-T 12 12-300 0.000 66085-VTG-Plate 2 0.056 3.699

B CB4-T 14 14-300 0.000 66085-VTG-Plate 2 0.056 3.699

B CB5-T 15 15-300 0.000 66085-VTG-Plate 2 0.056 3.699

B CB6-T 16 16-180 0.000 66085-VTG-Plate 8 0.036 2.179

B CB8-T 18 18-180 0.000 66085-VTG-Plate 8 0.036 2.179

C CC1-T 21 21-180 0.017 66085-VTG-Plate 3 0.033 2.451

C CC2-T 22 22-180 0.017 66085-VTG-Plate 3 0.033 2.451

C CC3-T 23 23-180 0.017 66085-VTG-Plate 3 0.033 2.451

C CC5-T 25 25-180 0.018 66085-VTG-Plate 5 0.036 2.492

C CC7-T 27 27-180 0.018 66085-VTG-Plate 5 0.036 2.492

C CC8-T 28 28-180 0.018 66085-VTG-Plate 5 0.036 2.492

C CC9-T 29 29-180 0.000 66085-VTG-Plate 7 0.037 2.545

D CD1-T 31 31-180 0.018 66085-VTG-Plate 7 0.037 2.545

D CD6-T 36 36-180 0.018 66085-VTG-Plate 7 0.037 2.545
ABC Study No. 66085 

Page 228 of 252 

Appendix M-2. Vitellogenin Data for Juvenile Males on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

Control E CE1-T 41 41-180 0.017 66085-VTG-Plate 3 0.033 2.451

E CE4-T 44 44-180 0.000 66085-VTG-Plate 4 0.037 2.505

E CE5-T 45 45-180 0.000 66085-VTG-Plate 6 0.036 2.405

E CE6-T 46 46-180 0.000 66085-VTG-Plate 6 0.036 2.405

E CE9-T 49 49-180 0.000 66085-VTG-Plate 4 0.037 2.505

E CE10-T 50 50-180 0.000 66085-VTG-Plate 4 0.037 2.505

F CF3-T 83 53-180 0.000 66085-VTG-Plate 8 0.036 2.179

F CF5-T 55 55-180 0.000 66085-VTG-Plate 8 0.036 2.179

F CF6-T 56 56-180 0.000 66085-VTG-Plate 8 0.036 2.179

F CF9-T 59 59-300 0.000 66085-VTG-Plate 2 0.056 3.699

F CF10-T 60 60-300 0.000 66085-VTG-Plate 2 0.056 3.699

G CG1-T 61 61-180 0.018 66085-VTG-Plate 5 0.036 2.492

G CG4-T 64 64-180 0.018 66085-VTG-Plate 5 0.036 2.492

G CG5-T 65 65-180 0.000 66085-VTG-Plate 6 0.036 2.405

G CG6-T 66 66-180 0.000 66085-VTG-Plate 6 0.036 2.405

G CG8-T 68 68-180 0.017 66085-VTG-Plate 3 0.033 2.451

G CG10-T 70 70-180 0.017 66085-VTG-Plate 3 0.033 2.451

H CH3-T 73 73-180 0.000 66085-VTG-Plate 4 0.037 2.505

H CH5-T 75 75-180 0.000 66085-VTG-Plate 4 0.037 2.505

H CH6-T 76 76-180 0.000 66085-VTG-Plate 4 0.037 2.505

H CH9-T 79 79-180 0.018 66085-VTG-Plate 7 0.037 2.545
ABC Study No. 66085 

Page 229 of 252 

Appendix M-2. Vitellogenin Data for Juvenile Males on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

6.27 A L1A1-T 81 81-180 0.044 66085-VTG-Plate 4 0.037 2.505

A L1A3-T 83 83-180 0.057 66085-VTG-Plate 4 0.037 2.505

A L1A4-T 84 84-180 0.000 66085-VTG-Plate 4 0.037 2.505

A L1A6-T 86 86-180 0.000 66085-VTG-Plate 6 0.036 2.405

A L1A7-T 87 87-180 0.000 66085-VTG-Plate 6 0.036 2.405

B L1B1-T 91 91-300 0.000 66085-VTG-Plate 1 0.062 4.042

B L1B2-T 92 92-300 0.000 66085-VTG-Plate 1 0.062 4.042

B L1B4-T 94 94-300 0.000 66085-VTG-Plate 1 0.062 4.042

B L1B5-T 95 95-180 0.000 66085-VTG-Plate 8 0.036 2.179

B L1B8-T 98 98-180 0.000 66085-VTG-Plate 8 0.036 2.179

C L1C1-T 101 101-300 0.000 66085-VTG-Plate 2 0.056 3.699

C L1C2-T 102 102-300 0.000 66085-VTG-Plate 2 0.056 3.699

C L1C3-T 103 103-300 0.000 66085-VTG-Plate 2 0.056 3.699

C L1C4-T 104 104-300 0.000 66085-VTG-Plate 2 0.056 3.699

C L1C5-T 105 105-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L1C9-T 109 109-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L1C10-T 110 110-180 0.018 66085-VTG-Plate 5 0.036 2.492

D L1D3-T 113 113-180 0.057 66085-VTG-Plate 3 0.033 2.451

D L1D6-T 116 116-180 0.018 66085-VTG-Plate 7 0.037 2.545

D L1D7-T 117 117-180 0.056 66085-VTG-Plate 7 0.037 2.545

D L1D8-T 118 118-180 0.018 66085-VTG-Plate 7 0.037 2.545

D L1D10-T 120 120-180 0.045 66085-VTG-Plate 7 0.037 2.545
ABC Study No. 66085 

Page 230 of 252 

Appendix M-2. Vitellogenin Data for Juvenile Males on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

15.4 A L2A4-T 124 124-180 0.017 66085-VTG-Plate 3 0.033 2.451

A L2A5-T 125 125-180 0.018 66085-VTG-Plate 7 0.037 2.545

A L2A8-T 128 128-180 0.000 66085-VTG-Plate 7 0.037 2.545

A L2A9-T 129 129-180 0.018 66085-VTG-Plate 7 0.037 2.545

B L2B1-T 131 131-300 0.000 66085-VTG-Plate 1 0.062 4.042

B L2B3-T 133 133-180 0.000 66085-VTG-Plate 4 0.037 2.505

B L2B5-T 135 135-180 0.000 66085-VTG-Plate 4 0.037 2.505

B L2B6-T 136 136-180 0.018 66085-VTG-Plate 5 0.036 2.492

B L2B9-T 139 139-180 0.018 66085-VTG-Plate 5 0.036 2.492

B L2B10-T 140 140-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L2C2-T 142 142-300 0.000 66085-VTG-Plate 1 0.062 4.042

C L2C3-T 143 143-300 0.000 66085-VTG-Plate 1 0.062 4.042

C L2C4-T 144 144-300 0.000 66085-VTG-Plate 1 0.062 4.042

C L2C5-T 145 145-180 0.000 66085-VTG-Plate 8 0.036 2.179

C L2C6-T 146 146-180 0.000 66085-VTG-Plate 8 0.036 2.179

C L2C8-T 148 148-180 0.000 66085-VTG-Plate 8 0.036 2.179

C L2C10-T 150 150-180 0.000 66085-VTG-Plate 8 0.036 2.179

D L2D1-T 151 151-300 0.000 66085-VTG-Plate 2 0.000 0.012

D L2D2-T 152 152-300 0.000 66085-VTG-Plate 2 0.056 3.699

D L2D3-T 153 153-300 0.000 66085-VTG-Plate 2 0.056 3.699

D L2D4-T 154 154-300 0.000 66085-VTG-Plate 2 0.056 3.699

D L2D5-T 155 155-180 0.000 66085-VTG-Plate 6 0.036 2.405
ABC Study No. 66085 

Page 231 of 252 

Appendix M-2. Vitellogenin Data for Juvenile Males on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

50.1 A L3A2-T 162 162-300 0.000 66085-VTG-Plate 2 0.056 3.699

A L3A5-T 165 165-180 0.000 66085-VTG-Plate 8 0.036 2.179

A L3A6-T 166 166-180 0.000 66085-VTG-Plate 8 0.036 2.179

A L3A9-T 169 169-180 0.000 66085-VTG-Plate 8 0.036 2.179

B L3B1-T 171 171-180 0.017 66085-VTG-Plate 3 0.033 2.451

B L3B3-T 173 173-180 0.017 66085-VTG-Plate 3 0.033 2.451

B L3B5-T 175 175-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L3C1-T 181 181-180 0.000 66085-VTG-Plate 4 0.037 2.505

C L3C3-T 183 183-180 0.000 66085-VTG-Plate 4 0.037 2.505

C L3C6-T 186 186-180 0.000 66085-VTG-Plate 6 0.036 2.405

C L3C8-T 188 188-180 0.000 66085-VTG-Plate 6 0.036 2.405

D L3D7-T 197 197-180 0.018 66085-VTG-Plate 7 0.037 2.545

D L3D10-T 200 200-180 0.000 66085-VTG-Plate 7 0.037 2.545
ABC Study No. 66085 

Page 232 of 252 

Appendix M-2. Vitellogenin Data for Juvenile Males on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

156 B L4B5-T 215 215-300 0.000 66085-VTG-Plate 2 0.056 3.699

B L4B8-T 218 218-300 0.000 66085-VTG-Plate 2 0.056 3.699

B L4B10-T 220 220-300 0.000 66085-VTG-Plate 2 0.056 3.699

C L4C2-T 222 222-180 0.017 66085-VTG-Plate 3 0.033 2.451

C L4C6-T 226 226-180 0.018 66085-VTG-Plate 5 0.036 2.492

D L4D3-T 233 233-180 0.000 66085-VTG-Plate 4 0.037 2.505

D L4D4-T 234 234-180 0.000 66085-VTG-Plate 4 0.037 2.505

D L4D5-T 235 235-180 0.000 66085-VTG-Plate 6 0.036 2.405

D L4D7-T 237 237-300 0.000 66085-VTG-Plate 1 0.062 4.042

D L4D7-T 238 238-180 0.000 66085-VTG-Plate 6 0.036 2.405

D L4D8-T 239 239-180 0.000 66085-VTG-Plate 6 0.036 2.405

D L4D10-T 240 240-180 0.018 66085-VTG-Plate 7 0.037 2.545
ABC Study No. 66085 

Page 233 of 252 

Appendix M-3. Vitellogenin Data for Juvenile Females on Study Day 110 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

Control A CA2-T 1 1-300 0.000 66085-VTG-Plate 1 0.062 4.042

A CA1-T 2 2-300 0.000 66085-VTG-Plate 1 0.062 4.042

B CB1-T 11 11-300 0.000 66085-VTG-Plate 2 0.056 3.699

B CB3-T 13 13-300 0.000 66085-VTG-Plate 1 0.062 4.042

B CB7-T 17 17-180 0.000 66085-VTG-Plate 8 0.036 2.179

B CB9-T 19 19-180 0.000 66085-VTG-Plate 8 0.036 2.179

B CB10-T 20 20-180 0.000 66085-VTG-Plate 8 0.036 2.179

C CC4-T 24 24-180 0.017 66085-VTG-Plate 3 0.033 2.451

C CC6-T 26 26-180 0.018 66085-VTG-Plate 5 0.036 2.492

C CC10-T 30 30-180 0.018 66085-VTG-Plate 7 0.037 2.545

D CD2-T 32 32-180 0.018 66085-VTG-Plate 7 0.037 2.545

D CD3-T 33 33-180 0.018 66085-VTG-Plate 7 0.037 2.545

D CD4-T 34 34-180 0.018 66085-VTG-Plate 7 0.037 2.545

D CD5-T 35 35-180 0.018 66085-VTG-Plate 7 0.037 2.545

D CD7-T 37 37-300 0.000 66085-VTG-Plate 2 0.056 3.699

D CD8-T 38 38-300 0.000 66085-VTG-Plate 2 0.056 3.699

D CD9-T 39 39-300 0.000 66085-VTG-Plate 2 0.056 3.699

D CD10-T 40 40-300 0.000 66085-VTG-Plate 2 0.056 3.699
ABC Study No. 66085 

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Appendix M-3. Vitellogenin Data for Juvenile Females on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

Control E CE2-T 42 42-180 0.017 66085-VTG-Plate 3 0.033 2.451

E CE3-T 43 43-180 0.000 66085-VTG-Plate 4 0.037 2.505

E CE7-T 47 47-180 0.000 66085-VTG-Plate 6 0.036 2.405

E CE8-T 48 48-180 0.000 66085-VTG-Plate 6 0.036 2.405

F CF1-T 51 51-180 0.000 66085-VTG-Plate 4 0.037 2.505

F CF2-T 52 52-180 0.000 66085-VTG-Plate 4 0.037 2.505

F CF4-T 54 54-180 0.000 66085-VTG-Plate 8 0.036 2.179

F CF7-T 57 57-300 0.000 66085-VTG-Plate 1 0.062 4.042

F CF8-T 58 58-300 0.000 66085-VTG-Plate 1 0.062 4.042

G CG2-T 62 62-180 0.018 66085-VTG-Plate 5 0.036 2.492

G CG3-T 63 63-180 0.018 66085-VTG-Plate 5 0.036 2.492

G CG7-T 67 67-180 0.017 66085-VTG-Plate 3 0.033 2.451

G CG9-T 69 69-180 0.017 66085-VTG-Plate 3 0.033 2.451

H CH1-T 71 71-180 0.041 66085-VTG-Plate 7 0.037 2.545

H CH2-T 72 72-180 0.018 66085-VTG-Plate 7 0.037 2.545

H CH4-T 74 74-180 0.000 66085-VTG-Plate 4 0.037 2.505

H CH7-T 77 77-180 0.018 66085-VTG-Plate 7 0.037 2.545

H CH8-T 78 78-180 0.000 66085-VTG-Plate 7 0.037 2.545

H CH10-T 80 80-180 0.088 66085-VTG-Plate 7 0.037 2.545
ABC Study No. 66085 

Page 235 of 252 

Appendix M-3. Vitellogenin Data for Juvenile Females on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

6.27 A L1A2-T 82 82-180 0.000 66085-VTG-Plate 4 0.037 2.505

A L1A5-T 85 85-180 0.000 66085-VTG-Plate 6 0.036 2.405

A L1A8-T 88 88-180 0.000 66085-VTG-Plate 6 0.036 2.405

A L1A9-T 89 89-180 0.133 66085-VTG-Plate 6 0.036 2.405

A L1A10-T 90 90-180 0.000 66085-VTG-Plate 6 0.036 2.405

B L1B3-T 93 93-300 0.000 66085-VTG-Plate 1 0.062 4.042

B L1B6-T 96 96-180 0.051 66085-VTG-Plate 8 0.036 2.179

B L1B7-T 97 97-180 0.000 66085-VTG-Plate 8 0.036 2.179

B L1B9-T 99 99-180 0.171 66085-VTG-Plate 8 0.036 2.179

B L1B10-T 100 100-180 0.000 66085-VTG-Plate 8 0.000 0.012

C L1C6-T 106 106-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L1C7-T 107 107-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L1C8-T 108 108-180 0.018 66085-VTG-Plate 5 0.036 2.492

D L1D1-T 111 111-180 0.017 66085-VTG-Plate 3 0.033 2.451

D L1D2-T 112 112-180 0.017 66085-VTG-Plate 3 0.033 2.451

D L1D4-T 114 114-180 0.047 66085-VTG-Plate 3 0.033 2.451

D L1D5-T 115 115-180 0.018 66085-VTG-Plate 7 0.037 2.545

D L1D9-T 119 119-180 0.078 66085-VTG-Plate 7 0.037 2.545
ABC Study No. 66085 

Page 236 of 252 

Appendix M-3. Vitellogenin Data for Juvenile Females on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

15.4 A L2A1-T 121 121-180 0.017 66085-VTG-Plate 3 0.033 2.451

A L2A2-T 122 122-180 0.017 66085-VTG-Plate 3 0.033 2.451

A L2A3-T 123 123-180 0.017 66085-VTG-Plate 3 0.033 2.451

A L2A6-T 126 126-180 0.000 66085-VTG-Plate 7 0.037 2.545

A L2A7-T 127 127-180 0.000 66085-VTG-Plate 7 0.037 2.545

B L2B2-T 132 132-180 0.000 66085-VTG-Plate 4 0.037 2.505

B L2B4-T 134 134-180 0.000 66085-VTG-Plate 4 0.037 2.505

B L2B7-T 137 137-180 0.018 66085-VTG-Plate 5 0.036 2.492

B L2B8-T 138 138-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L2C1-T 141 141-300 0.000 66085-VTG-Plate 1 0.062 4.042

C L2C7-T 147 147-180 0.000 66085-VTG-Plate 8 0.036 2.179

C L2C9-T 149 149-180 0.000 66085-VTG-Plate 8 0.036 2.179

D L2D6-T 156 156-180 0.000 66085-VTG-Plate 6 0.036 2.405

D L2D8-T 158 158-180 0.000 66085-VTG-Plate 6 0.036 2.405

D L2D9-T 159 159-180 0.000 66085-VTG-Plate 8 0.036 2.179

D L2D10-T 160 160-180 0.000 66085-VTG-Plate 8 0.036 2.179
ABC Study No. 66085 

Page 237 of 252 

Appendix M-3. Vitellogenin Data for Juvenile Females on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

50.1 A L3A1-T 161 161-300 0.000 66085-VTG-Plate 2 0.056 3.699

A L3A3-T 163 163-300 0.000 66085-VTG-Plate 2 0.056 3.699

A L3A4-T 164 164-300 0.028 66085-VTG-Plate 2 0.056 3.699

A L3A7-T 167 167-180 0.000 66085-VTG-Plate 8 0.036 2.179

A L3A8-T 168 168-180 0.000 66085-VTG-Plate 8 0.036 2.179

B L3B2-T 172 172-180 0.017 66085-VTG-Plate 3 0.033 2.451

B L3B4-T 174 174-180 0.017 66085-VTG-Plate 3 0.033 2.451

B L3B6-T 176 176-180 0.018 66085-VTG-Plate 5 0.036 2.492

B L3B7-T 177 177-180 0.018 66085-VTG-Plate 5 0.036 2.492

B L3B8-T 178 178-180 0.018 66085-VTG-Plate 5 0.036 2.492

B L3B9-T 179 179-180 0.018 66085-VTG-Plate 5 0.036 2.492

B L3B10-T 180 180-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L3C2-T 182 182-180 0.000 66085-VTG-Plate 4 0.037 2.505

C L3C4-T 184 184-180 0.000 66085-VTG-Plate 4 0.037 2.505

C L3C5-T 185 185-180 0.000 66085-VTG-Plate 6 0.036 2.405

C L3C7-T 187 187-180 0.000 66085-VTG-Plate 6 0.036 2.405

C L3C9-T 189 189-180 0.000 66085-VTG-Plate 6 0.036 2.405

C L3C10-T 190 190-180 0.000 66085-VTG-Plate 6 0.036 2.405

D L3D1-T 191 191-300 0.000 66085-VTG-Plate 1 0.062 4.042

D L3D2-T 192 192-300 0.000 66085-VTG-Plate 1 0.062 4.042

D L3D2-T 193 193-300 0.000 66085-VTG-Plate 1 0.062 4.042

D L3D4-T 194 194-300 0.000 66085-VTG-Plate 1 0.062 4.042

D L3D5-T 195 195-180 0.000 66085-VTG-Plate 7 0.037 2.545

D L3D6-T 196 196-180 0.018 66085-VTG-Plate 7 0.037 2.545

D L3D8-T 198 198-180 0.018 66085-VTG-Plate 7 0.037 2.545

D L3D9-T 199 199-180 0.000 66085-VTG-Plate 7 0.037 2.545
ABC Study No. 66085 

Page 238 of 252 

Appendix M-3. Vitellogenin Data for Juvenile Females on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID

Vtg Conc.

(mg/mL) Plate #

min. 

detection

max. 

detection 

156 A L4A1-T 201 201-300 0.000 66085-VTG-Plate 1 0.062 4.042

A L4A2-T 202 202-300 0.000 66085-VTG-Plate 1 0.062 4.042

A L4A6-T 206 206-180 0.018 66085-VTG-Plate 5 0.036 2.492

B L4B4-T 214 214-300 0.000 66085-VTG-Plate 2 0.056 3.699

B L4B9-T 219 219-300 0.000 66085-VTG-Plate 1 0.062 4.042

C L4C1-T 221 221-180 0.017 66085-VTG-Plate 3 0.033 2.451

C L4C3-T 223 223-180 0.017 66085-VTG-Plate 3 0.033 2.451

C L4C4-T 224 224-180 0.017 66085-VTG-Plate 3 0.033 2.451

C L4C5-T 225 225-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L4C7-T 227 227-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L4C8-T 228 228-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L4C9-T 229 229-180 0.018 66085-VTG-Plate 5 0.036 2.492

C L4C10-T 230 230-180 0.018 66085-VTG-Plate 5 0.036 2.492

D L4D1-T 231 231-180 0.000 66085-VTG-Plate 4 0.037 2.505

D L4D2-T 232 232-180 0.000 66085-VTG-Plate 4 0.037 2.505

D L4D6-T 236 236-180 0.000 66085-VTG-Plate 6 0.036 2.405
ABC Study No. 66085 

Page 239 of 252 

APPENDIX N – PLASMA T4 DATA
ABC Study No. 66085 

Page 240 of 252 

Appendix N-1. T4 Data for NF Stage 62 Pooled Plasma 

Treatment

(μg/L) REP Frog Code Microtube ID

T4 Conc.

(ng/mL) Plate #

min. 

detection

max. 

detection 

Control A CA-NF62 CA 88.60 13 30.00 7458

B CB-NF62 CB 56.66 13 30.00 7458

C CC-NF62 CC 315.0 7 33.30 7594

D CD-NF62 CD 182.0 7 33.30 7594

E CE-NF62 CE 71.92 3 3.300 552.3

F CF-NF62 CF 91.23 3 3.300 552.3

G CG-NF62 CG 165.7 15 16.20 7427

H CH-NF62 CH 149.7 15 16.20 7427

6.27 A L1A-NF62 L1A 88.05 13 30.00 7458

B L1B-NF62 L1B 55.02 15 16.20 7427

C L1C-NF62 L1C 177.2 7 33.30 7594

D L1D-NF62 L1D 249.0 3 3.300 552.3

15.4 A L2A-NF62 L2A 177.4 13 30.00 7458

B L2B-NF62 L2B 80.65 13 30.00 7458

C L2C-NF62 L2C 69.42 15 16.20 7427

D L2D-NF62 L2D 40.49 3 3.300 552.3

4

50.1 A L3A-NF62 L3A 154.5 7 33.30 7594

B L3B-NF62 L3B 243.1 3 3.300 552.3

C L3C-NF62 L3C 248.2 7 33.30 7594

D L3D-NF62 L3D 170.4 13 30.00 7458

156 A L4A-NF62 L4A 175.4 7 33.30 7594

B L4B-NF62 L4B 186.3 3 3.300 552.3

C L4C-NF62 L4C 90.06 15 16.20 7427

D L4D-NF62 L4D 35.23 13 30.00 7458
ABC Study No. 66085 

Page 241 of 252 

Appendix N-2. T4 Data for Juvenile Males on Study Day 110 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

Control A CA3-T 3 3 11 20.79 13.50 8450

A CA4-T 4 4 5 70.69 17.40 4619

A CA5-T 5 5 16 12.19 7.650 3640

A CA6-T 6 6 7 79.57 33.30 7594

A CA7-T 7 7 7 51.49 33.30 7594

A CA8-T 8 8 8 76.25 16.20 7427

A CA9-T 9 9 8 44.52 16.20 7427

A CA10-T 10 10 11 23.67 13.50 8450

B CB2-T 12 12 16 36.30 7.650 3640

B CB4-T 14 14 15 37.80 15.90 8702

B CB5-T 15 15 5 216.1 17.40 4619

B CB6-T 16 16 7 45.66 33.30 7594

B CB8-T 18 18 8 54.14 16.20 7427

C CC1-T 21 21 16 47.37 7.650 3640

C CC2-T 22 22

C CC3-T 23 23 16 16.07 7.650 3640

C CC5-T 25 25 4 20.62 3.000 7432

C CC7-T 27 27 4 26.22 3.000 7432

C CC8-T 28 28 4 21.65 3.000 7432

C CC9-T 29 29 9 44.79 17.10 6983

D CD1-T 31 31 9 31.33 17.10 6983

D CD6-T 36 36 9 32.57 17.10 6983
ABC Study No. 66085 

Page 242 of 252 

Appendix N-2. T4 Data for Juvenile Males on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

Control E CE1-T 41 41 16 28.21 7.650 3640

E CE4-T 44 44 3 87.35 3.300 8794

E CE5-T 45 45 13 11.34 15.00 3729

E CE6-T 46 46 9 43.31 17.10 6983

E CE9-T 49 49 3 60.31 3.300 8794

E CE10-T 50 50 3 15.63 3.300 8794

F CF3-T 53 53 8 53.70 16.20 7427

F CF5-T 55 55 11 14.38 13.50 8450

F CF6-T 56 56 8 36.74 16.20 7427

F CF9-T 59 59 5 100.9 17.40 4619

F CF10-T 60 60 13 6.340 15.00 3729

G CG1-T 61 61 4 24.89 3.000 7432

G CG4-T 64 64 4 17.85 3.000 7432

G CG5-T 65 65 16 23.91 7.650 3640

G CG6-T 66 66 8 36.05 16.20 7427

G CG8-T 68 68 3 12.66 3.300 8794

G CG10-T 70 70 3 14.67 3.300 8794

H CH3-T 73 73 13 9.860 15.00 3729

H CH5-T 75 75 4 46.84 3.000 7432

H CH6-T 76 76 4 40.83 3.000 7432

H CH9-T 79 79 7 58.73 33.30 7594
ABC Study No. 66085 

Page 243 of 252 

Appendix N-2. T4 Data for Juvenile Males on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

6.27 A L1A1-T 81 81 4 14.67 3.000 7432

A L1A3-T 83 83 4 30.04 3.000 7432

A L1A4-T 84 84 5 109.7 17.40 4619

A L1A6-T 86 86 9 44.41 17.10 6983

A L1A7-T 87 87 9 45.05 17.10 6983

B L1B1-T 91 91 5 159.5 17.40 4619

B L1B2-T 92 92 16 15.27 7.650 3640

B L1B4-T 94 94 5 128.7 17.40 4619

B L1B5-T 95 95 13 7.460 15.00 3729

B L1B8-T 98 98 8 28.22 16.20 7427

C L1C1-T 101 101 15 47.89 15.90 8702

C L1C2-T 102 102 15 59.50 15.90 8702

C L1C3-T 103 103 11 36.16 13.50 8450

C L1C4-T 104 104 15 148.2 15.90 8702

C L1C5-T 105 105 5 115.3 17.40 4619

C L1C9-T 109 109 4 39.94 3.000 7432

C L1C10-T 110 110 9 20.06 17.10 6983

D L1D3-T 113 113 3 50.87 3.300 8794

D L1D6-T 116 116 7 60.47 33.30 7594

D L1D7-T 117 117 7 86.85 33.30 7594

D L1D8-T 118 118 9 26.52 17.10 6983

D L1D10-T 120 120 13 3.840 15.00 3729
ABC Study No. 66085 

Page 244 of 252 

Appendix N-2. T4 Data for Juvenile Males on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

15.4 A L2A4-T 124 124 5 172.4 17.40 4619

A L2A5-T 125 125 7 52.40 33.30 7594

A L2A8-T 128 128 13 10.29 15.00 3729

A L2A9-T 129 129 9 50.21 17.10 6983

B L2B1-T 131 131 11 18.34 13.50 8450

B L2B3-T 133 133 5 148.6 17.40 4619

B L2B5-T 135 135 4 59.64 3.000 7432

B L2B6-T 136 136 13 3.980 15.00 3729

B L2B9-T 139 139 7 81.13 33.30 7594

B L2B10-T 140 140 13 4.530 15.00 3729

C L2C2-T 142 142 5 224.0 17.40 4619

C L2C3-T 143 143 15 33.59 15.90 8702

C L2C4-T 144 144 15 60.46 15.90 8702

C L25-T 145 145 8 40.28 16.20 7427

C L2C6-T 146 146 8 35.56 16.20 7427

C L2C8-T 148 148 11 15.11 13.50 8450

C L2C10-T 150 150 11 46.24 13.50 8450

D L2D1-T 151 151 16 54.48 7.650 3640

D L2D2-T 152 152 15 87.00 15.90 8702

D L2D3-T 153 153 16 33.17 7.650 3640

D L2D4-T 154 154 16 29.31 7.650 3640

D L2D5-T 155 155 7 88.09 33.30 7594

D L2D7-T 157 157 7 84.44 33.30 7594
ABC Study No. 66085 

Page 245 of 252 

Appendix N-2. T4 Data for Juvenile Males on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

50.1 A L3A2-T 162 162 15 38.63 15.90 8702

A L3A5-T 165 165 9 28.72 17.10 6983

A L3A6-T 166 166 9 32.21 17.10 6983

A L3A9-T 169 169 11 23.93 13.50 8450

B L3B1-T 171 171 3 78.26 3.300 8794

B L3B3-T 173 173 3 27.33 3.300 8794

B L3B5-T 175 175 11 25.47 13.50 8450

C L3C1-T 181 181 4 65.52 3.000 7432

C L3C3-T 183 183 4 59.30 3.000 7432

C L3C6-T 186 186 8 31.00 16.20 7427

C L3C8-T 188 188 8 21.33 16.20 7427

D L3D7-T 197 197 11 39.20 13.50 8450

D L3D10-T 200 200 11 53.12 13.50 8450
ABC Study No. 66085 

Page 246 of 252 

Appendix N-2. T4 Data for Juvenile Males on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

156 B L4B5-T 215 215 13 43.24 30.00 7458

B L4B8-T 218 218 16 79.36 7.650 3640

B L4B10-T 220 220 16 22.05 7.650 3640

C L4C2-T 222 222 3 44.72 3.300 8794

C L4C6-T 226 226 13 2.550 15.00 3729

D L4D3-T 233 233 4 46.72 3.000 7432

D L4D4-T 234 234 4 51.51 3.000 7432

D L4D5-T 235 235 9 157.8 17.10 6983

D L4D7-T 237 237 5 159.3 17.40 4619

D L4D8-T 238 238 15 174.0 15.90 8702

D L4D9-T 239 239 11 51.55 13.50 8450

D L4D10-T 240 240 11 45.56 13.50 8450
ABC Study No. 66085 

Page 247 of 252 

Appendix N-3. T4 Data for Juvenile Females on Study Day 110 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

Control A CA1-T 1 1 11 30.15 13.50 8450

A CA2-T 2 2 5 150.9 17.40 4619

B CB1-T 11 11 16 17.69 7.650 3640

B CB3-T 13 13 15 121.2 15.90 8702

B CB7-T 17 17 7 83.43 33.30 7594

B CB9-T 19 19 8 29.65 16.20 7427

B CB10-T 20 20 8 18.58 16.20 7427

C CC4-T 24 24 16 50.45 7.650 3640

C CC6-T 26 26 4 36.80 3.000 7432

C CC10-T 30 30 9 24.48 17.10 6983

D CD2-T 32 32 9 95.91 17.10 6983

D CD3-T 33 33 16 19.97 7.650 3640

D CD4-T 34 34 11 21.14 13.50 8450

D CD5-T 35 35 9 41.84 17.10 6983

D CD7-T 37 37 5 113.6 17.40 4619

D CD8-T 38 38 15 53.33 15.90 8702

D CD9-T 39 39 15 63.60 15.90 8702

D CD10-T 40 40 5 120.9 17.40 4619
ABC Study No. 66085 

Page 248 of 252 

Appendix N-3. T4 Data for Juvenile Females on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

Control E CE2-T 42 42 13 33.93 30.00 7458

E CE3-T 43 43 3 61.21 3.300 8794

E CE7-T 47 47 11 29.40 13.50 8450

E CE8-T 48 48 9 141.5 17.10 6983

F CF1-T 51 51 13 11.78 15.00 3729

F CF2-T 52 52 3 8.160 3.300 8794

F CF4-T 54 54 8 64.46 16.20 7427

F CF7-T 57 57 15 34.30 15.90 8702

F CF8-T 58 58 15 76.95 15.90 8702

G CG2-T 62 62 4 37.51 3.000 7432

G CG3-T 63 63 4 36.27 3.000 7432

G CG7-T 67 67 3 56.53 3.300 8794

G CG9-T 69 69 3 15.31 3.300 8794

H CH1-T 71 71 9 45.27 17.10 6983

H CH2-T 72 72 9 72.27 17.10 6983

H CH4-T 74 74 13 12.72 15.00 3729

H CH7-T 77 77 7 153.4 33.30 7594

H CH8-T 78 78 7 58.40 33.30 7594

H CH10-T 80 80 7 59.89 33.30 7594
ABC Study No. 66085 

Page 249 of 252 

Appendix N-3. T4 Data for Juvenile Females on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

6.27 A L1A2-T 82 82 4 11.02 3.000 7432

A L1A5-T 85 85 9 24.26 17.10 6983

A L1A8-T 88 88 9 62.13 17.10 6983

A L1A9-T 89 89 9 23.73 17.10 6983

A L1A10-T 90 90 9 33.57 17.10 6983

B L1B3-T 93 93 15 30.08 15.90 8702

B L1B6-T 96 96 8 78.35 16.20 7427

B L1B7-T 97 97 8 22.93 16.20 7427

B L1B9-T 99 99 8 38.44 16.20 7427

B L1B10-T 100 100 8 19.12 16.20 7427

C L1C6-T 106 106 4 45.13 3.000 7432

C L1C7-T 107 107 4 27.98 3.000 7432

C L1C8-T 108 108 4 11.46 3.000 7432

D L1D1-T 111 111 3 8.260 3.300 8794

D L1D2-T 112 112 3 28.96 3.300 8794

D L1D4-T 114 114 3 56.15 3.300 8794

D L1D5-T 115 115 3 162.2 3.300 8794

D L1D9-T 119 119 13 11.07 15.00 3729
ABC Study No. 66085 

Page 250 of 252 

Appendix N-3. T4 Data for Juvenile Females on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

15.4 A L2A1-T 121 121 3 43.56 3.300 8794

A L2A2-T 122 122 3 77.72 3.300 8794

A L2A3-T 123 123 3 87.33 3.300 8794

A L2A6-T 126 126 7 50.02 33.30 7594

A L2A7-T 127 127 7 55.00 33.30 7594

B L2B2-T 132 132 4 58.84 3.000 7432

B L2B4-T 134 134 4 27.84 3.000 7432

B L2B7-T 137 137 8 51.87 16.20 7427

B L2B8-T 138 138 8 32.12 16.20 7427

C L2C1-T 141 141 15 187.1 15.90 8702

C L2C7-T 147 147 8 63.51 16.20 7427

C L2C9-T 149 149 11 72.98 13.50 8450

D L2D6-T 156 156 13 4.570 15.00 3729

D L2D8-T 158 158 13 13.74 15.00 3729

D L2D9-T 159 159 11 28.03 13.50 8450

D L2D10-T 160 160 11 13.86 13.50 8450
ABC Study No. 66085 

Page 251 of 252 

Appendix N-3. T4 Data for Juvenile Females on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

50.1 A L3A1-T 161 161 15 117.4 15.90 8702

A L3A3-T 163 163 5 229.8 17.40 4619

A L3A4-T 164 164 16 59.85 7.650 3640

A L3A7-T 167 167 13 8.470 15.00 3729

A L3A8-T 168 168 13 3.120 15.00 3729

A L3A10-T 170 170 16 8.790 7.650 3640

B L3B2-T 172 172 3 63.17 3.300 8794

B L3B4-T 174 174 3 26.21 3.300 8794

B L3B6-T 176 176 7 66.05 33.30 7594

B L3B7-T 177 177 13 5.550 15.00 3729

B L3B8-T 178 178 7 40.55 33.30 7594

B L3B9-T 179 179 8 17.89 16.20 7427

B L3B10-T 180 180 8 27.34 16.20 7427

C L3C2-T 182 182 4 59.13 3.000 7432

C L3C4-T 184 184 4 73.29 3.000 7432

C L3C5-T 185 185 13 9.610 15.00 3729

C L3C7-T 187 187 8 29.51 16.20 7427

C L3C10-T 190 190 7 80.35 33.30 7594

D L3D1-T 191 191 5 318.4 17.40 4619

D L3D2-T 192 192 11 17.62 13.50 8450

D L3D2-T 193 193 11 24.00 13.50 8450

D L3D4-T 194 194 5 233.3 17.40 4619

D L3D5-T 195 195 16 9.830 7.650 3640

D L3D6-T 196 196 16 27.01 7.650 3640

D L3D8-T 198 198 11 45.68 13.50 8450

D L3D9-T 199 199 11 26.32 13.50 8450
ABC Study No. 66085 

Page 252 of 252 

Appendix N-3. T4 Data for Juvenile Females on Study Day 110 (cont.) 

Treatment

(μg/L) REP Frog Code

Frog 

ID Microtube ID Plate #

T4 Conc.

(ng/mL)

min. 

detection

max. 

detection 

156 A L4A1-T 201 201 15 53.71 15.90 8702

A L4A2-T 202 202 15 103.0 15.90 8702

A L4A6-T 206 206 8 27.53 16.20 7427

B L4B4-T 214 214 5 292.6 17.40 4619

B L4B9-T 219 219 11 40.83 13.50 8450

C L4C1-T 221 221 3 3.730 3.300 8794

C L4C3-T 223 223 13 1.710 15.00 3729

C L4C4-T 224 224 3 52.68 3.300 8794

C L4C5-T 225 225 7 142.5 33.30 7594

C L4C7-T 227 227 8 31.63 16.20 7427

C L4C8-T 228 228 16 13.33 7.650 3640

C L4C9-T 229 229 8 16.61 16.20 7427

C L4C10-T 230 230 13 12.94 15.00 3729

D L4D1-T 231 231 3 127.0 3.300 8794

D L4D2-T 232 232 4 25.09 3.000 7432

D L4D6-T 236 236 9 69.01 17.10 6983