### Page 1 of 252 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 Op erations

1529 West Sequim Bay Road

Sequim, Washington 98382

PERFORMING LABORATOR IES

ABC Laboratories, Inc. (In

- Life Phase)

7200 E. ABC Lane

Columbia, Missouri 65202

United States

Battelle

- Pacific Northwest Division ( Genetic Sexing & Statistics)

Marine Re search Operations

1529 West Sequim Bay Road

Sequim, Washington 98382

PROJECT IDENTIFICATION

ABC Study No. 66085

### AB C Study No. 66085

Page 6 of 252 TABLE OF CONTENTS

Page

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

STATEMENT OF GLP COM PLIANCE ........................................................................................2

QUALITY ASSURANCE ST ATEMEN T ......................................................................................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 METHOD S .......................................................................................13

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

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

2.3 D ilution 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 Vitello genin 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 In strument Conditions ................................................................................21

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

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

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

3.0 RESULTS AND DISCUSSI ON ........................................................................................24

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

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

3.3 Biological Results ..................................................................................................26

### AB C Study No. 66085

Page 7 of 252 3.4 Genotypic/Phenotypic Sex Ratios..........................................................................38

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

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

3.7 Plasma Thyroxin e (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 proc hloraz 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 Xe nopus 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 expo sure. ........36

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

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

Ta ble 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

### AB C Study No. 66085

Page 8 of 252 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 proc hloraz. ...........................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 mea n, 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 interquartil e 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. Th e 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

X

enopus 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 t he 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 terminatio n

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

### AB C Study No. 66085

Page 9 of 252 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 ci rcle 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 terminat ion

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 ANALY SIS ........................................................................50

APPENDIX B

- DILUTION WATER CHARA CTERIZATION .................................................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 D ATA ....................................................................177

APPENDIX H

– NF STAGE 62 DATA ......................................................................................186

APPENDIX I – N F STAGE 66 CULL DATA ............................................................................196

APPENDIX J

– TERMINATION DATA

– GROWTH AND GENDER ...................................200

APPENDIX K

– TERMINATION DATA

– NUPTIAL PAD AND LIV ER .............................207

APPENDIX L

– WATER QUALITY DATA .............................................................................215

### AB C Study No. 66085

Page 10 of 252 APPENDIX M

– PLASMA VITELLOGENIN DATA

............................................................... 225

APPENDIX N

– PLASMA T4 DATA ........................................................................................239

### AB C Study No. 66085

Page 11 of 252 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 nom inal 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 fr om 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 concentrati ons 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

Con centration (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

### AB C Study No. 66085

Page 12 of 252 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

### AB C Study No. 66085

Page 13 of 252 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 suppor t 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 obtain ing

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 endocrine

disrupting compounds. The chemical prochloraz is a known endoc rine

- 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 dis rupter 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 sample s of the test substance, prochloraz ( CAS No.: 67747 -09- 5; Lot No.: 450 -112B ), w ere received from Battelle Marine Sciences Laboratory, Sequim, Washington, on 22 September 2010

and w ere assigned ABC re ference number s 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 s ubstance 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 A frican 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 1 4- 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; N ASCO; Fort Atkinson, Wisconsin ) supplemented with a

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

### (S era North America; Montgomeryville, Pennsylvania ).

### AB C Study No. 66085

Page 14 of 252 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 di lution 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 CaCO 3 

### /L. The water was the n 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 analyz ed 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 deviation s ( 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 so luble 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), p acked with

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

end. A solution of p rochloraz 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 p rochloraz 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 p rochloraz 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 p rochloraz . The p rochloraz 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

### AB C Study No. 66085

Page 15 of 252 dilution water reservoir and collection vessel w ere 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 solut ions 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 t he U.S. EPA were 6.7, 20, 60, and 180 μg/L, along with a dilution

water 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 solut ion 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 ea ch 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 initiall y set to a minimum cycle rate of 5.0 cycles per hour. The cycle

rate was increased on 22 March 201 1 (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

rat e 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, an d 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 t est 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

### AB C Study No. 66085

Page 16 of 252 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 treat ment 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 3 0- 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 post

NF66 (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 i n a heparinized capillary tube. Immediately

following blood collection, each individual was euthanized by immersion in a MS

- 222 solution

### AB C Study No. 66085

Page 17 of 252 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 o f 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) th at 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

Biome dical was used to quantify Xenopus T4 in pooled plasma samples. V itellogenin 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, t en 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. Blott ed wet weight and snout

- to

- vent length of

each juvenile w ere recorded

, any gross morphological abnormalities or signs of toxicity w ere 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 trans fer 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.

### AB C Study No. 66085

Page 18 of 252 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 meas ured 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 concentrat ion w as 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 f ree

swimming 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: f ree

swimming stage

- day 7, 40 mg /larvae/day; day 8

- 10, 50 mg /larvae/day; day 11

- 14, 70 mg

/larvae/day; day 15- 21, 80 mg /larvae/day; day 22

- 70, 80 mg /larvae/day plus 1 mL of live brine

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

weekdays and once daily on weekends using th ree 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 decreas ed . Once animals

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

receive d 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 change d 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 ha d occurred, juveniles were fed only sinking frog food with each tank initially

receiving the amount appropriate for 10 individuals.

### AB C Study No. 66085

Page 19 of 252 2.4.3.7 Vitellogenin Analysis

Vitellogenin (V TG ) analysis of plasma samples was conducted acco rding 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 positi ve 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 Xen opus 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 spike d Xenopus samples we re 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 R 2 

### of 0.99 or

greater and the back calculate d standard concentrations of the standards used to prepare the

standard curve were between 70 and 120% of the nominal concentration.

Plates we re coated with coating solution containing V TG 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

wa s 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 1 M H 3 

### PO 4 

### 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 R 2



### 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 V TG 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 V TG value for a particular

sample was above the curve, the sample was re

- diluted to fal l 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.

### AB C Study No. 66085

Page 20 of 252 2.4.3.8 Thyroxine (T4 ) Analysis

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

quantify Xeno pus 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

sta ndards and samples. For each analysis, the calibration curve had an R 2 

### 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 fi ve 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 c alibration 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 cri teria

(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 pre

co ated 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 m ixed 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 n g/mL, were based on sample runs that had standard curve R 2



### 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 parti cular 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 r e- 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 dilut ion factor for that sample.

### AB C Study No. 66085

Page 21 of 252 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 meth od of analysis are described below.

2.5.1 Preparation of Analytical Standard and Matrix Spiking Solutions

P rimary stock solution s of prochloraz w ere prepared in methanol on 03 November 2010 and

14 April 2011 at concentrations of 3.37 and 0.617 m g/mL , respe ctively . Subsequent dilutions of

th ese stock solution s were prepared in 50:50 methanol: water for use as analytical reference

standards for high-performance liquid chromatography (HPLC).

P rimary stock solution s of prochloraz w ere prepared in methanol on 22 November 2010

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

, respectively . Subsequent dilutions of th ese stock solution s were prepared in methanol and used

to prepare quality control (QC) samples.

All analytical st andard 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. S ample s 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 samp les

. 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

. T he samples were placed into appropriately sized culture tube s. 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 n g/mL ). The

s amples were analyzed by a H PLC 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:

Column s: 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)

### AB C Study No. 66085

Page 22 of 252 Flow Rate: 1.0 m L/minute

Injection Volume: 15 0 μL

Wavelength: 220 nm

Detector: UV (VWD)

Stop Time: 8 minutes

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

the chromatographic conditions.

2.5.4 Calculations for Prochloraz

Ca lculation 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

mLin volume sample mLin volume analysis

curve standard 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 th e 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:



# ( )

a.i./L g 71 .7

mL 10 mL 20 a.i./L) g 857.3(

μ μ

=



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

115% 100x μg/L 6.7 a.i./L μg 7.71

=

### AB C Study No. 66085

Page 23 of 252 The minimum quantifiable limit (MQL) was determined from the equation:

μg/L as expressed MQL

(mL) sampled volume (mL) analysis

forvolume

L) (ion concentrat standard lowest

=

 

 

 

   

 

 

 

 

 

 

 

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 mL) (10 mL) (20 μg/L) (1.62

=



### 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 prese nce 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 withi n 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 Shap iro

- 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

h omogeneity 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

### AB C Study No. 66085

Page 24 of 252 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 t hose 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 altern atives. The Jonckheere

Terpstra 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 estim ated 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 homogeneit y 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 produ ct

- 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 p rovided in the results tables. However, statistical differences detected by the K

W test or the K

- W multiple comparisons test were between median values. When frog sex is

mentioned in statistical analyses, the genotypic sex was used. Analyses of all NF stage 62

parameters, both NF stage 66 cull parameters, and termination whole body weight and snout

- to

vent 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.

### AB C Study No. 66085

Page 25 of 252 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 <MQL a NA

Control 2 0 <MQL a NA

Control 3 0 <MQL a 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 sa mples collected during the definitive test. M easured

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 we re 8.17, 20.6,

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

m ean 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

w ith 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 sample s ranged from 82 to 111 % of the nominal

concentration with the exception of the day 0 sample which was 6 8%. No residues of the

prochloraz were detected in the control s above the MQL of 3.24 n g/mL

. Recoveries from the

prochloraz quality control fortifications ranged from 81 to 151 % of the nominal concentrations.

### AB C Study No. 66085

Page 26 of 252 Because the mean measured concentrations of prochloraz were not all within 20% of nominal

concentrations during the exposure, t he biological response results were reported based upon the

mean of the measured concent rations. 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 deliv ering 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 del ivery 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

(μ g/L ) Mean Measured

C oncentration (μ g/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 surv ival 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 compromise d (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 t est substance treatment. Spin al 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) spin al curvature in the con trol (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 th e 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 t wo juveniles (one in each of the 6.27 and 15.4 μg/L test substance treatments)

each had a missing eye. N o other sublethal effects were observed after study day 50.

### AB C Study No. 66085

Page 27 of 252 NF Stage 62 Sur v ival:

Survival through NF stage 62 was 92 % for the control treatment. Twelve of 15 9 controltreatment larvae died before reaching NF stage 62; one larvae w as 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 co ntrol- 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 μ g/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 r eached for each NF stage 62 larv a sampled are presented in Appendix H

.

Table 3.3.1

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

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

### AB C Study No. 66085

Page 28 of 252 156 50.1 15.4 6.27 <MQL 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 lar v ae measured are

presented in Appendix H

. M ean 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 hig h median weight and relatively low median weight for frogs in the

6.27 μg/L and 156 μ g/L exposures, respectively . M ean snout

- to

- vent length of NF stage 62

larvae in the control treatment was 2 1 m m and ranged from 20 to 21 m m 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 μ g/L

exposure.

### AB C Study No. 66085

Page 29 of 252 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

156 50.1 15.4 6.27 <MQL 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.

### AB C Study No. 66085

Page 30 of 252 156 50.1 15.4 6.27 <MQL 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 remov ed 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 2 9 m m in the control treatment and ranged from 27 to

31 m m in the prochloraz trea tments ( Table 3.3.3 ; Figure 3.3.5 ). No significant differences in

snout- to

- vent length among treatments were detected for these frogs.

### AB C Study No. 66085

Page 31 of 252 Table 3.3.3

. Mean (and standard deviation) whole body wei ght and snout

- to

- vent length at the NF stage

66 cull of the Xenopus laevis prochloraz exposure.

Mean Measured

Concentration (μ g/L ) Whole Body Weight

(g) Snout

- to

- Vent Length

( m m)

<MQL 3.18 (0.832) 2 9 ( 2.3 )

6.27 3.17 (0.624) 31 ( 1.4 )

15.4 2.37 (0.752) 2 8 ( 2.5 )

50.1 2.36 (0.449) 2 8 ( 1.6 )

156 2.10 (0.467) 27 ( 1.3 )

p

- value 0.068 0. 079

Test: 1- Way ANOVA K-W

156 50.1 15.4 6.27 <MQL 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.

### AB C Study No. 66085

Page 32 of 252 156 50.1 15.4 6.27 <MQL 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

proc hloraz 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 pro chloraz 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 4 5 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 Measu red

Concentration (μg/L) Whole Body Weight

(g) Snout

- to

- Vent Length

( m m)

<MQL 12.6 (1.75) 4 5 ( 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

### AB C Study No. 66085

Page 33 of 252 156 50.1 15.4 6.27 <MQL 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.

156 50.1 15.4 6.27 <MQL 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.



### Nuptia l Pad Development at Termination :

The status of nuptial pads (i.e., presen t or absen t) 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.

### AB C Study No. 66085

Page 34 of 252 ( 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

hav ing 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 laev is 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)

.

### AB C Study No. 66085

Page 35 of 252 156 50.1 15.4 6.27 <MQL 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 i s the median.

156 50.1 15.4 6.27 <MQL 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.

### AB C Study No. 66085

Page 36 of 252 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 P ads 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 l iver 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 ( T able 3.3.7 ; Figure 3.3.1 1 ).

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 fo r 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

### AB C Study No. 66085

Page 37 of 252 156 50.1 15.4 6.27 <MQL 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 pro chloraz exposure. The box represents the interquartile range, the

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

156 50.1 15.4 6.27 <MQL 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 t he Xenopus

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

mean, and the horizontal line is the median.

### AB C Study No. 66085

Page 38 of 252 3.4 Genotypic/Phenotypic Sex Ratios

The genetic sexing of the juvenile frogs using PCR followed by gel sepa ration 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 Xen opus 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 10 8 %

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 day s 91 and 100

. The conti nuous 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 replic ate 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 CaC O 3 

### /L, from 152 to 164 mg CaCO 3 

### /L, and from 315 to 356 μ S, respectively. All

individual water parameter measurements during the 110

- day exposure are presented in

Appendix L

.

### AB C Study No. 66085

Page 39 of 252 Table 3.5.1

. Ranges for Water Quality Me asurements During the 110

- Day Exposure with the African

Clawed Frog, Xenopus laevis, Exposed to Prochloraz

Mean Measured

Concentration

(μ g/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

-- -- --

15 6 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 Vitell ogenin Results

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

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

V TG was below the concentration of the lowest standa rd (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 V TG 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 V TG or below the lowest standard (i.e., 0.03 3

to 0.037 mg/mL) than those males with a quantifiable concentration of V TG

. The measured

V TG 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 V TG or below the lowest standard (i.e., 0.034 or 0.036 mg/mL) than

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

for fr ogs 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 co ncentrations 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

.

### AB C Study No. 66085

Page 40 of 252 Table 3.6.1

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

Xenopus laevis prochloraz exposure.

Mean M easured

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

156 50.1 15.4 6.27 <MQL 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.

### AB C Study No. 66085

Page 41 of 252 156 50.1 15.4 6.27 <MQL 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 e xposure. 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 9 2.0 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 3 7.8 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.

### AB C Study No. 66085

Page 42 of 252 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

### AB C Study No. 66085

Page 43 of 252 156 50.1 15.4 6.27 <MQL 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 Xenop us

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

mean, and the horizontal line is the median

156 50.1 15.4 6.27 <MQL 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 termina tion 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.

### AB C Study No. 66085

Page 44 of 252 156 50.1 15.4 6.27 <MQL 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 thyroxi ne 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

V alidity and p erformance criteria state d 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 t est 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. De livery 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

tr eatments. T ime to NF stage 62 among control larvae ranged from 41 to 46 days

. The m ean 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 μ g/L prochloraz and >156 μ g/L prochloraz

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

ratio (Table 4.0.1 ).

### AB C Study No. 66085

Page 45 of 252 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.

Param eter 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

Whole Body Weight 156 >156

Snout

- to

- Vent Lengt h 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 >1 56

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.

### AB C Study No. 66085

Page 46 of 252 PROTOCOL DEVIATION S

Item 1 Section 10.0 Test Subst ance 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 pum p had seized and this stopped

delivery of the test substance to the diluter system.

Effect on Study: No adverse e ffect on study integrity

. The duration the diluter system had

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

a nd <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 restor ing test substance delivery w ere ≤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 replicat e 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 re

circulation of water within the wate r bath) were not sufficient to achieve

maintain all test solutions at this temperature.

Effect on Study: No adverse e ffect on study integrity

. With the above noted exceptions, test

solution temperatures were 21 ± 1 oC and the continuous temperature re cord

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

### AB C Study No. 66085

Page 47 of 252 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 μ g/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 acro ss 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 s tudy.

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

solution temperatures across treatments and replicates on a g iven day were

consistent (i.e., ≤0.5 oC 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 treatm ent 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 thes e days.

Effect on Study: No adverse e ffect 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. Samp les 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 St udy: None. Analytical samples were collected at least weekly throughout the

110 day exposure.

### AB C Study No. 66085

Page 48 of 252 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: Eclips e 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.

### AB C Study No. 66085

Page 49 of 252 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. Wa ter 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 . 20 th 

### ed. Washington, D.C.

### ABC Study No. 66085

Page 50 of 252 A PPENDIX A - CERTIFICATE OF ANALY SIS

### ABC Study No. 66085

Page 51 of 252

### ABC Study No. 66085

Page 52 of 252 A PPENDIX B - DILUTION WATER CHARACTERIZATION

### ABC Study No. 66085

Page 53 of 252 Chemical Characteristics of ABC Well Water Used by

ABC Laboratories' Chemical Servic es 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 ethyl 5, 7

--- <1.0 Aluminum 4 <0.0500 <0.0500

Azinphos

- methyl 3 <1.00 <0.200

- <1.00 Antimony 4 <0.0500 <0.0500

Bolstar 4 <1.00 <0.200

- <1.00 Arsenic <0.0030 <0.0030

- <0.050

Chloropyrifos 4 <1.00 <0.200

- <1.00 Barium 4 0.0206 0.0189

- 0.0206

Coumaphos 4 <1.00 <0.400

- <1.00 Beryllium 4 <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

Dichlorvos 4 <1.00 <0.200

- <1.00 Calcium 2 78.4 52

- 83.1

Dimethoate 4 <1.00 <1.00 Chromium <0.0100 <0.010

Disulfoton <1.00 <0.200

- <1.0 Cobalt 4 <0.0100 <0.0100

EPN 4 <1.00 <0.200

- <1.00 Copper <0.0100 <0.010

Ethion 5, 7

--- <1.0 Iron 0.0294 <0.0059

- 0.16

Ethoprop 4 <1.00 <0.200

- <1.00 Lead <0.0020 <0.0020

- 0.0400

Famphur 6 <1.00 <1.00 Magnesium 2 33.3 27

- 33.3

Fensulfothio n 4 <1.00 <1.00 Manganese 4 <0.0050 <0.0050

Fenthion 4 <1.00 <0.200

- <1.00 Molybdenum 4 <0.0100 <0.0100

Malathion <1.00 <0.200

- <1.0 Mercury 5 <0.00020 <0.00020

-

<0.00060

Merphos 4, 7

--- <0.200 Nickel <0.0100 <0.0100

- <0.020

Mevinphos 4 <1.0 0 <1.00 Potassium 2 8.42 6.6

- 8.42

Monocrotophos 4, 7

--- <1.00 Selenium <0.0500 <0.050

Naled 4, 7

--- <2.00 Silver <0.0100 <0.010

O,O,O

- Triethyl

phosphorothioate 6 <1.00 <1.00 Sodium 2 30.0 27

- 32.2

Thallium 4 <0.0500 <0.0500

Parat hion 5, 7

--- <1.0 Tin 4 <0.0200 <0.0200

Parathion, ethyl 3 <1.00 <0.200

- <1.00 Vanadium 4 <0.0100 <0.0100

Parathion, methyl 3 <1.00 <0.200

- <1.00 Zinc 0.0198 0.0118

- 0.078

Phorate 4 <1.00 <0.200

- <1.00

Ronnel 4 <1.00 <0.200

- <1.00

Stirophos 4 <1.00 <0.200

- <1.00 Polychlorinated

Biphenyls ( μ g/L) Sulfotepp 4 <1.00 <0.200

- <1.00

TEPP 4, 7

--- <0.200 Aroclor 1016 <1.00 <1.00

Thionazin 6 <1.00 1.00 Aroclor 1221 <1.00 <1.00

Tokuthion 4 <1.00 <0.200

- <1.00 Aro clor 1232 <1.00 <1.00

Trichloronate 4 <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

### ABC Study No. 66085

Page 54 of 252 Chemical Characteristics of ABC Well Water Used by

ABC Laboratories' Chemical Services Group (continued)

February 2011 ABC Well Water Screen (non

- GLP)

Chlorinated

Herbicides ( μ g/L) 2011 Historical

Range

1998

- 2011 1 Chlorinated

Hydrocarbons ( μ g/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 f or ABC Aged

- Blended Water.

### ABC Study No. 66085

Page 55 of 252 A PPENDIX C - PROTOCOL , A MENDMENTS, AND DEVIATION S

### ABC Study No. 66085

Page 56 of 252

### ABC Study No. 66085

Page 57 of 252

### ABC Study No. 66085

Page 58 of 252

### ABC Study No. 66085

Page 59 of 252

### ABC Study No. 66085

Page 60 of 252

### ABC Study No. 66085

Page 61 of 252

### ABC Study No. 66085

Page 62 of 252

### ABC Study No. 66085

Page 63 of 252

### ABC Study No. 66085

Page 64 of 252

### ABC Study No. 66085

Page 65 of 252

### ABC Study No. 66085

Page 66 of 252

### ABC Study No. 66085

Page 67 of 252

### ABC Study No. 66085

Page 68 of 252

### ABC Study No. 66085

Page 69 of 252

### ABC Study No. 66085

Page 70 of 252

### ABC Study No. 66085

Page 71 of 252

### ABC Study No. 66085

Page 72 of 252

### ABC Study No. 66085

Page 73 of 252

### ABC Study No. 66085

Page 74 of 252

### ABC Study No. 66085

Page 75 of 252

### ABC Study No. 66085

Page 76 of 252

### ABC Study No. 66085

Page 77 of 252

### ABC Study No. 66085

Page 78 of 252

### ABC Study No. 66085

Page 79 of 252

### ABC Study No. 66085

Page 80 of 252

### ABC Study No. 66085

Page 81 of 252

### ABC Study No. 66085

Page 82 of 252

### ABC Study No. 66085

Page 83 of 252

### ABC Study No. 66085

Page 84 of 252

### ABC Study No. 66085

Page 85 of 252

### ABC Study No. 66085

Page 86 of 252

### ABC Study No. 66085

Page 87 of 252

### ABC Study No. 66085

Page 88 of 252

### ABC Study No. 66085

Page 89 of 252

### ABC Study No. 66085

Page 90 of 252

### ABC Study No. 66085

Page 91 of 252

### ABC Study No. 66085

Page 92 of 252

### ABC Study No. 66085

Page 93 of 252

### ABC Study No. 66085

Page 94 of 252

### ABC Study No. 66085

Page 95 of 252

### ABC Study No. 66085

Page 96 of 252

### ABC Study No. 66085

Page 97 of 252

### ABC Study No. 66085

Page 98 of 252

### ABC Study No. 66085

Page 99 of 252 APPENDIX D – GENDER REPORT

### ABC Study No. 66085

Page 100 of 252

### ABC Study No. 66085

Page 101 of 252

### ABC Study No. 66085

Page 102 of 252

### ABC Study No. 66085

Page 103 of 252

### ABC Study No. 66085

Page 104 of 252

### ABC Study No. 66085

Page 105 of 252

### ABC Study No. 66085

Page 106 of 252

### ABC Study No. 66085

Page 107 of 252

### ABC Study No. 66085

Page 108 of 252

### ABC Study No. 66085

Page 109 of 252

### ABC Study No. 66085

Page 110 of 252

### ABC Study No. 66085

Page 111 of 252

### ABC Study No. 66085

Page 112 of 252

### ABC Study No. 66085

Page 113 of 252

### ABC Study No. 66085

Page 114 of 252

### ABC Study No. 66085

Page 115 of 252

### ABC Study No. 66085

Page 116 of 252

### ABC Study No. 66085

Page 117 of 252

### ABC Study No. 66085

Page 118 of 252

### ABC Study No. 66085

Page 119 of 252

### ABC Study No. 66085

Page 120 of 252

### ABC Study No. 66085

Page 121 of 252

### ABC Study No. 66085

Page 122 of 252

### ABC Study No. 66085

Page 123 of 252

### ABC Study No. 66085

Page 124 of 252

### ABC Study No. 66085

Page 125 of 252

### ABC Study No. 66085

Page 126 of 252

### ABC Study No. 66085

Page 127 of 252

### ABC Study No. 66085

Page 128 of 252

### ABC Study No. 66085

Page 129 of 252

### ABC Study No. 66085

Page 130 of 252

### ABC Study No. 66085

Page 131 of 252

### ABC Study No. 66085

Page 132 of 252

### ABC Study No. 66085

Page 133 of 252

### ABC Study No. 66085

Page 134 of 252

### ABC Study No. 66085

Page 135 of 252

### ABC Study No. 66085

Page 136 of 252

### ABC Study No. 66085

Page 137 of 252

### ABC Study No. 66085

Page 138 of 252

### ABC Study No. 66085

Page 139 of 252

### ABC Study No. 66085

Page 140 of 252

### ABC Study No. 66085

Page 141 of 252

### ABC Study No. 66085

Page 142 of 252

### ABC Study No. 66085

Page 143 of 252

### ABC Study No. 66085

Page 144 of 252

### ABC Study No. 66085

Page 145 of 252 APPENDIX E – STATISTICAL REPORT

### ABC Study No. 66085

Page 146 of 252

### ABC Study No. 66085

Page 147 of 252

### ABC Study No. 66085

Page 148 of 252

### ABC Study No. 66085

Page 149 of 252

### ABC Study No. 66085

Page 150 of 252

### ABC Study No. 66085

Page 151 of 252

### ABC Study No. 66085

Page 152 of 252

### ABC Study No. 66085

Page 153 of 252

### ABC Study No. 66085

Page 154 of 252

### ABC Study No. 66085

Page 155 of 252

### ABC Study No. 66085

Page 156 of 252

### ABC Study No. 66085

Page 157 of 252

### ABC Study No. 66085

Page 158 of 252

### ABC Study No. 66085

Page 159 of 252

### ABC Study No. 66085

Page 160 of 252

### ABC Study No. 66085

Page 161 of 252

### ABC Study No. 66085

Page 162 of 252

### ABC Study No. 66085

Page 163 of 252

### ABC Study No. 66085

Page 164 of 252

### ABC Study No. 66085

Page 165 of 252

### ABC Study No. 66085

Page 166 of 252

### ABC Study No. 66085

Page 167 of 252

### ABC Study No. 66085

Page 168 of 252

### ABC Study No. 66085

Page 169 of 252

### ABC Study No. 66085

Page 170 of 252

### ABC Study No. 66085

Page 171 of 252

### ABC Study No. 66085

Page 172 of 252

### ABC Study No. 66085

Page 173 of 252

### ABC Study No. 66085

Page 174 of 252 APPENDIX F – ANALYTICAL DATA SUMMARY

### ABC Study No. 66085

Page 175 of 252 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 μ g/L

Control

0 6.7 20 60 180 Stock

15,200

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

- 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 7 6 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 6 4 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 ini tiation. Day

– N values not included in overall mean calculation.

b Average of duplicate re

- analyses.

### ABC Study No. 66085

Page 176 of 252 Table F-2. Measured Concentrations of Prochloraz Quality Control Samples During the

Larval Growth and Development Assay with the African Clawed Frog,

Xe nopus laevis

Sample Day Measured Concentration Expressed as μ g/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 μ g/L.

b 

### Average of duplicate re

- analyses.

c 

### Value confirmed by reanalysis.

### ABC Study No. 66085

Page 177 of 252 APPENDIX G - REPLICATE SURVIVAL DATA

### ABC Study No. 66085

Page 178 of 252 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to Prochloraz for 110- Days

Mean

Measured

Concentration

(μ g/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.

### ABC Study No. 66085

Page 179 of 252 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to Prochloraz for 110-Days (continued)

Mean

Measured

Concentration

(μ g/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 Cumulati ve

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 10 0

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

### ABC Study No. 66085

Page 180 of 252 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to Prochloraz for 110-Days (continued)

Mean

Measured

Concentration

(μ g/L ) Replicate Day 0

Initial

Number Number

Removed for

Samp ling 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

### ABC Study No. 66085

Page 181 of 252 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to Prochloraz for 110-Days (continued)

Mean

Measured

Concentration

(μ g/L ) Replicate Day 0

Initial

Numbe r 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 1 3 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 da y and was approximately

NF stage 44. The loss of this organism was considered to be an exposure

- related mortalit y and was added to the cumulative number dead.

### ABC Study No. 66085

Page 182 of 252 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to Prochloraz for 110-Days (continued)

Mean

Measured

Concentration

(μ g/L ) Replicate Day 0

Initial

Number Number

Removed fo r

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

Page 183 of 252 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to Prochloraz for 110-Days (continued)

Mean

Measured

Concentration

(μ g/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 1 0 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

Page 184 of 252 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to Prochloraz for 110-Days (continued)

Mean

Measured

Concentration

(μ g/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 9 2

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 2 3 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

Page 185 of 252 Table G -2. Survival of the African Clawed Frog, Xenopus laevis , Exposed to Prochloraz for 110 Days

Mean

Measured

Concentration

(μ g/L ) Rep Day 0

Initial

Number NF Stage 62

Survivors NF Stage 6 2

% 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 exposur e- 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

(μ g/L ) Replicate Tadpole# Study Day NF 62

Achieved Weight (g) Snout

- to

- Vent

Len gth (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 (Contro l) 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 (C ontrol) 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

(μ g/ 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

1 5.4 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

(μ g/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

Page 190 of 252 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 (Con trol) 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

Page 191 of 252 Appendix H-2. Replicate Data for Number of Day s 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 Num ber 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 2 0

--

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 4 4

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) R eplicate 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 DAT A

### ABC Study No. 66085

Page 197 of 252 Appendix I-1. Replicate Weight, Length, and Phenotypic Sex D ata at NF Stage 66 Cull

Treatment

(μ g/L ) Replicate Juvenile Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic S ex

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 3.43 0 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 (C ontrol) 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 (C ontrol) H 4

--- --- ---

0 (Control) H 5

--- --- ---



### ‘--- ’ denotes a juvenile was not available for this sample

### ABC Study No. 66085

Page 198 of 252 Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull (cont.)

Treatment

(μ g/L ) Replicate Juvenile Weight

(g) Snou t

- to

- Vent

Length (mm) Phenotypic S ex

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 F EMALE

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

Page 199 of 252 Appendix I-1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull (cont.)

Treatment

(μ g/L ) Replicate Juvenile Weight

(g) Snout

- to

- Vent

Length (mm) Phenotypic S ex

50.1 A 1 2.256 28 FEMALE

50.1 A 2 3.186 29 M ALE

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

15 6 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

– GROWT H AND GENDER

### ABC Study No. 66085

Page 201 of 252 Appendix J-1. Replicate Weight, Length, and Sex Data at Termination

Treatment

(μ g/L ) Replicate Juvenile Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic /Genotypic

S ex a

0 (Control) A CA1

- T 10.449 49 FEMALE

0 (Control) A CA2 -T 7.216 39 FEMA LE

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 7.15 0 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 (Con trol) B CB7 -T 9.404 42 FEMALE

0 (Control) B CB8

- T 15.856 49 MALE

0 (Control) B CB9

- T 12.98 0 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 (Cont rol) 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 11.78 0 43 FEMALE

0 (Co ntrol) 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

Page 202 of 252 Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μ g/L ) Replicate Juvenile Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic /Genotypic

S ex 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 14.5 49 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 C H10

- 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

(μ g/L ) Replicate Juvenile Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic /Genotypic

S ex 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 F EMALE

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

Page 204 of 252 Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μ g/L ) Replicate Juvenile Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic /Genotypic

S ex 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 4 2 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

Page 205 of 252 Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μ g/L ) Replicate Juvenile Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic /Genotypic

S ex 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 L 3 B9

- T 7.311 38 FEMALE

50.1 B L3B10

- T 6.687 36 FEMALE

50.1 C L3 C1

- 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 12.16 9 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

Page 206 of 252 Appendix J-1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μ g/L ) Replica te Juvenile Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic /Genotypic

S ex 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 L4 B5

- 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 

### G enotypic sex confirmed the phenotypic sex

### ABC Study No. 66085

Page 207 of 252 APPENDIX K – TERMINATION DATA

– NUPTIAL PAD AND LIVE R

### 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 Casse tte

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 3.97 40 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 ab sent 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

CE 8

- 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 0.98 36

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 4.0 081 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

(Pr esent/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

L 1A3

- 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 4.0 206 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 pre sent 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 1.16 50

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 3.45 50 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

Page 211 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)

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 0.5 279

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

L2 C4

- 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



### ‘--- ’ denot es 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)

L2 D7

- 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 pre sent 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 3.636 8 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 Nupti al 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

S ex 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 μ g/L ) Level 2 (15.4 μ g/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 μ g/L ) Level 4 (156 μ g/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 Re p 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 7.8 6 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 μ g/L ) Level 2 (15.4 μ g/L )

St udy 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 7.3 9 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 oxyg en 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 μ g/L ) Level 4 (156 μ g/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

5 7 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% disso lved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21

, and 22°C, respectively.

### ABC Study No. 66085

Page 222 of 252 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 7.9 0 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 7.6 4 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 7.7 5 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 μ g/L ) Level 2 (15.4 μ g/L )

St udy 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 8.1 0 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 Ch amber (continued)

pH

Level 3 (50.1 μ g/L ) Level 4 (156 μ g/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 8.1 0 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 A PPENDIX M – PLASMA VITELLOGENIN DATA

### ABC Study No. 66085

Page 226 of 252 Appendix M-1. Vitellogenin Data for NF Stage 62 Pooled Plasma

Treatment

( μ g/L ) REP Frog Code Microtube ID Vtg Conc.

(mg/mL) Plate # min.

detection max.

detection

Control A CA

- NF62 CA

- 1 80 0.01 7 66085

- VTG

- Plate 3 0.033 2.451

B CB

- NF62 CB

- 180 0.01 7 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 2.5 05

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

- NF6 2 L1B

- 180 0.01 7 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

- 18 0 0.01 7 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 0.01 7 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

- V TG

- Plate 4 0.037 2.505

C L4C

- NF62 L4C

- 180 0.01 7 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

( μ g/L ) REP Frog Code F rog

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 0.01 7 66085

- VTG

- Plate 3 0.033 2.451

C CC2

- T 22 22

- 180 0.01 7 66085

- VTG

- Plate 3 0.033 2.451

C CC3

- T 23 23

- 180 0.0 17 66085

- VTG

- Plate 3 0.033 2.451

C CC5

- T 25 25

- 180 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

C CC7

- T 27 27

- 180 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

C CC8

- T 28 28

- 180 0.0 18 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 0.01 8 66085

- VTG

- Plate 7 0.037 2.545

D CD6

- T 36 36

- 180 0.0 18 66085

- V TG

- 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

( μ g/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

- Pla te 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 5 0

- 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

( μ g/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

- V TG

- 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 L 1B5

- 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

( μ g/L ) REP Frog Code Frog

ID Microtube ID Vtg Conc.

(mg/mL) Plate # min.

detection max.

detection

15.4 A L2A4

- T 12 4 124

- 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 6608 5

- 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 14 5 145

- 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 0.000 660 85

- 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

( μ g/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 3.69 9

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 S tudy Day 110 (cont.)

Treatment

( μ g/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

- Plat e 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

( μ g/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 66 085

- 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 0.0 17 66085

- VTG

- Plate 3 0.033 2.451

C CC6

- T 26 26

- 180 0.01 8 66085

- VTG

- Plate 5 0.036 2.492

C CC10

- T 30 30

- 180 0.0 18 66085

- VTG

- Plate 7 0.037 2.545

D CD2

- T 32 32

- 180 0.0 18 66085

- VTG

- Plate 7 0.037 2.545

D CD3

- T 33 33

- 180 0.0 18 66085

- VTG

- Plate 7 0.037 2.545

D CD4

- T 34 34

- 180 0.0 18 66085

- VTG

- Plate 7 0.037 2.545

D CD5

- T 35 35

- 180 0.0 18 66085

- VT G

- 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

Page 234 of 252 Appendix M-3. Vitellogenin Data for Juvenile Females on Study Day 110 (cont.)

Treatment

( μ g/L ) REP Frog Code Frog

ID Microtube ID Vtg Conc.

(mg/mL) Plate # min.

detection max.

detection

Control E CE2

- T 42 42

- 180 0.01 7 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 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

G CG3

- T 63 63

- 180 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

G CG7

- T 67 67

- 180 0.0 17 66085

- VTG

- Plate 3 0.033 2.451

G CG9

- T 69 69

- 180 0.01 7 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 0.01 8 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 0.0 18 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

( μ g/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 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

C L1C7

- T 107 107

- 180 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

C L1C8

- T 108 108

- 180 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

D L1D1

- T 111 111

- 180 0.0 17 66085

- VTG

- Plate 3 0.033 2.451

D L1D2

- T 112 112

- 180 0.0 17 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 0.0 18 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

( μ g/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 0.0 1 7 66085

- VTG

- Plate 3 0.033 2.451

A L2A2

- T 122 122

- 180 0.01 7 66085

- VTG

- Plate 3 0.033 2.451

A L2A3

- T 1 23 123

- 180 0.01 7 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 0.000 660 85

- VTG

- Plate 4 0.037 2.505

B L2B7

- T 137 137

- 180 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

B L2B8

- T 138 138

- 180 0.0 18 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 L2D 8

- T 15 8 15 8

- 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 1 60 160

- 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

( μ g/L ) REP Frog Code Frog

ID Microtube ID Vtg Conc.

(mg/mL) Plate # min.

detection max.

detection

50.1 A L3A1

- T 161 1 61

- 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

- V TG

- 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 2.4 92

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 1 84

- 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

( μ g/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

- 30 0 0.000 66085

- VTG

- Plate 1 0.062 4.042

A L4A6

- T 206 206

- 180 0.01 8 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 0.0 17 66085

- VTG

- Pl ate 3 0.033 2.451

C L4C3

- T 223 223

- 180 0.0 17 66085

- VTG

- Plate 3 0.033 2.451

C L4C4

- T 224 224

- 180 0.01 7 66085

- VTG

- Plate 3 0.033 2.451

C L4C5

- T 225 225

- 180 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

C L4C7

- T 227 227

- 180 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

C L4C8

- T 228 228

- 180 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

C L4C9

- T 229 229

- 180 0.0 18 66085

- VTG

- Plate 5 0.036 2.492

C L4C10

- T 230 230

- 180 0.0 18 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

- 18 0 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 – P LASMA T4 DATA

### ABC Study No. 66085

Page 240 of 252 Appendix N-1. T4 Data for NF Stage 62 Pooled Plasma

Treatment

( μ g/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 91.2 3 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

( μ g/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 2 7 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

( μ g/L ) REP Fro g 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

( μ g/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 9 4 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

( μ g/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 2 9.31 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

( μ g/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

( μ g/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 7.65 0 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

( μ g/L ) REP Frog Code Frog

ID Microtube ID Plat e # 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.)

T reatment

( μ g/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

( μ g/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 1 14 114 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

( μ g/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 3.000 743 2

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

( μ g/L ) REP Frog Code Frog

ID Microtube ID P late # 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 1 6.20 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

( μ g/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 87 02

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 5 2.68 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