### Page 1 of 261 STUDY TITLE

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

AUTHOR

Simon Dinehart, Ph.D.

STUDY INITIATION DATE

30 June 2011

STUDY COMPLETION DATE

28 December 2012

SPONSOR

Battelle

- Pacific Northwest Division

Marine Resea rch Operations

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 Research Operations

1529 West Sequim Bay Road

Sequim, Washington 98382

PROJECT IDENTIFICATION

ABC Study No. 66086

### AB C Study No. 66086

Page 6 of 261 TABLE OF CONTENTS

Page

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

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

QUALITY ASSURANCE ST ATEMENT ......................................................................................3

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

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

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

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

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

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

E XECUTIVE SUMMARY ...........................................................................................................11

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

2.0 MATERIALS AND METHOD S .......................................................................................13

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

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

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

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

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

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

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

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

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

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

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

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

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

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

3.0 RESULTS AND DISCUSSI ON ........................................................................................25

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

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

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

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

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

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

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

4.0 CONCLUSIONS

................................ ................................ ................................................42

### AB C Study No. 66086

Page 7 of 261 5.0 RECORDS .........................................................................................................................43

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

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

LIST OF TABLES

Page

Table 3.1.1. Summary of analytical results for the Xenopus laevis 4- tert

- octylphenol in

life exposure. ..........................................................................................................26

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

laevis 4- tert

- octylphenol exposure. ........................................................................27

Table 3.2.2. Mean (and standard deviation) whole body weight and snout

- to

- vent length

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

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

Table 3.2.3. Mean (and standard deviation) whole body weight and snout

- to

- vent length

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

Table 3.2.4. Mean (and standard deviation) whole body weight and snout

- to

- vent length

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

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

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

4- t ert -octylphenol exposure. ..................................................................................35

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

termination o f the Xenopus laevis 4- tert -octylphenol exposure. ...........................36

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

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

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

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

laevis 4- tert

- octylphenol exposure. ........................................................................38

Table 3.4.1. Ranges for Water Quality Measurements During the 113

- Day Exposure

with the African Clawed Frog, Xenopus laevis , Exposed to 4

- tert

octylphenol .............................................................................................................39

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

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

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

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

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

- Observable

Effect Concentration based on mean measured 4

- tert

- octylphenol

concentrati ons for parameter measured for Xenopus laevis exposed to 4

- tert

octylphenol. ............................................................................................................43

### AB C Study No. 66086

Page 8 of 261 LIST OF FIGURES

Page

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

exposure based on the replicate analysis. The box represents the

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

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

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

exposure based on the Kaplan

- Meier product

- limit esti mator analysis. The

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

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

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

laevis 4- tert

- octylphenol exposure. The box represents the interquartile

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

Figure 3.2.4. Boxplot of snout

- to

- vent length for NF stage 62 larvae during the Xenopus

laevis 4- tert

- octylphenol exposure. The b ox represents the interquartile

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

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

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

Xenopus laevis 4- tert

- octylphenol exposure. The box represents the

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

horizontal line is t he median. .................................................................................30

Figure 3.2.6. Boxplot of snout

- to

- vent length for frogs at the NF stage 66 cull of the

Xenopus laevis 4- tert

- octylp henol exposure. The box represents the

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

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

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

Xenopus laevis 4- tert

- octylphenol exposure. The box represents the

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

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

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

Xenopus laevis 4- tert

- octylphenol exposure. The box represents the

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

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

Figure 3.2.9. Boxplot of snout

- to

- vent length for male frogs at the termination of the

Xenopus laevis 4

- tert

- octylphenol exposure. The box represents the

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

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

Figure 3.2.10. Boxplot of snout

- to

- vent length for female frogs at the termination of the

Xenopus laevis 4- tert

- octylphenol exposure. The box represents the

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

median. Gender is b ased on genotypic sex. ..........................................................34

### AB C Study No. 66086

Page 9 of 261 Figure 3.2.11. Boxplot of the proportion of male frogs having nuptial pads at the

termination of the Xenopus laevis 4- tert

- octylphenol exposure. The box

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

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

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

termination of the Xenopus laevis 4- tert

- octylphenol exposure. The box

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

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

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

of the Xenopus laevis 4- tert

- octylphenol exposure. The box represents the

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

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

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

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

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

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

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

during the Xenopus laevis 4- tert

- octylphenol exposure. The box represents

the interquartile rang e, the circle is the mean, and the horizontal line is the

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

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

of the Xenopus laevis 4- tert

- octylphenol exposure. The box represents the

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

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

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

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

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

line is the median. Gender is based on genotypic sex. ..........................................42

### AB C Study No. 66086

Page 10 of 261 LIST OF APPENDICES

Page

APPENDIX A

- CERTIFICATES OF ANAL YSIS ......................................................................49

APPENDIX B

- DILUTION WATER CHARA CTERIZATION .................................................52

APPENDIX C

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

APPENDIX D

– GENDER REPORT ...........................................................................................97

APPENDIX E

– STATISTICAL REPORT .................................................................................146

APPENDIX F

– ANALYTICAL DATA SUMMARY ...............................................................175

APPENDIX G

- REPLICATE SURVIVAL D ATA ....................................................................180

APPENDIX H

– NF STAGE 62 DATA ......................................................................................188

APPENDIX I

– NF STAGE 66 CULL D ATA ............................................................................200

APPENDIX J

– TERMINATION DATA

– GROWTH AND GENDER ...................................208

APPENDIX K

– TERMINATION DATA

– NUPTIAL PAD AND LIVER WEIGHT .............216

APPENDIX L

– WATER QUALITY DATA .............................................................................224

APPENDIX M

– PLASMA VITELLOGENIN DATA ..............................................................234

APPENDIX N

– PLASMA T4 DATA ........................................................................................248

### AB C Study No. 66086

Page 11 of 261 EXECUTIVE SUMMARY

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

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

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

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

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

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

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

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

, respectively,

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

all test substance treatments at study initiation (ranging from 136

- 150% of nominal), measured

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

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

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

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

the 4

- tert

- octyl phenol quality control fortifications ranged from 88 to 118% of the nominal

concentrations. Because the mean measured concentrations of 4

- tert -o ctylphenol were not all

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

reported based upon the mean of the measured concentrations.

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

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

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

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

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

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

differences exceeding 0.5 oC among treatments ranged from 0.6

– 1.0 oC with the following

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

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

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

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

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

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

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

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

criteria stated in the study protocol. The no-observable-e ffect concentration and l owest

observable-e ffect concentration were calculated as mean measured concentrations of 38.0 μg/L

4- tert

- octylphenol and >38.0 μg/L 4

- tert

- octylphenol, respectively, for each Xenopus laevis

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

### AB C Study No. 66086

Page 12 of 261 Parameter No

- Observable

- Effect

Concentration (μg/L) Lowest

- Observable

Effect Concentration

(μg/L)



### NF stage 62

Time to Metamorphosis (Replicate Analysis) 38.0 >38.0

Time to Metamorphosis (Kaplan

- Meier) 38.0 >38.0

Whole B ody Weight 38.0 >38.0

Snout

- to

- Vent Length 38.0 >38.0

Plasma Thyroxine Level 38.0 >38.0

Plasma Vitellogenin Level NR 1 NR



### NF stage 66 Cull

Whole Body Weight 38.0 >38.0

Snout

- to

- Vent Length 38.0 >38.0



### Frogs at Termination

Whole Body Weight (Male ) 38 .0 >38 .0

Whole Body Weight (Female) 38 .0 >38 .0

Snout

- to

- Vent Length (Male) 38 .0 >38 .0

Snout

- to

- Vent Length (Female) 38 .0 >38 .0

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

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

Nuptial Pad Develop ment (Male) 38.0 >38.0

Nuptial Pad Development (Female) 38.0 >38.0

Plasma Thyroxine Level (Male) 38.0 >38.0

Plasma Thyroxine Level (Female) 38.0 >38.0

Plasma Vitellogenin Level (Male, Female) NR NR

1 

### NR = no result.

### AB C Study No. 66086

Page 13 of 261 1.0 INTRODUCTION

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

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

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

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

standardizing, and validating an ecotoxicological test for identifying and characterizing

endocrine effects through exposure to pesticides, industrial chemicals, and envir onmental

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

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

being considered for the Endocrine Disruptor Screening Program to chem icals 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 compoun ds. The chemical 4- tert -octylphenol is a known endocrine

- disruptor and was

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

mechanistic biomarker responses should be indicative of potential adverse effects on popul ation

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

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

the lowest

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

2.0 MATERIALS AND METHODS

2.1 Test Substance and Reference Substance s

Pre

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

05130KD ), w ere received from the U.S. EPA, Duluth, Minnesota, on 3 1 May 2011 and w ere assigned ABC reference number s MM-2628-00001 and MM -2628-00002

. The expiration date

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

- loaded saturator column

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

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

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

The do not use beyond date for this saturator column was 0 8 February 201 4. A sample of the

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

31 May 2011 and was assigned ABC reference number MM

- 2631. The do not use beyond date

assigned to the test substance was 31 May 2013

. This sample of the test substance was used to

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

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

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

13 June 2012 and was assigned ABC reference number MM

- 5729. The expiration date assigned

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

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

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

sampl es were stored at room temperature

. The test substance was described by the Material

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

substance was used to prepare all matrix spiking solutions as well as anal ytical reference

### AB C Study No. 66086

Page 14 of 261 standards. Any remaining test substance will be returned to the EDSP Chemical Repository

. A

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

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

2.2 Test Species

The test species for this study was the African clawed frog

, Xenopus laevis

. Xenopus laevis

embryos were obtained from an in

- house culture of adults . The adults exhibited no signs of

disease nor were any prophylactic or therapeutic treatments utilized during the 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; NASCO; Fort Atkinson, Wisconsin) supplemented with a

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

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

viability and developmental stage. All adult frogs in culture were maintained on a 16

- hr light:

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

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

2.3 Dilution Water

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

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

a total hardness of approximately 140 to 160 mg CaCO 3 

### /L. The water was then passed through a

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

blended water is provided in Appendix B

. Analysis of a dilution water sample collected prior to

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

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

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

2.4 Test Methods

Test procedures followed the ABC test protocol entitled “ 4- tert

- Octylphenol : Larval Amphibian

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

( Appendix C ). This protocol was based upon the draft proposal for a new OECD guidelin e “Larval Amphibian Growth and Development Assay (LAGDA)” ( 1 ).

2.4.1 Saturator Solution Preparations

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

stock sol ution

. T herefore

, a saturator column was used for the preparation of the

4- tert -octylphenol solution. Initially

, columns prepared by the U.S. EPA were used . Additional

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

June 2012 as follows.

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

in outer diameter, bent into a U

- shape. The bent section of the column was open and the walls

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

and 10 cm apart. Each column was packed tightly with glass wool using a dowel rod, until an 8

### AB C Study No. 66086

Page 15 of 261 to 10 cm void remained at the top of each leg. The packed column was rinsed by drawing

acetone through the column, followed by clean air to dry. A column

- coating solution was

prepared by dissolving approximately 5 g of 4

- tert -o ctylphenol in 100 mL of acetone. The

solution was divided into two equal aliquots, one for coating each leg of the saturator colum n. One aliquot was added to each leg and drawn down with vacuum pressure until it nearly reached

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

an attempt to maximize 4

- tert

- octylphenol concentrations in test solutions r elative to target

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

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

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

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

sampling

.

2.4.2 Exposure System

A 2

- L proportional equal solvent diluter system

, similar to that described by Mou nt and Brungs

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

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

concentratio ns selected by the U.S. EPA were 0 ( dilution

- water control ), 6.25, 12.5, 25 .0 , and

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

adhesive. Each mixing/flow

- splitting cell divided each 2

- L volume four way s resulting in a

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

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

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

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

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

with the study numb er

.

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

metering pump introduced approximately 20

- mL volumes of saturator solution outflow to the

### AB C Study No. 66086

Page 16 of 261 diluter system where it was mixed with approximately 3.9 L of dilution water to generate the

highest test substance treatment solution

. The three lower test substance treatment solutions

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

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

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

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

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

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

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

increased again on 29 May 201 2 ( study day 26) to approximately 7.6 cycles per hour in an

attempt to improve test substance recoveries from test solution s relative to target nominal

concentrations

. The diluter delivered approximately 6 0 L of dilution water control and test

substance treatment solution to each chamber daily at 5. 0 cycles per hour, 70 L at 5.8 cycles per

hour, and 9 1 L at 7.6 cycles per hour, which was sufficient to provide approximately 6.0, 7.0,

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

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

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

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

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

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

stee l screen to prevent the tadpoles from escaping. Start ing on study day 75, test chambers were

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

chambers were arranged in a water bath using a computer

- generated rando m number table. The

temperature

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

treatment solutions were maintained at 2 1 ± 1 ° C during the exposure. Test chambers were

labeled with the treatment and replicate.

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

12- hour dark photoperiod with 30

- minute simulated dawn and dusk transition periods. Light

intensity on day 111

, as measured with a LI

- COR Model LI

- 189 light meter equipp ed with a

photometric sensor, ranged from 608 to 613 lux.

2.4.3 Biological Methods

Twelve male

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

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

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

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

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

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

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

impartially selected groups of 2 5 eg gs were reexamined to confirm that all eggs were at

developmental NF stage 9.

### AB C Study No. 66086

Page 17 of 261 2.4.3.1 Test Initiation

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

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

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

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

Embryos within a container we re 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) ( 4 ).

2.4.3.2 Larval Sub- Sampling

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

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

larva from every four that attained NF stage 62 was selected for larval sub

- sampling. Subsampled individuals were anesthetized by immersion in a MS

- 222 solution that had been

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

- to -vent

length (to the nearest mm) of each sub

- sampled individual were recor ded and any gross

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

hemorrhage) prior to dissection.

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

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

following blood collection, each individual was euthanized by immersion in a 2000 mg/L MS

222 solution that had been buffered to approximately pH 7.0. The upper torso from just below

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

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

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

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

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

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

the results of these analyses are reported separately.

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

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

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

analyses. The plasma samples were stored at approximately

- 80oC following collection and

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

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

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

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

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

of T4 and vitellogenin levels

. The ELISA kit Total T4 (Thyrox ine) EIA (cat# 07BC1007 ) by

### AB C Study No. 66086

Page 18 of 261 MP Biomedical 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 70. A cull of surviving individuals

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

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

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

randomly identified and euthanized by immersion in a buffered MS

- 222 solution. The excess

individuals were weighed (i.e., blotted wet weight), measured (i.e., snout

- to

- vent length), and

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

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

- crowding resulting

from juvenile growth.

2.4.3.4 Juvenile Sampling

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

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

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

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

injection of a buffered 10% MS

- 222 solution. Blotted wet weight and snout

- to

- vent length of

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

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

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

placed on ice. Following blood collection, the fo relimbs were exa mined under

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

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

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

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

freezer. The frozen hind limbs were shipped to Battelle

- Pacific Northwest Division, Sequim,

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

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

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

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

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

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

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

approximately

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

Section 2.4.3.7 (VTG) and 2.4.3.8 (T4).

2.4.3.5 Water Chemistry

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

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

was measured with a WTW pH 330i pH meter

, a WTW OXi 330 dissolved oxygen meter

, or

WTW Multi 3500i or 3430 multimeter. Solution pH was measured with a WTW pH 330i

### AB C Study No. 66086

Page 19 of 261 pH meter, WTW Multi 3500i or 3430 multimeter. Dissolved oxygen concentration was

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

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

the test. A continuous recording of temperature in replicate B of the 12.5 μg/L treatment was made using a data lo gger and thermistor probe. Alkalinity, hardness

, and conductivity were

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

. Conductivity was

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

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

2.4.3.6 Feeding

Larvae received a diet consisting of Sera Micron ® 

### solution beginning when larvae were free

swimming

, with live brine shrimp added to the diet starting on study day 22. The total amount of

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

Sera Micron added to each tank was initially calculated based on 2 5 larvae and was adjusted for

changing larval d ensity

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

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

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

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

among controls as follows: weeks 0

- 1, 0.05 mL powder + 0.05 mL crushed pellets; weeks 2 -3,

0.2 mL crushed pellets; weeks 4

- 5, 0.3 mL crushed pellets; wee ks 6

- 9, 0.4 mL crushed pellets.

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

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

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

amount appropriate for 10 individuals.

2.4.3.7 Vitellogenin Analysis

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

by EPA

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

antigens. For this purpose, X. laevis VTG standards, “anti

- X. tropicalis VTG” antibody, as well

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

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

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

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

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

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

samples were to be 70 to 1 5 0% of the nominal concentratio n 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 calculated standard

concentrations of the standards used to prepare the standard curve were between 70 and 1 5 0% of

the nominal concentration.

### AB C Study No. 66086

Page 20 of 261 Plates were coated with coating solution containing VTG standard and coating buffer (50 mM

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

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

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

approximately 37 ° C. Pre

- adsorbed primary antibody serum was diluted, combined with samples

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

washed three times with washing buffer (PBS + 0.05% Tween

- 20 pH 7.4), and after all buffer

was removed

, appropriate wells were loaded with d iluted standards and samples and incubated at

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

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

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

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

least 10 minutes. The reaction was stopped with 1M H 3 

### PO 4 

### and the absorbance read by a

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

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

5.3 software.

The reported results, expressed as ng/mL, were based on sample runs that had standard curve 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 VTG value for a particular sample was

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

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

sample was above the curve, the sample was re

- diluted to fall within the standard curve. If

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

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

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

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

2.4.3.8 Thyroxine ( T4 ) Analysis

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

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

August 2013) obtained from Sigma was used to prepare T4 standards. A 1x Tris

- buffer (0.01M

Tris, 0.15M NaCl) pH

- adjusted to approximately 7.6 2 was prepared as dilution buffer for

standards 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 five standards in the

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

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

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

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

QC sample was freshly prepared for each analysis by spiking pooled non

- study plasma with T4

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

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

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

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

CV between replicates was to be ≤20% for the plate results to be considered acceptable unless

### AB C Study No. 66086

Page 21 of 261 other evidence indicated the data were reliable . Fluctuations in calibration curves between plates

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

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

plate(s).

Dilution buffer, diluted standards, and plasma were added to the appropriate wells on the pre

coated plates

. C onjugate reagent was added to each well, plates were gently mixed for

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

then washed (Molecular Devices AquaMax 2000 plate washer) five time s with water

. TMB

reagent was added to the wells, plate s were gently mixed for five seconds, and the n incubated

again at approximately 22oC for approximately 3 0 minutes in the dark. After incubation, the

reaction was stopped with 1M HCl and the plate cont ents were gently m ixed for 30 seconds.

Within 15 minutes after stopping the reaction, the absorbance was read using a spectrophotometer (Molecular Devices SpectraMax 190) at a wavelength of 450 nm. All

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

5.3 software.

The reported results, expressed as ng/mL, were based on sample runs that had standard curve R 2



### values >0.99. The curves for the reported data all were based upon the four

- parameter logistic

model equat ion: y = ((A

- D)/(1+ ((x/C)^B))) + D. If the T4 value for a particular sample was

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

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

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

2.5 Analytical Method

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

liquid chromatography w ith ultraviolet detection ( HPLC

- UV )

. Details of the sample collection,

preparation, and method of analysis are described below.

2.5.1 Preparation of Analytical Standard and Matrix Spiking Solutions

P rimary stock solution s of 4- tert -octylphenol w ere prepared in methanol on 17 August 2011 and

23 July 2012 at concentration s of 0.614 and 0.765 m g/mL , respectively . Subsequent dilutions of

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

standards.

P rimary stock solu tion s of 4- tert -octylphenol w ere prepared in methanol on 17 August 2011 and

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

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

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

2.5.2 Test Solution Analysis

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

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

weekly throughout the definitive test. S ample s collected on day -1 were from one of the four

### AB C Study No. 66086

Page 22 of 261 replicates for all treatments. All replicates of all treatments were sampled on day 0 an d at

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

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

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

the test solution samples

.

A 10

- mL sample was collected from the control

, each test substance treatment solution , and the

saturator outflow solution (s) . T he samples were placed into appropriately sized culture tub es. Samples were diluted with methanol and further diluted, if necessary, with 50:50 methanol:water

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

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

2.5.3 Instrument Conditions

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

an UV detector using the fo llowing analytical parameters:

Column: Alltima C18, 4.6 mm × 150 mm, 5 μ m

Temperature: 40 ° C

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

Flow Rate: 1.1 mL/minute

Injection Volume: 300 μL

Wavelength: 220 nm

Detector: UV

Stop Time: 10 minutes

Attenuat ion: 500 mAU

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

the chromatographic conditions.

2.5.4 Calculations for 4- tert -o ctylphenol

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

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

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



### μg/L

mLin volume sample mLin volume analysis

curve standard fromμg/L

=  

 

 

 

 

 

 

 

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

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

where:

### AB C Study No. 66086

Page 23 of 261 y = peak area units

m = slope of the standard curve

x = μg 4-tert octylphenol /L

b = y -intercept of the standard curve

Standard Curve: y = 549.7048 x – 29.69535

Sample Peak Area: = 2,301

μg/L from Standard Curve = 4.239 μ g/L

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



# ( )

a.i./L μg 8.48

mL 5 mL 10 a.i./L) μg 239.4(

=



### Recovery of 6.25 μg/L nominal treatment

, replicate D, sample on day 0:

136% 100x μg/L 6.25 a.i./L μg 8.48

=



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

μg/L as expressed MQL

(mL) sampled volume (mL) analysis

forvolume

L) /(ion concentrat standard lowest

=

 

 

 

   

 

 

 

 

 

 

 

μg



### Lowest standard concentration: 1.54 μ g/L

Volume for analysis: 10 mL

Volume of sample: 5 mL

therefore:

MQL = a.i./L μg 3.08 mL) (5 mL) (10 μg/L) (1.54

=



### 2.6 Genetic Sexing

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

dissection. The hind f oo t from each specimen was stored at

- 20°C until DNA isolation. Testing

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

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

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

small tissue sa mple 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 includ ed

### AB C Study No. 66086

Page 24 of 261 primers for the DMRT1 gene as a positive control to be amplified in both ZW and ZZ types.

Because the DM

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

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

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

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

.

2.7 Statis tical Analyses

Parametric assumptions were tested using the residuals from a one

- way analysis of variance

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

from normality were tested with the Shapiro

- Wilks’ test (α= 0.05) and homogeneity of within

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

root, proportional data were transformed to the arcsine square root, and continuous data were transformed to the natural logarithm. If the responses were monotonic, the William ’ s test of

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

the no

- observed

- effects concentration (NOEC) when the assumptions of normality and

homogeneity of within class variances were met, otherwise the step

- down Jonckheere

- Terpstra

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

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

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

were met. When these assumptions were not met, the Kruskal

- Wallis (K

- W) test followed by a

step

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

bounds determined by Q1

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

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

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

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

treatments (n=4), the LOEC/NOEC was estimated from a K

- W multiple comparisons test of the

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

estimate the LOEC/NOEC for non

- monotonic responses when the assumptions of normality and

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

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

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

and homogeneity of within class variances were not met. When the K

- W (a comparison among

all treatments) was significant, a K

- W multiple comparison test was used to evaluate the median

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

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

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

Kaplan

- Meier product

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

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

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

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

treatments. The mean and standard deviation of the treatment

- related responses for each

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

by the K

- W test or the K

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

sex is mentioned in statistical analyses, the genotypic sex was used. Samples CE6

- T and CE7

- T

### AB C Study No. 66086

Page 25 of 261 produced weak bands and the genetic results are equivocal. Individual CE6

- T was a phenotypic

and genotypic male; individual CE7

- T was a phenotypic male but a genotypic female. Analyses

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

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

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

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

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

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

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

Appendix E

.

3.0 RESULTS AND DISCUSSION

3.1 Analytical Results

M easured 4- tert -octylphenol concentrations in the 6.25, 12.5, 25 .0 , and 50 .0 μg /L nominal test

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

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

on day 0 ranged from 136 to 150 % o f the nominal treatment concentrations. Overall m ean

measured concentrations of 4- tert -octylphenol in the 6.25, 12.5, 25 .0 , and 50 .0 μg/L nominal test

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

, respectively, which represented 76 to

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

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

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

77, 91, and 97 sample s from the 6.25 μg/L nominal treatment w ere 126, 65, 66, 62, 129, 126,

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

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

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

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

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

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

residues of the 4- tert -octylphenol were detected in the control s above the MQL of 3.08 n g/mL

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

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

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

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

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

.

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

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

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

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

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

above pump malfunction was addressed indicated recoverie s ranging from 52 -73% of nominal.

### AB C Study No. 66086

Page 26 of 261 Table 3. 1 .1

. Summary of analytical results for the Xenopus laevis 4- tert -o ctylphenol in

- life exposure.



### Nominal

Concentration

(μ g/L ) Mean Measured

Concentration (μ g/L ) StDev CV Recovery

0 <MQL a 

### NA NA NA

6.25 4.97 1.48 30% 80%

12.5 10.3 2.2 2 22% 82%

25 .0 19.5 4.72 24% 78%

50 .0 38.0 10.4 27% 76%

a MQL = Minimum Quantifiable Limit = 3. 08 μg/L



### 3.2 Biological Results

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

NF Stage 62 Survival:

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

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

exposure (i.e., day 70). These individuals were not counted in the total number of control

treatment larvae. Total survival in the 4

- tert -o ctylphenol treatments ranged from 85 to 9 4%

( Appendix G-2 ).

Time to NF Stage 62 :

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

Xenopus laevis larvae to reach NF s tage 62 based on the within

- replicate average analysis was

### AB C Study No. 66086

Page 27 of 261 43 days in the control treatment and ranged from 43 to 44 days in the 4

- tert -octylphenol

treatments ( Table 3. 2.1

, Figure_3.2.1 ). No significant differences among treatments were

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

- Meier product

- limit

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

4- tert

- octylphenol treatments ( Table 3. 2.1

, Figure_3.2.2 ). No significant differences among

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

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

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

H

.

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

4- tert -o ctylphenol exposure.

Mean Measured

Concentration (μg/L) Time to 

### NF stage 62 (d)

Within Replicate Average Kaplan

- Meier Estimate

<MQL 

### 43 (1.6) 42 (1.8)

4.97 

### 43 (1.1) 42 (1.5)

10.3 

### 44 (2.0) 44 (2.0)

19.5 

### 44 (1.1) 43 (1.6)

38.0 

### 44 (1.0) 43 (0.9)

p

- value 0.472 0.491

Test: 1

- way ANOVA K

- W

38.0 19.5 10.3 4.97 <MQL 48

47

46

45

44

43

42

Mean Measured Dose (μg/L)Time to NF Stage 62 (d)

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

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

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

### AB C Study No. 66086

Page 28 of 261 38.0 19.5 10.3 4.97 <MQL 47

46

45

44

43

42

41

40

Mean Measured Dose (μg/L)Time to NF Stage 62 (d)

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

- octylphenol exposure based

on the Kaplan

- Meier product

- limit esti mator analysis. The box represents the interquartile

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



### NF Stage 62 Growth:

The whole body (wet) weight and snout

- to

- vent length for all NF stage 62 larvae me asured are

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

the control treatment and ranged from 2.04 to 2.19 g in the 4

- tert

- octylphenol treatments

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

were detected at α = 0.05. Mean snout

- to

- vent length of NF stage 62 larvae in the control

treatment was 23 mm and ran ged from 23 to 24 mm in the 4

- tert

- octylphenol treatments

( Table 3.2.2 ; Figure 3.2. 4 ). No significant differences in snout

- to

- vent length among treatments

were detected at α = 0.05.

Table 3.2.2. Mean (and standard deviation) whole body weight and snout

- to

- vent length for NF stage 62

larvae during the Xenopus laevis 4- tert

- octylphenol exposure.

Mean Measured

Concentration (μg/L) Whole Body Weight

(g) Snout

- to

- Vent Length

(mm)

<MQL 

### 2.07 (0.1 73) 23 (0.5)

4.97 

### 2.04 (0.131) 24 (0.5)

10.3 

### 2.17 (0.125) 23 (0.8)

19.5 

### 2.11 (0.241) 23 (0.8)

38.0 

### 2.19 (0.113) 24 (0.9)

p

- value 0.614 0.220

Test: K-W 1- way ANOVA

### AB C Study No. 66086

Page 29 of 261 38.0 19.5 10.3 4.97 <MQL 2.4

2.3

2.2

2.1

2.0

1.9

1.8

1.7

Mean Measured Dose (μg/L)Whole Body Weight (g)

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

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

mean, and the horizontal line is the median.

38.0 19.5 10.3 4.97 <MQL 25.0

24.5

24.0

23.5

23.0

22.5

22.0

Mean Measured Dose (μg/L)Snout-to-Vent Length (mm)

Figure 3.2.4. Boxplot of snout

- to

- vent leng th for NF stage 62 larvae during the Xenopus laevis

4- tert

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

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



### Juvenile Survival:

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

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

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

as exposure

- related mortalities. Cumulative mean survival from Day 0 to Day 113 was

### AB C Study No. 66086

Page 30 of 261 93 percent in the control treatment and ranged from 84 to 93 percent in the 4

- tert -octylphenol

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

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

Growth of NF Stage 66 Culled Juveniles:

The whole body (wet) weight, snout

- to

- vent length, and phenotypic sex of all NF stage 66 culled

frogs is present ed in Appendix I

. Mean whole body (wet) weight of frogs measured at the NF

stage 66 cull was 3.71 g in the control treatment and ranged from 3.44 to 4.02 g in the 4

- tert

octylphenol treatments ( Table 3.2.3 ; Figure 3.2. 5 ) . No significant differences in whole body

weight among treatments were detected for these frogs at α = 0.05. Mean snout

- to

- vent length of

frogs measured at the NF stage 66 cull was 3 0 mm in the control treatment and ranged from 30

to 31 mm in the 4

- tert

- octylphenol treatments ( Table 3.2.3 ; Figure 3.2. 6 )

. No significant

differences in snout- to

- vent length among treatments were detected for these frogs at α = 0.05.

Table 3.2.3. Mean (and standard deviation) whole body weight and snout

- to

- vent length at the NF

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

Mean Measured

Concentration (μg/L) Whole Body Weight

(g) Snout

- to

- Vent Length

(mm)

< MQL 

### 3.71 (0.303) 30 (0.8)

4.97 

### 4.02 (0.295) 31 (0.7)

10.3 

### 3.44 (0.075) 30 (0.8)

19.5 

### 3.66 (0.325) 30 (1.7)

38.0 

### 3.61 (0.435) 30 (1.1)

p

- value 0.078 0.208

Test: K

- W K

- W

38.0 19.5 10.3 4.97 <MQL 4.4

4.2

4.0

3.8

3.6

3.4

3.2

3.0

Mean Measured Dose (μg/L)Whole Body Weight (g)

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

4- tert

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

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

### AB C Study No. 66086

Page 31 of 261 38.0 19.5 10.3 4.97 <MQL 32

31

30

29

28

27

Mean Measured Dose (μg/L)Snout-to-Vent Length (mm)

F igure 3.2.6. Boxplot of snout

- to

- vent length for frogs at the NF stage 66 cull of the Xenopus laevis

4- tert

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

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



### Growth at Termination :

The whole body (wet) weight, snout

- to

- vent length, phenotypic sex, and genotypic sex of all

frogs at termination are presented in Appendix J

. Mean whole body (wet) weight of male frogs

measured at the termination of the 4

- tert

- octylphenol exposure was 10.0 g in the control

treatment and ranged from 9.3 to 10.8 g in the 4

- tert

- octylphenol treatments ( Table 3.2.4 ; Figure 3.2.7 ). Mean whole body (wet) weight of female frogs measured at the termination of the

4- tert

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

the 4

- tert

- octylphenol treatments ( Ta ble 3.2.4 ; Figure 3.2. 8 ). No significant differences among

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

exposure at α = 0.05. Mean snout

- to

- vent length of male frogs mea sured at the termination of

the 4

- tert

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

44 mm in the 4

- tert

- octylphenol treatments ( Table 3.2.4 ; Figure 3.2. 9 ). M ean snout

- to

- vent

length of female frogs measu red at the termination of the 4

- tert

- octylphenol exposure was

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

- tert -octylphenol

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

treatments were detected for male or female frogs at the termination of the exposure at α = 0.05.

### AB C Study No. 66086

Page 32 of 261 Table 3.2.4. Mean (and standard deviation) whole body weight and snout

- to

- vent length at the

termination of the Xenopus laevis 4

- tert

- octylphenol exposure.

Mean Measured

Concentration

(μg/L) Male Whole Body

Weight (g) Female Whole

Body Weight (g) Male Snout

- to

Vent Length (mm) Female Snout

- to

Vent Length (mm)

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

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

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

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

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

p

- value 0.36 2 0.708 0.337 0.327

Test: K

- W K

- W K

- W K

- W

Note: Gender is based on genotypic sex.

38.0 19.5 10.3 4.97 <MQL 12

11

10

9

8

Mean Measured Dose (μg/L)Whole Body Weight (g)

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

4- tert

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

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

### AB C Study No. 66086

Page 33 of 261 38.0 19.5 10.3 4.97 <MQL 14

13

12

11

10

9

8

7

Mean Measured Dose (μg/L)Whole Body Weight (g)

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

4- tert

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

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

38.0 19.5 10.3 4.97 <MQL 46

45

44

43

42

41

40

39

38

37

Mean Measured Dose (μg/L)Snout-to-Vent Length (mm)

Figure 3.2. 9

. Boxplot of snout

- to

- vent length for male frogs at the termination of the Xenopus laevis 4

- tert

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

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

### AB C Study No. 66086

Page 34 of 261 38.0 19.5 10.3 4.97 <MQL 47

46

45

44

43

42

41

40

39

38

Mean Measured Dose (μg/L)Snout-to-Vent Length (mm)

Figure 3.2.10

. Boxplot of snout

- to

- vent length for female frogs at the termination of the Xenopus laevis 4-

tert

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

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



### Nuptial Pad Development at Termination :

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

Appendix K

. The mean prop ortions of male and female frogs in the control treatment that had

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

proportions of male and female frogs in the 4

- tert

- octylphenol treatments that had nuptial pads a t

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

( Table 3.2.5 ; Figure 3. 2.11 ; Figure 3. 2.12 ). No significant differences among treatment medians

were detected for females or males at α = 0.05.

In addition to the Kruskal

- Wallis test of the null hypothesis of equal medians for all exposure

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

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

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

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

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

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

concentrati on. The proportion of males in the 10.3

- μg/L 4

- tert

- octylphenol concentration having

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

proportions of females in the 4.97

- and 38.0

- μg/L 4- tert -octylphenol conce ntrations having

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

chance ( p = 0.008 and 0.005).

### AB C Study No. 66086

Page 35 of 261 Table 3. 2 .5. Mean (standard deviation) proportion of female and male frogs and total number of frogs

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

Mean Measured

Concentration (μg/L) Nuptial Pads

(proportion of females) Nuptial Pads

(proportion of males) Total Number of

Frogs with Nuptial

Pads

<MQL 

### 78 0.23 (0.24) 0.19 (0 .16)

4.97 

### 39 0.25 (0.18) 0.47 (0.19)

10.3 

### 40 0.57 (0.18) 0.36 (0.33)

19.5 

### 39 1 

### 0.26 (0.19) 0.35 (0.25)

38.0 

### 40 0.28 (0.28) 0.45 (0.33)

p

- value

– 0.184 0.288

Test:

– 1- way ANOVA K-W

1 One female in replicate C was missing forelimbs

Note: Gende r is based on genotypic sex.

38.0 19.5 10.3 4.97 <MQL 0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Mean Measured Dose (μg/L)Proportion of Males with Nuptial Pads

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

Xenopus laevis 4- tert

- octylphenol exposure. The box represents the interquartile range, the

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

### AB C Study No. 66086

Page 36 of 261 38.0 19.5 10.3 4.97 <MQL 0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Mean Measured Dose (μg/L)Proportion of Females with Nuptial Pads

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

Xenopus laevis 4- t ert -o ctylphenol exposure. The box represents the interquartile range, the

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

Table 3. 2 .6. Proportion of total frogs within each treatment having nuptial pads at t he termination of the

Xenopus laevis 4

- tert -o ctylphenol exposure



### .

Mean Measured

Concentration

(μg/L) N Proportion of Total

Males with Nuptial

Pads Binomial

Probability Proportion of

Total Females

with Nuptial Pads Binomial

Probability

<MQL 78 0.22

– 0.19

–

4.97 39 0.27 0.349 0.47 0.008

10.3 40 0.57 <0.001 0.35 0.085

19.5 39 1 0.2 8 0. 359 0.33 0.085

38.0 40 0.29 0.262 0.50 0.005

1 One female in replicate C was missing forelimbs

Note: Gender is based on genotypic sex.



### Liver Weight at Termination :

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

. Mean liver

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

the 4

- tert

- octylphenol expos ure were 0.058 for males and 0.059 for females. The ratio for males

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

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

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

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

α = 0.05.

### AB C Study No. 66086

Page 37 of 261 Table 3.2.7. 

### Mean (and standard deviation) liver weight:whole body weight for male and female

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

Mean Measured

Conc entration (μg/L) Liver

Weight:Whole Body

Weight (Males) Liver

Weight:Whole Body

Weight (Females)

<MQL 0.058 (0.003) 0.059 (0.006)

4.97 0.057 (0.003) 0.058 (0.002)

10.3 0.059 (0.005) 0.061 (0.005)

19.5 0.059 (0.003) 0.060 (0.005)

38.0 0.058 (0.004) 0.0 56 (0.002)

p

- value 0.906 0.322

Test: 1

- way ANOVA K

- W

Note: Gender is based on genotypic sex.

38.0 19.5 10.3 4.97 <MQL 0.064

0.062

0.060

0.058

0.056

0.054

0.052

Mean Measured Dose (μg/L)Liver Weight:Whole Body Weight

Figure 3.2.13. 

### Boxplot of liver weight:whole body weight for male frogs at the termination of the

Xenopus laevis 4- ter t -o ctylphenol exposure. The box represents the interquartile

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

on genotypic sex.

### AB C Study No. 66086

Page 38 of 261 38.0 19.5 10.3 4.97 <MQL 0.070

0.065

0.060

0.055

Mean Measured Dose (μg/L)Liver Weight:Whole Body Weight

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

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

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



### 3.3 Genotypic/Phen otypic Sex Ratios

Detailed results of genetic sexing of the juvenile frogs using PCR followed by gel separation are presented in Appendix D and Appendix J . The phenotypic and genotypic sex ratios at the

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

45 individuals differed from their phenotypic determinations.

Table 3. 3 .1. Frog phenotypic and genoty pic sex ratios at the termination of the Xenopus laevis 4- tert

octylphenol exposure.

Mean Measured

Concentration (μg/L) N Phenotype Genotype Number of

Mismatches 1 Females Males Females Males

<MQL 

### 78 33 45 37 41 4

4.97 

### 39 21 18 17 22 10

10.3 

### 40 18 2 2 17 23 13

19.5 

### 40 18 22 22 18 16

38.0 

### 40 18 22 16 24 2

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



### 3.4 Water Quality

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

limits throughout the course of the 113

- day exposure ( Table 3.4.1 ). The dissolved oxygen

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

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

to 21.9°C during the test. The continuous temperature data collected from a centrally located test

### AB C Study No. 66086

Page 39 of 261 chamber indicated that, with two exceptions (05July12 and 30July12) , the temperature remaine d

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

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

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

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

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

dilution water ranged from 136 to 154 mg Ca CO 3 

### /L, from 150 to 160 mg CaCO 3 

### /L, and from

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

dissolved oxygen concentration, and pH during the 113

- day exposure are presented in

Appendix L

.

Table 3. 4 .1

. Ranges for Water Quality Measurements During the 113

- Day Exposure with the African

Clawed Frog, Xenopus laevis , Exposed to 4- tert -o ctylphenol

Mean Measured

Concentration

( μ g/L ) Temperature

( ° C) a Dissolved

Oxygen

(mg/L) a ,b pH a C onductivity

(μS) a Alkalinity

(mg/L) a Hardness

(mg/L) a

Control 20.1

– 21.8 4.01

– 8.70 7.47

– 8.46 340

– 357 150

– 160 136

– 154

4.97 20.2

– 21.7 4.33

– 8.72 7.59

– 8.26 340

– 357 152

– 1 6 0 140

– 15 0

10.3 20.2

– 21.7 4.55

– 8.71 7.62

– 8.28

-- -- --

19.5 20.5

– 21.9 4.31

– 8.69 7.63

– 8.24

-- -- --

38.0 19.9

– 21.7 4.38

– 8.67 7.33

– 8.46

-- -- --

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

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



### 3.5 Vitellogenin Results

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

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

No statistical analyses were conducted for this parameter.

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

in Appendix M

. P lasm a VTG concentrations for 117 of 128 males and 103 of 109 females from

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

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

3.6 Pla sma Thyroxine ( T4 ) Results

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

control treatment and ranged from 203 to 287 ng/mL in the 4

- tert

- octylphenol treatments

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

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

samples in presented in Appendix N

.

### AB C Study No. 66086

Page 40 of 261 The mean plasma T4 concentrations for control

- treatment male and female frogs at the

termination of the 4

- tert

- octylphenol exposure were 61.0 and 76. 8 ng/mL, respectively

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

- tert -octylphenol

treatments ranged from 55.4 to 7 9 .4 ng/mL for males and from 57.2 to 72.6 ng/mL for females

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

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

samples from juveniles at study termination are presented in Appendix N

.

Table 3. 6 .1

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

Xenopus laevis 4- tert -o ctylphenol exposure.

Mean Measured

Concentration (μg/L) Plasma Thyroxine (T4)

(ng/mL)

<MQL 227 (72.6)

4.97 283 (125)

10.3 203 (94.4)

19.5 280 (171)

38.0 287 (84.8)

p

- value 0.739

Test: K

- W

Table 3. 6.2

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

Xenopus laevis 4- tert

- octylphenol exposure.

Mean Measured

Conce ntration (μg/L) Male Plasma

Thyroxine (T4)

(ng/mL) Female Plasma

Thyroxine (T4)

(ng/mL)

<MQL 61.0 (24.0) 76. 8 (28.6)

4.97 66.2 (19.3) 60.1 (14.2)

10.3 79.4 (23.5) 57.2 (21.6)

19.5 59.1 (20.5) 61.6 (15.6)

38.0 55.4 (12.1) 72.6 (11.6)

p

- value 0.544 0.550

Test: 1

- Way ANOVA K

- W

Note : Gender is based on genotypic sex.

### AB C Study No. 66086

Page 41 of 261 38.0 19.5 10.3 4.97 0.0 500

400

300

200

100

Mean Measured Dose (μg/L)Thyroxine Concentration (ng/mL)

Figure 3. 6.1. Boxplot of plasma thyroxine (T4) concentration for NF Stage 62 larvae during the Xenopus

laevis 4- tert

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

the mean, and the horizontal line is the median

.

38.0 19.5 10.3 4.97 0.0 110

100

90

80

70

60

50

40

30

Mean Measured Dose (μg/L)Thyroxine Concentration (ng/mL)

Figure 3. 6.2. Boxplot of plasma thyroxine concentration for male frogs at the termination of the

Xenopus laevis 4- tert

- octylphenol exposure. The box represents the interquartile range, the

circle is the mean, 

### the asterisk is an outlier, and the horizontal line is the median. Gender

is based on genotypic sex.

### AB C Study No. 66086

Page 42 of 261 38.0 19.5 10.3 4.97 0.0 120

100

80

60

40

20

Mean Measured Dose (μg/L)Thyroxine Concentration (ng/mL)

Figure 3. 6.3. Boxplot of plasma thyroxine concentration for female frogs at the termination of the

Xenopus laevis 4- tert

- octylp henol exposure. The box represents the interquartile range, the

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



### 3.7 Validity and Performance Criteria

With several exceptions, validity and performance criteria stated in the study protocol were met

for this study. Water quality characteristics remained within the tolerance limits outlined in the

protocol with the exception of temperature. The t est solution temperature deviated from the

acceptable range (i.e., 2 1 ± 1 oC) on 05July12 and 30July12 (study day 63 and 88, respectively)

. On

05July12, test solution temperature was outside the acceptable range for approximately 3 hours

and reached a maximum of 22.2 oC. On 30July12, test solution temperature was outside t he

acceptable range for approximately 45 minutes and reached a minimum of 19.5 oC. With the

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

solution temperature differences among treatments on a given study day exceeded the acceptable

level of variation stated in the protocol (i.e., 0.5 oC). Temperature differences exceeding 0.5 oC

among treatments ranged from 0.6

– 1.0 oC with the following exceptions (differences of 1.3, 1.2,

1.2 oC on day 41, 85, and 88, respectively). Delivery of chemical to the test organisms (e.g.,

concentrations of the chemical in test water) was not maintained at ≤20% CV over the entire test

(CV values ranged from 22 to 30% for the test substance treatments). Survival of test organisms

was ≥90% in all control replicates and mean survival was ≥84 % in all test substance treatments.

Two test replicates ( replicate B of the 4.79 and 10.3 μg/L treatment s) were compromised (i.e.,

>20% mortality). This number of compromised replicates did not exceed the acceptable number

as per the study protocol. Time to NF stage 62 among control larvae ranged from 38 to 44 days.

The mean weight of culled NF stage 66 control larvae was 3.71 g.

4.0 CONCLUSIONS

With the above not ed exceptions, this study satisfied acceptability and performance criteria

stated in the study protocol. The no-o bservable -e ffect concentration and l owest -observablee ffect concentration were calculated as mean measured concentrations of 38.0 μg/L 4

- tert

-

### AB C Study No. 66086

Page 43 of 261 octylphenol and >38.0 μg/L 4

- tert

- octylphenol, respectively, for each Xenopus laevis parameter.

( Table 4.0.1 ).

T able 4.0.1. Calculated No

- Observable

- Effect Concentration and Lowest

- Observable

- Effect

Concentration based on mean measured 4- tert -o ctylphenol concentrations for parameter

measured for Xenopus laevis exposed to 4

- tert -o ctylphenol.

Parameter No

- Observable -E ffect

Concentration (μg/L) Lowest

- Observable

Effect Concentration

(μg/L)



### NF stage 62

Time to Metamorphosis (Replicate Analysis) 38.0 >38.0

Time to Metamorphosis (Kaplan

- Meier) 38.0 >38.0

Whole Body Weight 38.0 >38.0

Snout

- to

- Vent Length 38.0 >38.0

Plasma Thyroxine Level 38.0 >38.0

Plasma Vitellogenin Level NR 1 NR



### NF stage 66 Cull

Whole Body Weight 38.0 >38.0

Snout

- to

- Vent Length 38.0 >38.0



### Frogs at Termination

Whole Body Weight (Male) 38 .0 >38 .0

Whole Body Weight (Female) 38 .0 >38 .0

Sno ut

- to

- Vent Length (Male) 38 .0 >38 .0

Snout

- to

- Vent Length (Female) 38 .0 >38 .0

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

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

Nuptial Pad Development (Male) 38.0 >38.0

Nuptial Pad Development (Female) 38.0 >38.0

Plasma Thyroxine Level (Male) 38.0 >38.0

Plasma Thyroxine Level (Female) 38.0 >38.0

Plasma Vitellogenin Level (Male, Female) NR NR

1 

### NR = no result.

5.0 RECORDS

The original raw data and the study plan were provided to the U.S. EPA with the final report.

Copies of all data in support of this report were retained at ABC Laboratories, Inc., along with

facility records and a copy of the final report and the study plan.

### AB C Study No. 66086

Page 44 of 261 PROTOCOL DEVIATION S

Item 1 Protocol Section: 11.0 Exposure System

D eviation: Only a single diluter function check was performed on 23May12.

Reason for

Deviation: Technical oversight.

Effect on Study: None. Diluter function checks were performed twice daily throughout the

remainder of the study.

Item 2 Section 13. 0 Temperature

Deviation: Test solution temperature deviated from the acceptable range (i.e., 21 ± 1oC) in

two instances during the course of the test (05July12 and 30July12).

Reason for

Deviation: On 05July12, the chiller units used to regulate wa ter bath (and therefore test

solution) temperature were inadequate to counteract occasional fluctuations

in the temperature of the ambient dilution water supplied to the test system.

On 30July12, a diluter malfunction caused the diluter to not cycle for

a

pproximately 80- 85 minutes. During this malfunction, no fresh test solution

was supplied to chambers and test solution temperature declined to a

temperature similar to that of the water bath.

Effect on Study: No adverse effect on study integrity. On 05July12, test solution temperature

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

maximum of 22.2 oC. On 30July12, test solution temperature was outside the

acceptable range for approximately 45 minutes and reached a minimum of

1 9.5 oC.

Item 3 Section 13.0 Temperature

Deviation: With the exception of the following study days (0, 6, 8, 12, 19, 21, 32, 39, 50,

60, 97, 106, 112, and 113), test solution temperature differences among

treatments on a given study day exceeded 0.5 oC (n = 22). Temperature

differences exceeding 0.5 oC among treatments ranged from 0.6

– 1.0 oC with

the following exceptions (differences of 1.3, 1.2, 1.2 oC on day 41, 85, and 88,

respectively).

Reason for

Deviation: Efforts made to limit test sol ution temperature variation to ≤ 0.5 oC across

treatments within a given day (e.g., appropriate placement of re

- circulating

pumps in the water bath relative to chiller inflow/outflow tubes) were not

sufficient to achieve this criterion throughout the course of the study.

### AB C Study No. 66086

Page 45 of 261 Effect on Study: No adverse effect on study integrity. With the above noted exceptions, test

solution temperatures across treatments on a given day were consistent (i.e.,

≤ 1.0 oC variation) throughout the test.

Item 4 Section 15.0 P hysical Parameters Monitored

Deviation: The alkalinity, hardness, and conductivity were measured in one replicate of

the control and one concentration of the test substance at test initiation

(03May12) and monthly thereafter (31May12, 27Jun12, 31Jul12) with the

following exception: these parameters were not measured during the month of

August.

Reason for

Deviation: Technical oversight

Effect on Study: None. Available data indicates that these parameters were consistent

throughout the study (conduc tivity = 340

- 357 μS, alkalinity = 150

- 160,

hardness = 136

- 154).

Item 5 Protocol Section: 17.0 Experimental Procedure

Deviation: Test Initiation

Embryos at Nieuwkoop

- Faber (NF) stage 9 were randomly distributed into

exposure treatment tanks.

En d of Larval Exposure

At the end of the larval exposure, one additional NF stage 66 frog (i.e., six in

total) was inadvertently removed from the control, replicate C test chamber (a

total of nine animals were present in this chamber at completion of the cul l).

Reason for

Deviation: Test Initiation

Communication with EPA Duluth indicated that examining embryos at a later

development stage may help to minimize the number of non

- viable/abnormal

embryos selected for test initiation. In an attempt to improve l arval hatch,

embryos were examined as they approached NF stage 9 prior to distributing

embryos to exposure treatment tanks.

End of Larval Exposure

Technical oversight.

### AB C Study No. 66086

Page 46 of 261 Effect on Study: Test Initiation

None. No meaningful change in test exposure per iod resulted from using NF

stage 9 embryos to initiate the test.

End of Larval Exposure

No adverse effect on study integrity. The above noted error was discovered

on study day 78 (i.e., eight days after the end of the larval exposure) and the

volume of fee d added to the control, replicate C test chamber was corrected

for this day and throughout the remainder of the study (i.e., study day 78

- 112). While an additional 10% of feed was added to the above mentioned

test chamber for seven days, data collected at test termination indicates that

this did not have a meaningful effect on growth related endpoints (the range

of data values for control, replicate C growth related endpoints at test

termination are similar to those of other control replicates).

Item 6 Protocol Section: 18.0 Diet

Deviation Whole Frog Brittle pellets were ground briefly with a mortar and pestle to

produce coarse chunks of feed.

During weeks 2

- 3 after the median metamorphosis date among controls,

juveniles were fed 0.2 mL of sink ing frog food/animal/day as per the protocol.

The total amount of sinking frog food offered was calculated by multiplying

the above feeding rate by the numbers of juveniles (i.e., NF stage 66 animals)

present in a particular test chamber on a given day. In several instances

(below), the incorrect volume of sinking frog food was offered to juveniles.

a) 6.25 μg a.i./L nominal treatment, replicate A: Instead of 3.0 and 3.2 mL of

food, 3.2 and 3.4 mL were offered on study days 57

- 59 and 60

- 61, respectively

(r epresenting approximately 7 and 6% of additional food, respectively).

b) 12.5 μg a.i./L nominal treatment, replicate C: Instead of 3.0 and 3.2 mL of

food, 3.2 and 3.4 mL were offered on study days 57 and 58

- 61, respectively

(representing approximately 7 an d 6% of additional food, respectively).

c) 50 μg a.i./L nominal treatment, replicate B: Instead of 3.0 and 3.2 mL of

food, 3.4 and 3.6 mL were offered on study days 57 and 58

- 61, respectively

(representing approximately 13% of additional food).

Reason for

Deviation: Coarse chunks of Frog Brittle were used in place of whole pellets to improve

the accuracy of food measurement and therefore achieve the post

- NF stage 62

feeding rates stated in protocol section 18.0.

Incorrect feeding volumes were offered as no ted above because of calculation

error.

### AB C Study No. 66086

Page 47 of 261 Effect on Study: No adverse effect on study integrity. Frog Brittle was provided at the

feeding rate stated in protocol section 18.0.

The above described incorrect feeding volumes did not have any adverse

effect on study integrity because of the short duration of the feeding error

(i.e., 5 days) relative to the duration of the j uvenile exposure (from 46 to

69 days).

### AB C Study No. 66086

Page 48 of 261 REFERENCES

1. 

### Organization for Economic Co

- operation and Development. 2010 . Guideline for the

Testing of chemicals; Draft

– The Larval Amphibian Growth and Development Assay. 28 pp.

October 2010.

2. Porter, K.L., A.W. Olmstead, D.M. Kumsher, W.E. Dennis, R.L. Sprando., G.W. Holcombe,

J.J. Korte, A. Lindberg

- Livingston, and S.J. Degitz. 2011. Effects of 4

- tert

- octylphenol on

Xenopus tropicalis in a long term exposure, Aquatic Toxicology 103, PP 159-169



### 3. Mount, D.I. and W.A. Brungs. 1967. A Simplified Dosing Apparatus for Fish

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

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

New York

5. American Public Health Association. 1998. Methods 2320 and 2340 In: Standard

Methods for the Examination for Water and Wastewater . 20 th 

### ed. Washington, D.C.

### ABC Study No. 66086

Page 49 of 261 A PPENDIX A - CERTIFICATE S OF ANALYSIS

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Page 52 of 261 A PPENDIX B - DILUTION WATER CHARACTERIZATION

### ABC Study No. 66086

Page 53 of 261 Chemical Characteristics of ABC Well Water Used by

ABC Laboratories' Chemical Services Group

February 2012 ABC Well Water Screen (non

- GLP)

Or ganophosphate

(μg/L) 2012 Historical

Range

1998

- 2012 1 Elements (mg/L) 2012 Historical Range

1998

- 2012 1

Azinphos ethyl 5, 7, 9

--- <1.0 Aluminum 4 <0.0500 <0.0500

Azinphos

- methyl 3 <1 <0.200

- <1 Antimony 4 <0.0500 <0.0500

Bolstar 4 <1 <0.2 00

- <1 Arsenic <0.0030 <0.0030

- <0.050

Chloropyrifos 4 <1 <0.200

- <1 Barium 4 0.0188 0.0188

- 0.0206

Coumaphos 4 <1 <0.400

- <1 Beryllium 4 <0.0010 <0.0010

Demeton, Total

--- <0.200

- <1.0 Boron 6 0.396 0.370

- 0.432

Demeton

- O 8 <1 <1 Cadmium <0.0 020 <0.0020

- <0.0050

Demeton

- S 8 <1 <1 Calcium 2 65.2 52.0

- 83.1

Diazinon <1 <0.200

- <1 Chromium <0.0100 <0.010

Dichlorvos 4 <1 <0.200

- <1 Cobalt 4 <0.0100 <0.0100

Dimethoate 4 <1 <1 Copper <0.0100 <0.010

Disulfoton <1 <0.200

- <1 Iron 0 .107 <0.0059

- 0.16

EPN 4 <1 <0.200

- <1 Lead <0.0020 <0.0020

- 0.0400

Ethion 5, 7, 9

--- <1.0 Magnesium 2 30.3 27.0

- 33.3

Ethoprop 4 <1 <0.200

- <1 Manganese 4 <0.0050 <0.0050

Famphur 6 <1 <1 Molybdenum 4 <0.0100 <0.0100

Fensulfothion 4 <1 <1 Mercury 5 <0.00020 <0.00020

-

<0.00060

Fenthion 4 <1 <0.200

- <1 Nickel <0.0100 <0.0100

- <0.020

Malathion <1 <0.200

- <1 Potassium 2 7.99 6.6

- 8.42

Merphos 4, 7 <1 <0.200

- <1 Selenium <0.0500 <0.050

Mevinphos 4 <1 <1 Silver <0.0100 <0.010

Mono crotophos 4, 7 <1 <1 Sodium 2 29.5 27.0

- 32.2

Naled 4, 7 <1 <1

- <2.00 Thallium 4 <0.0500 <0.0500

O,O,O

- Triethyl

phosphorothioate 6 <1 <1 Tin 4 <0.0200 <0.0200

Vanadium 4 <0.0100 <0.0100

Parathion 5, 7, 9

--- <1.0 Zinc 0.0975 0.0118

- 0. 0975

Parathion, ethyl 3 <1 <0.200

- <1 Polychlorinated

Biphenyls ( μ g/L) Parathion, methyl 3 <1 <0.200

- <1

Phorate 4 <1 <0.200

- <1 Aroclor 1016 <1.00 <1.00

Ronnel 4 <1 <0.200

- <1 Aroclor 1221 <1.00 <1.00

Stirophos 4 <1 <0.200

- <1 A roclor 1232 <1.00 <1.00

Sulfotepp 4 <1 <0.200

- <1 Aroclor 1242 <1.00 <1.00

TEPP 4, 7 <1 <0.200

- <1 Aroclor 1248 <1.00 <1.00

Thionazin 6 <1 <1 Aroclor 1254 <1.00 <1.00

Tokuthion 4 <1 <0.200

- <1 Aroclor 1260 <1.00 <1.00

Trichloron ate 4 <1 <0.200

- <1

### ABC Study No. 66086

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ABC Laboratories' Chemical Services Group (continued)

February 2012 ABC Well Water Screen (non

- GLP)

Chlorinated

Herbicides ( μ g/L) 2012 Historical

Range

1998

- 2012 1 Chlorinated

Hydrocarbons ( μ g/L) 2012 Historical

Range

1998

- 2012 1

2,4,5

- TP (Silvex) <0.200 <0.200

- <50 4,4’

- DDD <0.05 <0.04

- <0.05

2,4

- D <0.200 <0.200

- <250 4,4’

- DDE <0.05 <0.04

- <0.05

4,4’

- DDT <0.05 <0.04

- <0.05

Aldrin <0.05 <0.04

- <0.05

Miscellaneous (mg/L) α

- BHC <0.05 <0.04

- <0.05

β

- BHC <0.05 <0.04

- <0.05

∆

- BHC <0.05 <0.04

- <0.05

Nitrite N <0.05 <0.01

- ≤0.05 Chlordane <0.05 <0.05

- <0.48

Nitrate N 0.307 <0.110

- 0.329 Dieldrin <0.05 <0.04

- <0.05

Total Phosphorus as P <0.050 <0.050

- 0.640 Endosulfan I <0.05 <0.04

- <0.05

Endosulfan II <0.05 <0.04

- <0.05

Endosulfan sulfate <0.0 5 <0.04

- <0.05

Endrin <0.05 <0.04

- <0.05

Endrin aldehyde <0.05 <0.04

- <0.05

Endrin ketone <0.05 <0.04

- <0.05

γ

- BHC <0.05 <0.04

- <0.05

Heptachlor <0.05 <0.04

- <0.05

Heptachlor epoxide <0.05 <0.04

- <0.05

Methoxychlor <0.05 <0.04

- <0.095

Toxaphene <0.50 <0.50

- <3.80

1 Data supporting these values are on file at ABC Laboratories. Less than (<) values indicate recovery was below

the greatest limit of detection during these analyses.

2 Historic al Range is from 2003.

3 Historical Range is from 2008.

4 Historical Range is from 2009.

5 Historical Range does not include 2009.

6 Historical Range is from 2011.

7 Historical Range does not include 2011 and 2012.

8 Historical Range is from 2012.

9 Histo rical Range does not include 2012



### Note: ABC well water is the base water for ABC Aged

- Blended Water.

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### ABC Study No. 66086

Page 176 of 261 Table F-1. Measured Concentrations of 4- tert -octylphenol During the Larval Growth and Development Assay with the African

Clawed Frog, Xenopus laevis

Control Level 1 Level 2 Level 3 Level 4

(0 μg/L) Nominal (μg/L): 6.25 Nominal (μg/L): 12.5 Nominal (μg/L): 25 .0 Nominal (μg/L): 50.0

Sample Measured Measured % of % of Measured % of % of Measured % of % of Measured % of % of

Day / Rep. (μg/L) (μg/L) Nominal Mean (μg/L) Nominal Mean (μg/L) Nominal Mean (μg/L) Nominal Mean

Study

Initiation

Day 0 Rep. A <MQL a 8.98 144 181 18.0 144 175 36.1 144 185 74.9 150 197

Rep. B <MQL a 9.04 145 182 17.5 140 170 35.8 143 184 74.2 148 195

Rep. C <MQL a 8.49 136 171 18.0 144 175 33.9 136 174 74.3 149 196

Rep. D <MQL a 8.48 136 171 17.4 139 169 36.9 148 189 73.9 148 194

Rep. E <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. F <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. G <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. H <M QL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Daily Mean <MQL a 8.75 140 176 17.7 142 172 35.7 143 183 74.3 149 196

3 <MQL a 6.25 100 126 12.0 96 117 26.0 104 133 49.4 99 130

7 <MQL a 4.77 76 96 8.87 71 86 19.1 76 98 36.5 73 96

1 3 <MQL a 5.15 82 104 8.91 71 87 15.6 62 80 32.4 65 85

18 <MQL a 4.09 65 82 7.97 64 77 16.7 67 86 32.3 65 85

28 <MQL a 3.21 51 65 7.26 58 70 15.5 62 79 28.7 57 76

32 <MQL a 3.27 52 66 8.00 64 78 13.6 54 70 24.9 50 66

39 <MQL a 3.10 50 62 7.44 60 72 16.4 66 84 31.7 63 83

47 <MQL a 5.51 88 111 11.3 90 109 20.9 84 107 46.9 94 123

54 <MQL a 6.42 103 129 11.1 89 108 20.2 81 104 44.2 88 116

60 <MQL a 4.26 68 86 10.9 87 106 18.9 76 97 36.8 74 97

61 <MQL a 4.58 73 f 92 9.50 76 f 92 15.7 63 f 81 26.2 52 f 69

64 <MQL a 5.12 82 103 10.4 83 101 22.8 91 117 40.4 81 106

a MQL =[ Low STD Conc. * Analysis Volume (mL)] / Sample Volume (mL) = (1.54 ng/mL * 20 mL) / 10 mL = 3.08 ng/mL

b Not Applicable

c The higher measured concentrations are suspected to have resulted from the animals being removed prior to the collection of the samples.

d The actual concentration for Day 88, Level 1 was <MQL. 1/2 MQL (1.54 ng/mL) was used for calculating the Overall Mean.

e When applicable, the Daily Mean was used for calculating the Overall Mean

f Due to a pump malfunction, solution was delivered to the chemical mixing box from one of two 4

- tert

- octylphenol saturator columns prior to collection of day 61. This

malfunction may have cont r ibuted to lower test substance re coveries from some of the samples.

### ABC Study No. 66086

Page 177 of 261 Table F -1. Measured Concentrations of 4

- tert -octylphenol During the Larval Growth and Development Assay with the African

Clawed Frog, Xenopus laevis (continued)

Control Level 1 Level 2 Level 3 Level 4

(0 μg/ L) Nominal (μg/L): 6.25 Nominal (μg/L): 12.5 Nominal (μg/L): 25.0 Nominal (μg/L): 50.0

Sample Measured Measured % of % of Measured % of % of Measured % of % of Measured % of % of

Day / Rep. (μg/L) (μg/L) Nominal Mean (μg/L) Nominal Mean (μg/L) Nomi nal Mean (μg/L) Nominal Mean

70 <MQL a 5.31 85 107 10.7 86 104 17.1 68 88 33.8 68 89

73 <MQL a 5.11 82 103 9.17 73 89 16.8 67 86 34.2 68 90

77 <MQL a 5.46 87 110 11.0 88 107 17.8 71 91 31.1 62 82

81 <MQL a 5.01 80 101 11.7 94 114 24.1 96 124 38.8 78 102

88 <MQL a 1.54 d 25 b 31 b 8.71 70 85 17.6 70 90 32.8 66 86

91 <MQL a 6.68 107 134 12.1 97 117 21.8 87 112 44.4 89 117

97 <MQL a 6.10 98 123 11.0 88 107 20.6 82 106 45.4 91 119

104 <MQL a 4.48 72 90 10.3 82 100 17.8 71 91 35.4 71 93

Rep. A <MQL a 4.46 71 90 10.30 82 c 100 19.3 77 99 36.5 73 96

Study

Termination

Days 112 and

113 Rep. B <MQL a 6.62 106 c 133 9.65 77 94 16.3 65 84 39.3 79 103

Rep. C <MQL a 5.60 90 c 113 9.66 77 94 18.9 76 97 36.0 72 95

Rep. D <MQL a 3.77 6 0 76 9.25 74 90 18.4 74 94 32.9 66 87

Rep. E <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. F <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. G <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Rep. H <MQL a NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b NA b

Daily

Mean <MQL a 5.11 82 103 9.72 78 95 18.2 73 94 36.2 73 95

Overall

Mean e <MQL a 4.97 80 10.3 82 19.5 78 38.0 76

SD 1.479 2.21 4.72 10.44

a MQL =[ Low STD Conc. * Analysis Volume (mL)] / Sample Volume (mL) = (1.54 ng/mL * 20 mL) / 10 mL = 3.08 ng/mL

b Not Applicable

c The higher measured concentrations are suspected to have resulted from the animals being removed prior to the collection o f the samples.

d The actual concentration for Day 88, Level 1 was <MQL. 1/2 MQL (1.54 ng/mL) was used for calculating the Overall Mean.

e When applicable, the Daily Mean was used for calculating the Overall Mean

f Due to a pump malfunction, solution was delivered to the chemical mixing box from one of two 4

- tert

- octylphenol saturator columns prior to collection of day 61. This

malfunction may have cont r ibuted to lower test substance recoveries from some of the samples.

### ABC Study No. 66086

Page 178 of 261 Table F -2. Measured Conc entrations of 4

- tert -octylphenol from the Saturator Column

Outflows During the Larval Growth and Development Assay with the African

Clawed Frog, Xenopus laevis

Sample Day Measured Concentration Expressed as μg/L

(Percent of Nominal Concentration)

Satura tor Outflow A

( 11,000 ) a Saturator Outflow B

( 11,000 ) a

0 12,500 (114) NA

3 13,200 (120) NA

7 12,400 (113) NA

13 10,800 (98) NA

18 10,800 (98) NA

28 7,050 (64) 12,600 (115) a

32 7,990 (73) 15, 0 00 ( 136 )

39 12,200 (111) 12,100 (110)

47 12,200 (111) 11,700 (106)

54 12,000 (109) 11,800 (107)

60 11,200 (102) 12,500 (114)

64 12,700 (115) 11,200 (102)

70 12,600 (115) 9,750 (89)

73 11,200 ( 102 ) 9270 ( 84 )

77 9,570 (87) 12,900 (117)

81 9,230 (84) 14,500 (132)

88 10,800 (98) 12,500 (114)

91 12,700 (115) 12,600 (115)

97 11,600 (105) 13,000 (118)

104 13,800 (125) 16,400 (149)

112 11,600 (105) 9,650 (88)

113 10,900 (99) 9,450 (86)

NA = Not applicable

a A second saturator column was placed on the diluter system on day 26 in an attempt to improve t est substance

recoveries relative to target nominal concentrations.

### ABC Study No. 66086

Page 179 of 261 Table F-3. Measured Concentrations of 4- tert -octylphenol Quality Control Samples During

the Larval Growth and Development Assay with the African Clawed Frog,

Xenopus laevis

Sample Day Mea sured Concentration Expressed as μ g/L

(Percent of Nominal Concentration)

Low Spike

( 5.06 ) a High Spike

( 61.8 ) a

0 4.72 (93) 68.1 (110)

3 4.43 (88) 61.6 (100)

7 5.31 (105) 67.4 (109)

13 5.10 (101) 65.9 (107)

18 b 5.24 (104) 63.2 (102)

18 c 5.32 (105) 66.3 (107)

28 5.24 (104) 62.2 (101)

32 5.98 (118) 67.1 (109)

39 5.44 (108) 68.2 (110)

47 5.82 (115) 65.0 (105)

54 5.18 (102) 68.3 (111)

60 5.43 (107) 66.2 (107)

61 5.17 (102) 65.4 (106)

64 5.24 (104) 65.7 (106)

70 5.45 (108) 66.7 (108)

73 5.09 (1 01) 65.3 (106)

77 5.15 (102) 65.4 (106)

81 5.71 (113) 65.6 (106)

88 5.77 (114) 65.6 (106)

91 5.13 (101) 65.8 (106)

97 5.64 (111) 72.9 (118)

104 5.22 (103) 65.8 (106)

112 5.09 (100) 62.8 (102)

113 5.48 (108) 60.9 (99)

a Nominal concentration as μg /L

. b Non

- centrifuged

.

c Centrifuged

.

### ABC Study No. 66086

Page 180 of 261 APPENDIX G - REPLICATE SURVIVAL DATA

### ABC Study No. 66086

Page 181 of 261 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to 4- tert -octylphenol for 113- Days

Mean

Measured

Concentra tion

(μ g/L ) R

e

p Day 0

Initial

Number Day 5 Day 12 Day 14

Number

Hatched Cumulative

Number

Dead Not

Found a %

Survival Number

Surviving Cumulative

Number

Dead % Survival Number

Surviving Cumulative

Number

Dead % Survival

0 (Control) A 25 25 0 0 100 25 0 100 25 0 100

B 25 24 1 0 96 24 1 96 24 1 96

C 25 24 0 1 96 25 0 100 25 0 100

D 25 23 2 0 92 23 2 92 23 2 92

E 25 25 0 0 100 25 0 100 25 0 100

F 25 24 1 0 96 23 2 92 23 2 92

G 25 24 1 0 96 23 2 92 23 2 92

H 25 22 2 1 88 22 3 88 22 3 88

4.97 A 25 22 3 0 88 22 3 88 22 3 88

B 25 18 6 1 72 18 7 72 18 7 72

C 25 24 1 0 96 24 1 96 24 1 96

D 25 25 0 0 100 23 2 92 23 2 92

10.3 A 25 21 4 0 84 21 4 84 21 4 84

B 25 21 3 1 84 21 4 84 21 4 84

C 25 24 1 0 96 23 2 92 23 2 92

D 25 23 2 0 9 2 23 2 92 23 2 92

19.5 A 25 24 0 1 96 24 1 96 24 1 96

B 25 24 0 1 96 25 0 100 25 0 100

C 25 25 0 0 100 25 0 100 25 0 100

D 25 23 2 0 92 23 2 92 23 2 92

38.0 A 25 23 0 2 92 24 1 96 24 1 96

B 25 21 3 1 84 21 4 84 21 4 84

C 25 24 1 0 96 24 1 96 24 1 96

D 25 23 1 1 92 24 1 96 24 1 96

a All "Not Found" considered dead on study day 9 and added to cumulative number dead

.

### ABC Study No. 66086

Page 182 of 261 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to 4- tert -o ctylphenol for 113- Days

(continu ed)

Mean

Measured

Concentration

(μg/L) R

E

P Day 0 Initial

Number Day 2 1 a Day 28 Day 35

Number

Surviving Cumulative

Number

Dead %

Survival Number

Surviving Cumulative

Number

Dead %

Survival Number

Surviving Cumulative

Number

Dead %

Survival

0 (Contr ol) A 25 25 0 100 25 0 100 25 0 100

B 25 24 1 96 24 1 96 24 1 96

C 25 24 0 100 23 1 96 23 1 96

D 25 23 2 92 23 2 92 23 2 92

E 25 25 0 100 25 0 100 25 0 100

F 25 23 2 92 23 2 92 23 2 92

G 25 23 2 92 23 2 92 23 2 92

H 25 22 3 88 22 3 88 22 3 88

4.97 A 25 21 4 84 21 4 84 21 4 84

B 25 18 7 72 18 7 72 18 7 72

C 25 24 1 96 24 1 96 24 1 96

D 25 22 3 88 22 3 88 22 3 88

10.3 A 25 21 4 84 21 4 84 21 4 84

B 25 21 4 84 20 5 80 20 5 80

C 25 23 2 92 23 2 92 23 2 92

D 25 23 2 92 23 2 92 23 2 92

19.5 A 25 24 1 96 24 1 96 24 1 96

B 25 24 1 96 24 1 96 24 1 96

C 25 24 1 96 24 1 96 24 1 96

D 25 22 3 88 22 3 88 22 3 88

38.0 A 25 24 1 96 24 1 96 24 1 96

B 25 21 4 84 21 4 84 21 4 84

C 25 24 1 96 24 1 96 24 1 96

D 25 24 1 96 24 1 96 24 1 96

a One larv a in the control, replicate C chamber was inadvertently killed during cleaning on study day 17 and was not counted as an exposure related mortality.

### ABC Study No. 66086

Page 183 of 261 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to 4- tert -o ctylphenol for 113- Days

(continued)

Mean

Measured

Concentration

(μg/L) R

E

P Day 0

Initial

Number Number

Removed

for

Sampling Day 56 Day 63 Day 70

Number

Alive Cumulative

Number

Dead %

Survival Number

Alive Cumulative

Number

Dead %

Survival Number

Alive Cumulative

Number

Dead %

Survival

0 (Control) A 25 5 20 0 100 20 0 100 20 0 100

B 25 5 19 1 96 19 1 96 19 1 96

C 25 5 17 2 92 17 2 92 17 2 92

D 25 5 17 3 88 17 3 88 17 3 88

E 25 5 20 0 100 20 0 100 20 0 100

F 25 5 17 3 88 17 3 8 8 17 3 88

G 25 5 18 2 92 18 2 92 18 2 92

H 25 5 17 3 88 17 3 88 17 3 88

4.97 A 25 5 16 4 84 16 4 84 16 4 84

B 25 5 13 7 72 13 7 72 13 7 72

C 25 5 19 1 96 19 1 96 19 1 96

D 25 5 16 4 84 16 4 84 16 4 84

10.3 A 25 5 15 5 80 15 5 80 15 5 80

B 25 5 14 6 76 14 6 76 14 6 76

C 25 5 18 2 92 18 2 92 18 2 92

D 25 5 18 2 92 18 2 92 18 2 92

19.5 A 25 5 19 1 96 19 1 96 19 1 96

B 25 5 19 1 96 19 1 96 19 1 96

C 25 5 19 1 96 19 1 96 19 1 96

D 25 5 17 3 88 17 3 88 16 4 84

38.0 A 25 5 19 1 96 19 1 9 6 19 1 96

B 25 5 16 4 84 16 4 84 16 4 84

C 25 5 19 1 96 19 1 96 19 1 96

D 25 5 18 2 92 18 2 92 18 2 92

### ABC Study No. 66086

Page 184 of 261 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to 4- tert -o ctylphenol for 113- Days

(continued)

Mean

Measure d

Concentration

(μg/L) R

E

P Day 0 Initial

Number Number

Removed at

juvenile cull Day 77 a Day 84

Number

Alive Cumulative

Number Dead %

Survival Number

Alive Cumulative

Number Dead %

Survival

0 (Control) A 25 10 10 0 100 10 0 100

B 25 8 10 1 b 96 10 1 96

C 25 8 9 2 92 9 2 92

D 25 7 10 3 88 10 3 88

E 25 10 10 0 100 10 0 100

F 25 7 10 3 88 10 3 88

G 25 8 10 2 92 10 2 92

H 25 7 10 3 88 10 3 88

4.97 A 25 6 10 4 84 10 4 84

B 25 3 10 7 72 10 7 72

C 25 9 10 1 96 10 1 96

D 25 6 10 4 84 10 4 84

10.3 A 25 5 10 5 80 10 5 80

B 25 4 10 6 76 10 6 76

C 25 7 10 2 b 92 10 2 92

D 25 8 10 2 92 10 2 92

19.5 A 25 9 10 1 96 10 1 96

B 25 9 10 1 96 10 1 96

C 25 9 10 1 96 10 1 96

D 25 6 10 4 84 10 4 84

38.0 A 25 9 10 1 96 10 1 96

B 2 5 6 10 4 84 10 4 84

C 25 9 10 1 96 10 1 96

D 25 8 10 2 92 10 2 92

a One extra juvenile in the control, replicate C chamber was inadvertently removed at the juvenile cull. This was not counted as an exposure related mortality.

b One larva did not att ain NF stage 62 prior to the day 70. This individual was removed at the juvenile cull and was not counted as an exposure rela ted mortality.

### ABC Study No. 66086

Page 185 of 261 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to 4- tert -o ctylphenol for 113-D ays (continued)

Mean

Measured

Concentration

(μg/L) R

E

P Day 0

Initial

Number Day 91 Day 98

Number

Alive Cumulative

Number Dead %

Survival Number

Alive Cumulative

Number Dead %

Survival

0 (Control) A 25 

### 10 0 100 10 0 100

B 25 

### 10 1 96 10 1 96

C 25 

### 9 2 92 9 2 92

D 25 

### 10 3 88 10 3 88

E 25 

### 10 0 100 10 0 100

F 25 

### 10 3 88 10 3 88

G 25 

### 10 2 92 10 2 92

H 25 

### 10 3 88 10 3 88

4.97 A 25 

### 10 4 84 10 4 84

B 25 

### 10 7 72 10 7 72

C 25 

### 10 1 96 10 1 96

D 25 

### 10 4 84 10 4 84

10.3 A 25 

### 10 5 80 10 5 80

B 25 

### 10 6 76 10 6 76

C 25 

### 10 2 92 10 2 92

D 25 

### 10 2 92 10 2 92

19.5 A 25 

### 10 1 96 10 1 96

B 25 

### 10 1 96 10 1 96

C 25 

### 10 1 96 10 1 96

D 25 

### 10 4 84 10 4 84

38.0 A 25 

### 10 1 96 10 1 96

B 25 

### 10 4 84 10 4 84

C 25 

### 10 1 96 10 1 96

D 25 

### 10 2 92 10 2 92

### ABC Study No. 66086

Page 186 of 261 Appendix G-1. Replicate Survival of the African Clawed Frog, Xenopus laevis , Exposed to 4- tert -o ctylphenol for 113- Days

(continued)

Mean

Measured

Concentration

(μg/L) R

E

P Day 0

Initial

Number Day 105 Termination

Number

Alive Cumulative

Number Dead a %

Survival Number

Alive Cumulative

Number Dead %

Survival

0 (Control) A 25 10 0 100 10 0 100

B 25 10 1 96 10 1 96

C 25 9 2 92 9 2 92

D 25 9 3 88 9 3 88

E 25 10 0 100 10 0 100

F 25 10 3 88 10 3 88

G 25 10 2 92 10 2 92

H 25 10 3 88 10 3 88

4.97 A 25 10 4 84 10 4 84

B 25 10 7 72 10 7 72

C 25 10 1 96 10 1 96

D 25 9 4 83 9 4 83

10.3 A 25 10 5 80 10 5 80

B 25 10 6 76 10 6 76

C 25 10 2 92 10 2 92

D 25 10 2 92 10 2 92

19.5 A 25 10 1 96 10 1 96

B 25 10 1 96 10 1 96

C 25 10 1 96 10 1 96

D 25 10 4 84 10 4 84

38.0 A 25 10 1 96 10 1 96

B 25 10 4 84 10 4 84

C 25 10 1 96 10 1 96

D 25 10 2 92 10 2 92

a One animal escaped from R ep

. D of the control and 4.97 μg/L treatment during routine daily activities and wa s not counted as an exposure related mortality.

### ABC Study No. 66086

Page 187 of 261 Appendix G-2. Survival Summary for the African Clawed Frog, Xenopus laevis , Exposed to 4- tert -octylphenol for 113- Days

Mean

Measured

Concentration

(μg/L) R

E

P Day 0

Initial

number of

Organisms Number

re moved d Day 71

initial

number Day 113

NF Stage

62

survivors NF Stage 62

% Survival Treatment

mean Cumulative number

juveniles dead e %

survival Treatment

mean juvenile

% survival

Control A 25 25 100 94 15 10 0 100 100

B 25 23 a 96 14 10 0 100

C 25 23 b 96 13 9 0 f 100

D 25 23 92 12 10 0 g 100

E 25 25 100 15 10 0 100

F 25 22 88 12 10 0 100

G 25 23 92 13 10 0 100

H 25 22 88 12 10 0 100

4.97 A 25 21 84 85 11 10 0 100 100

B 25 18 72 8 10 0 100

C 25 24 96 14 10 0 100

D 25 22 88 11 10 0 g 100

10.3 A 25 20 80 85 10 10 0 100 100

B 25 19 c 76 9 10 0 100

C 25 22 a 92 13 10 0 100

D 25 23 92 13 10 0 100

19.5 A 25 24 96 94 14 10 0 100 100

B 25 24 96 14 10 0 100

C 25 24 96 14 10 0 100

D 25 22 88 11 10 0 100

38.0 A 25 24 96 93 14 10 0 100 100

B 25 21 84 11 10 0 100

C 25 24 96 14 10 0 100

D 25 24 96 13 10 0 100

a One larva did not attain NF stage 62 prior to the day 70. This individual was removed at the juvenile cull and wa s not counted as an exposure related mortality.

b One larva was inadvertently killed during test chamber cleaning on study day 17. This individual was not counted as an expos ure related mortality.

c Included three larvae for which the date achieved NF s tage 62 was not documented.

d Five NF stage 62 larvae were removed for larval sub

- sampling and remainder were removed at cull to 10 animals on study day 70.

e Indicates exposure

- related mortalities following juvenile cull.

f One extra juvenile was inadver tently removed at the juvenile cull. This individual was not counted as an exposure related mortality.

g One juvenile escaped during routine daily activities. This individ ual was not counted as an exposure related mortality.

### ABC Study No. 66086

Page 188 of 261 APPENDIX H – NF STAGE 62 DATA

### ABC Study No. 66086

Page 189 of 261 Appendix H-1. Replicate Data for Number of Days to NF Stage 62, Weight and Length

Treatment

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

Achieved Weight (g) Snout

- to

- Vent

Length (mm)

0 (Control) A 1 44 1.483 20

0 (Contr ol) A 2 44 2.328 22

0 (Control) A 3 44 1.893 23

0 (Control) A 4 44 2.35 25

0 (Control) A 5 44 2.462 25

0 (Control) B 1 39 1.842 21

0 (Control) B 2 40 2.097 24

0 (Control) B 3 40 2.578 25

0 (Control) B 4 41 2.666 25

0 (Control) B 5 41 2.121 23

0 (C ontrol) C 1 40 2.044 22

0 (Control) C 2 41 1.882 23

0 (Control) C 3 42 1.584 21

0 (Control) C 4 42 1.614 22

0 (Control) C 5 42 1.405 22

0 (Control) D 1 38 1.990 23

0 (Control) D 2 40 2.161 23

0 (Control) D 3 40 2.396 25

0 (Control) D 4 40 1.484 22

0 (Control) D 5 40 1.817 23

0 (Control) E 1 39 1.935 22

0 (Control) E 2 40 2.132 23

0 (Control) E 3 40 2.135 24

0 (Control) E 4 40 1.680 23

0 (Control) E 5 41 2.189 23

0 (Control) F 1 40 1.884 22

0 (Control) F 2 40 1.851 23

0 (Control) F 3 41 2.34 2 25

0 (Control) F 4 41 2.112 23

0 (Control) F 5 41 2.502 23

0 (Control) G 1 40 2.224 23

0 (Control) G 2 40 2.289 24

0 (Control) G 3 40 2.076 23

0 (Control) G 4 40 2.352 25

0 (Control) G 5 40 2.018 22

0 (Control) H 1 40 1.632 22

0 (Control) H 2 40 1.930 24

0 (Control) H 3 40 2.760 25

0 (Control) H 4 40 2.348 24

0 (Control) H 5 40 2.067 23

### ABC Study No. 66086

Page 190 of 261 Appendix H-1. Replicate Data for Number of Days to NF Stage 62, Weight and Length

(continued)

Treatment

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

Achieved W eight (g) Snout

- to

- Vent

Length (mm)

4.97 A 1 40 1.983 23

4.97 A 2 41 1.868 23

4.97 A 3 41 1.622 23

4.97 A 4 41 1.834 23

4.97 A 5 43 2.629 26

4.97 B 1 40 2.098 25

4.97 B 2 40 2.336 25

4.97 B 3 40 1.709 23

4.97 B 4 40 1.987 24

4.97 B 5 41 2.243 24

4.97 C 1 40 1.992 24

4.97 C 2 40 2.295 24

4.97 C 3 40 2.774 24

4.97 C 4 41 1.754 22

4.97 C 5 41 2.211 24

4.97 D 1 40 1.674 21

4.97 D 2 40 1.783 23

4.97 D 3 40 2.127 24

4.97 D 4 40 1.932 24

4.97 D 5 40 1.980 23

10.3 A 1 40 1.893 23

10.3 A 2 40 1 .670 23

10.3 A 3 41 2.542 27

10.3 A 4 41 2.982 25

10.3 A 5 41 2.443 24

10.3 B 1 40 2.097 22

10.3 B 2 40 1.562 22

10.3 B 3 41 1.411 21

10.3 B 4 41 1.743 22

10.3 B 5 41 3.263 27

10.3 C 1 44 2.085 24

10.3 C 2 44 2.295 24

10.3 C 3 44 1.972 23

10.3 C 4 45 2.103 23

10.3 C 5 45 2.668 24

10.3 D 1 41 2.105 23

10.3 D 2 41 1.980 23

10.3 D 3 41 2.588 23

10.3 D 4 41 2.508 24

10.3 D 5 41 1.496 21

### ABC Study No. 66086

Page 191 of 261 Appendix H-1. Replicate Data for Number of Days to NF Stage 62, Weight and Length

(continued)

Treatment

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

Achieved Weight (g) Snout

- to

- Vent

Length (mm)

19.5 A 1 40 1.893 21

19.5 A 2 40 2.038 23

19.5 A 3 43 2.071 24

19.5 A 4 43 1.927 22

19.5 A 5 43 1.814 22

19.5 B 1 41 1.864 23

19.5 B 2 41 2.370 23

19.5 B 3 43 2.4 04 24

19.5 B 4 44 2.403 24

19.5 B 5 44 2.695 25

19.5 C 1 40 2.216 22

19.5 C 2 40 2.485 23

19.5 C 3 40 1.697 23

19.5 C 4 41 2.663 26

19.5 C 5 41 2.352 25

19.5 D 1 40 2.173 24

19.5 D 2 40 1.830 22

19.5 D 3 41 1.589 22

19.5 D 4 41 1.700 20

19.5 D 5 41 2.017 24

38.0 A 1 38 1.890 23

38.0 A 2 40 1.805 25

38.0 A 3 40 2.475 24

38.0 A 4 41 2.238 25

38.0 A 5 41 2.233 25

38.0 B 1 40 1.856 21

38.0 B 2 40 2.639 24

38.0 B 3 40 2.474 25

38.0 B 4 41 2.607 25

38.0 B 5 42 1.331 20

38.0 C 1 42 2.335 26

38.0 C 2 42 2.526 25

38.0 C 3 42 2.281 24

38.0 C 4 42 2.394 25

38.0 C 5 43 2.202 25

38.0 D 1 41 2.063 24

38.0 D 2 41 2.117 24

38.0 D 3 42 2.016 24

38.0 D 4 42 2.006 23

38.0 D 5 42 

### 2.263 24

### ABC Study No. 66086

Page 192 of 261 Appendix H-2. Replicate Data for Number of Days to NF Sta ge 62

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62

Achieved Tadpole# Study Day NF 62

Achieved

0 (Control) A 1 44 14 47

0 (Control) A 2 44 15 48

0 (Control) A 3 44 16 48

0 (Control) A 4 44 17 48

0 (Control) A 5 44 18 48

0 (Control) A 6 44 19 4 8

0 (Control) A 7 45 20 49

0 (Control) A 8 45 21 49

0 (Control) A 9 46 22 49

0 (Control) A 10 46 23 50

0 (Control) A 11 47 24 51

0 (Control) A 1 2 47 25 56

0 (Control) A 1 3 47

0 (Control) B 1 39 14 43

0 (Control) B 2 40 15 43

0 (Control) B 3 40 16 43

0 (Control) B 4 41 17 43

0 (Control) B 5 41 18 43

0 (Control) B 6 41 19 44

0 (Control) B 7 42 20 45

0 (Control) B 8 42 21 45

0 (Control) B 9 42 22 45

0 (Control) B 10 42 23 45

0 (Control) B 11 42 24

--

0 (Control) B 1 2 43 25

--

0 (Control) B 1 3 43

0 (Control) C 1 40 14 43

0 (Control) C 2 41 15 43

0 (Control) C 3 42 16 43

0 (Control) C 4 42 17 44

0 (Control) C 5 42 18 44

0 (Control) C 6 42 19 45

0 (Control) C 7 42 20 45

0 (Control) C 8 42 21 46

0 (Control) C 9 43 22 47

0 (Control) C 10 43 23 48

0 (Control) C 11 43 24

--

0 (Control) C 1 2 43 25

--

0 (Control) C 13 43



### ‘-- ’ denotes a mortality that occurred before NF stage 62

.

### ABC Study No. 66086

Page 193 of 261 Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued)

Treatment

(μg/L) Replicate T adpole# Study Day NF 62

Achieved Tadpole# Study Day NF 62

Achieved

0 (Control) D 1 38 14 41

0 (Control) D 2 40 15 42

0 (Control) D 3 40 16 42

0 (Control) D 4 40 17 43

0 (Control) D 5 40 18 43

0 (Control) D 6 40 19 43

0 (Control) D 7 40 20 45

0 (Con trol) D 8 41 21 47

0 (Control) D 9 41 22 48

0 (Control) D 10 41 23 54

0 (Control) D 11 41 24

--

0 (Control) D 1 2 41 25

--

0 (Control) D 1 3 41

0 (Control) E 1 39 14 44

0 (Control) E 2 40 15 44

0 (Control) E 3 40 16 44

0 (Control) E 4 40 17 44

0 (Control) E 5 41 18 44

0 (Control) E 6 41 19 44

0 (Control) E 7 41 20 45

0 (Control) E 8 42 21 45

0 (Control) E 9 42 22 45

0 (Control) E 10 42 23 47

0 (Control) E 11 43 24 47

0 (Control) E 1 2 43 25 48

0 (Control) E 1 3 44

0 (Control) F 1 40 14 43

0 (Control) F 2 40 15 43

0 (Control) F 3 41 16 43

0 (Control) F 4 41 17 44

0 (Control) F 5 41 18 44

0 (Control) F 6 41 19 44

0 (Control) F 7 41 20 44

0 (Control) F 8 42 21 44

0 (Control) F 9 42 22 47

0 (Control) F 10 42 23

--

0 (Control) F 11 42 24

--

0 (Control) F 1 2 43 25

--

0 (Control) F 1 3 43



### ‘

-- ’ denotes a mortality that occurred before NF stage 62

.

### ABC Study No. 66086

Page 194 of 261 Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued)

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62

Achieved Ta dpole# Study Day NF 62

Achieved

0 (Control) G 1 40 14 43

0 (Control) G 2 40 15 44

0 (Control) G 3 40 16 44

0 (Control) G 4 40 17 44

0 (Control) G 5 40 18 45

0 (Control) G 6 40 19 45

0 (Control) G 7 41 20 45

0 (Control) G 8 41 21 49

0 (Control) G 9 41 22 49

0 (Control) G 10 41 23 49

0 (Control) G 11 41 24

--

0 (Control) G 1 2 42 25

--

0 (Control) G 1 3 43

0 (Control) H 1 40 14 42

0 (Control) H 2 40 15 42

0 (Control) H 3 40 16 42

0 (Control) H 4 40 17 43

0 (Control) H 5 40 18 43

0 (Control) H 6 41 19 43

0 (Control) H 7 41 20 44

0 (Control) H 8 41 21 45

0 (Control) H 9 42 22 45

0 (Control) H 10 42 23

--

0 (Control) H 11 42 24

--

0 (Control) H 1 2 42 25

--

0 (Control) H 1 3

4.97 A 1 40 14 45

4.97 A 2 41 15 45

4.97 A 3 41 16 45

4.97 A 4 41 17 47

4.97 A 5 43 18 47

4.97 A 6 43 19 47

4.97 A 7 43 20 47

4.97 A 8 43 21 54

4.97 A 9 43 22

--

4.97 A 10 44 23

--

4.97 A 11 45 24

--

4.97 A 1 2 45 25

--

4.97 A 1 3 45



### ‘

-- ’ denotes a mortality that occurred before NF stage 62

.

### ABC Study No. 66086

Page 195 of 261 Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued)

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62

Achieved Tadpole# Study Day NF 62

Achieved

4.97 B 1 40 14 44

4.97 B 2 40 15 44

4.97 B 3 40 16 44

4.97 B 4 40 17 46

4.97 B 5 41 18 46

4.97 B 6 41 19

--

4.97 B 7 41 20

--

4.97 B 8 41 21

--

4.97 B 9 41 22

--

4.97 B 10 41 23

--

4.97 B 11 41 24

--

4.97 B 1 2 42 25

--

4.97 B 1 3 42

4.97 C 1 40 14 42

4.97 C 2 40 15 43

4.97 C 3 40 16 43

4.97 C 4 41 17 43

4.97 C 5 41 18 43

4.97 C 6 41 19 44

4.97 C 7 41 20 44

4.97 C 8 42 21 45

4.97 C 9 42 22 45

4.97 C 10 42 23 45

4.97 C 11 42 24 46

4.97 C 1 2 42 25 47

4.97 C 1 3 42

4.97 D 1 40 14 44

4.97 D 2 40 15 44

4.97 D 3 40 16 44

4.97 D 4 40 17 44

4.97 D 5 40 18 45

4.97 D 6 42 19 45

4.97 D 7 42 20 47

4.97 D 8 42 21 49

4.97 D 9 42 22 49

4.97 D 10 43 23

--

4.97 D 11 43 24

--

4.97 D 1 2 44 25

--

4.97 D 1 3 44



### ‘

-- ’ denotes a mortality that occurred before NF stage 62

.

### ABC Study No. 66086

Page 196 of 261 Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued)

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62

Achieved Tadpole# Study Day NF 62

Achieved

10.3 A 1 

### 40 14 

### 44

10.3 A 2 

### 40 15 

### 44

10.3 A 3 

### 41 16 

### 44

10.3 A 4 

### 41 17 

### 45

10.3 A 5 

### 42 18 

### 48

10.3 A 6 

### 42 19 

### 49

10.3 A 7 

### 42 20 

### 52

10.3 A 8 

### 42 21



### --

10.3 A 9 

### 43 22



### --

10.3 A 10 

### 43 23



### --

10.3 A 11 

### 43 24



### --

10.3 A 1 2 

### 43 25



### --

10.3 A 1 3 

### 43

10.3 B 1 

### 40 14 

### 45

10.3 B 2 

### 40 15 

### 48

10.3 B 3 

### 41 16 

### 52

10.3 B 4 

### 41 17



### - a

10.3 B 5 

### 41 18



### - a

10.3 B 6 

### 41 19



### - a

10.3 B 7 

### 42 20



### --

10.3 B 8 

### 42 21



### --

10.3 B 9 

### 43 22



### --

10.3 B 10 

### 43 23



### --

10.3 B 11 

### 44 24



### --

10.3 B 1 2 

### 44 25



### --

10.3 B 1 3 

### 44

10.3 C 1 

### 44 14 

### 48

10.3 C 2 

### 44 15 

### 48

10.3 C 3 

### 44 16 

### 48

10.3 C 4 

### 45 17 

### 49

10.3 C 5 

### 45 18 

### 50

10.3 C 6 

### 45 19 

### 50

10.3 C 7 

### 46 20 

### 50

10.3 C 8 

### 46 21 

### 51

10.3 C 9 

### 46 2 2 

### 54

10.3 C 10 

### 47 23



### --

10.3 C 11 

### 47 24



### --

10.3 C 1 2 

### 47 25



### --

10.3 C 1 3 

### 47

‘

-- ’ denotes a mortality that occurred before NF stage 62

.

a 

### Date NF62 attained not recorded.

### ABC Study No. 66086

Page 197 of 261 Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued)

Trea tment

(μg/L) Replicate Tadpole# Study Day NF 62

Achieved Tadpole# Study Day NF 62

Achieved

10.3 D 1 41 14 44

10.3 D 2 41 15 45

10.3 D 3 41 16 45

10.3 D 4 41 17 45

10.3 D 5 41 18 45

10.3 D 6 42 19 45

10.3 D 7 42 20 46

10.3 D 8 42 21 47

10.3 D 9 42 22 47

10.3 D 10 42 23 47

10.3 D 11 43 24

--

10.3 D 1 2 44 25

--

10.3 D 1 3 44

19.5 A 1 40 14 44

19.5 A 2 40 15 45

19.5 A 3 43 16 45

19.5 A 4 43 17 45

19.5 A 5 43 18 45

19.5 A 6 43 19 45

19.5 A 7 43 20 45

19.5 A 8 43 21 46

19.5 A 9 43 22 46

19 .5 A 10 43 23 47

19.5 A 11 43 24 49

19.5 A 1 2 44 25

--

19.5 A 1 3 44

19.5 B 1 41 14 47

19.5 B 2 41 15 47

19.5 B 3 43 16 47

19.5 B 4 44 17 47

19.5 B 5 44 18 48

19.5 B 6 44 19 48

19.5 B 7 44 20 48

19.5 B 8 44 21 49

19.5 B 9 45 22 49

19.5 B 10 4 5 23 49

19.5 B 11 45 24 54

19.5 B 1 2 46 25

--

19.5 B 1 3 46



### ‘

-- ’ denotes a mortality that occurred before NF stage 62

.

### ABC Study No. 66086

Page 198 of 261 Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued)

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62

Achi eved Tadpole# Study Day NF 62

Achieved

19.5 C 1 40 14 43

19.5 C 2 40 15 44

19.5 C 3 40 16 44

19.5 C 4 41 17 44

19.5 C 5 41 18 44

19.5 C 6 41 19 44

19.5 C 7 41 20 45

19.5 C 8 41 21 47

19.5 C 9 42 22 47

19.5 C 10 42 23 56

19.5 C 11 42 24 62

19.5 C 1 2 43 25

--

19.5 C 1 3 43

19.5 D 1 40 14 44

19.5 D 2 40 15 44

19.5 D 3 41 16 44

19.5 D 4 41 17 44

19.5 D 5 41 18 44

19.5 D 6 41 19 44

19.5 D 7 42 20 46

19.5 D 8 42 21 46

19.5 D 9 42 22 62

19.5 D 10 42 23

--

19.5 D 11 42 24

--

19.5 D 1 2 43 2 5

--

19.5 D 1 3 44

38.0 A 1 38 14 42

38.0 A 2 40 15 42

38.0 A 3 40 16 43

38.0 A 4 41 17 43

38.0 A 5 41 18 43

38.0 A 6 41 19 43

38.0 A 7 41 20 43

38.0 A 8 42 21 44

38.0 A 9 42 22 45

38.0 A 10 42 23 45

38.0 A 11 42 24 46

38.0 A 1 2 42 25

--

38. 0 A 1 3 42



### ‘

-- ’ denotes a mortality that occurred before NF stage 62

.

### ABC Study No. 66086

Page 199 of 261 Appendix H-2. Replicate Data for Number of Days to NF Stage 62 (continued)

Treatment

(μg/L) Replicate Tadpole# Study Day NF 62

Achieved Tadpole# Study Day NF 62

Achieved

38.0 B 1 40 14 45

38.0 B 2 40 15 45

38.0 B 3 40 16 45

38.0 B 4 41 17 46

38.0 B 5 42 18 47

38.0 B 6 42 19 47

38.0 B 7 43 20 48

38.0 B 8 43 21 52

38.0 B 9 43 22

--

38.0 B 10 44 23

--

38.0 B 11 44 24

--

38.0 B 1 2 44 25

--

38.0 B 1 3 44

38.0 C 1 42 14 45

38 .0 C 2 42 15 45

38.0 C 3 42 16 45

38.0 C 4 42 17 45

38.0 C 5 43 18 45

38.0 C 6 43 19 46

38.0 C 7 43 20 46

38.0 C 8 43 21 46

38.0 C 9 43 22 47

38.0 C 10 44 23 47

38.0 C 11 44 24 48

38.0 C 1 2 44 25

--

38.0 C 1 3 44

38.0 D 1 41 14 44

38.0 D 2 41 15 44

38.0 D 3 42 16 44

38.0 D 4 42 17 44

38.0 D 5 42 18 45

38.0 D 6 42 19 45

38.0 D 7 42 20 46

38.0 D 8 43 21 46

38.0 D 9 43 22 46

38.0 D 10 43 23 48

38.0 D 11 43 24 50

38.0 D 1 2 43 25

--

38.0 D 1 3 44



### ‘

-- ’ denotes a mortality that occurred before NF stage 62

.

### ABC Study No. 66086

Page 200 of 261 APPENDIX I – NF STAGE 66 CULL DAT A

### ABC Study No. 66086

Page 201 of 261 Appendix I -1. Replicate Weight, Length, and Phenotypic Sex D ata at NF Stage 66 Cull

Treatment

(μg/L) Replicate Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phen otypic Sex



### 0 (Control) A CA1

- 66 4.825 33 Female



### 0 (Control) A CA2

- 66 3.683 30 Female



### 0 (Control) A CA3

- 66 2.575 28 Female



### 0 (Control) A CA4

- 66 3.870 31 Male



### 0 (Control) A CA5

- 66 2.603 27 Male



### 0 (Control) A CA6

- 66 1.906 26 Male



### 0 (Control) A CA7

- 66 2 .732 27 Female



### 0 (Control) A CA8

- 66 2.367 27 Female



### 0 (Control) A CA9

- 66 4.532 31 Female



### 0 (Control) A CA10

- 66 1.184 23 Indeterminate



### 0 (Control) B CB1

- 66 4.877 32 Female



### 0 (Control) B CB2

- 66 4.470 33 Female



### 0 (Control) B CB3

- 66 2.224 25 Male



### 0 (Con trol) B CB4

- 66 3.747 30 Male



### 0 (Control) B CB5

- 66 4.230 31 Male



### 0 (Control) B CB6

- 66 3.405 29 Female



### 0 (Control) B CB7

- 66 3.499 28 Male



### 0 (Control) B CB8

- 66 4.258 31 Male



### 0 (Control) B CB9

- 66

-- -- -- 

### 0 (Control) B CB10

- 66

-- -- -- 

### 0 (Control) C CC1

- 66 2.939 28 Male



### 0 (Control) C CC2

- 66 4.678 31 Male



### 0 (Control) C CC3

- 66 4.251 31 Female



### 0 (Control) C CC4

- 66 4.396 32 Female



### 0 (Control) C CC5

- 66 3.785 31 Male



### 0 (Control) C CC6

- 66 3.485 30 Male



### 0 (Control) C CC7

- 66 5.243 35 Female



### 0 (Control) C C C8

- 66 3.704 30 Male



### 0 (Control) C CC9

- 66

-- -- -- 

### 0 (Control) C CC10

- 66

-- -- -- 

### 0 (Control) D CD1

- 66 5.086 33 Female



### 0 (Control) D CD2

- 66 3.571 31 Female



### 0 (Control) D CD3

- 66 2.085 26 Male



### 0 (Control) D CD4

- 66 5.216 33 Male



### 0 (Control) D CD5

- 66 2.5 32 26 Female



### 0 (Control) D CD6

- 66 2.928 28 Male



### 0 (Control) D CD7

- 66 4.140 32 Male



### 0 (Control) D CD8

- 66

-- -- -- 

### 0 (Control) D CD9

- 66

-- -- -- 

### 0 (Control) D CD10

- 66

-- -- --



### ‘

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

.

### ABC Study No. 66086

Page 202 of 261 Appendix I -1. Rep licate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull

(continued)

Treatment

(μg/L) Replicate Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex



### 0 (Control) E CE1

- 66 4.276 32 Male



### 0 (Control) E CE2

- 66 3.735 30 Male



### 0 (Control ) E CE3

- 66 3.215 29 Female



### 0 (Control) E CE4

- 66 3.705 30 Male



### 0 (Control) E CE5

- 66 3.498 30 Male



### 0 (Control) E CE6

- 66 3.956 31 Female



### 0 (Control) E CE7

- 66 2.628 26 Female



### 0 (Control) E CE8

- 66 3.382 29 Male



### 0 (Control) E CE9

- 66 3.820 30 Female



### 0 (Con trol) E CE10

- 66 4.722 37 Male



### 0 (Control) F CF1

- 66 4.158 30 Male



### 0 (Control) F CF2

- 66 3.793 31 Male



### 0 (Control) F CF3

- 66 3.250 29 Female



### 0 (Control) F CF4

- 66 3.777 30 Female



### 0 (Control) F CF5

- 66 2.979 28 Male



### 0 (Control) F CF6

- 66 4.925 33 Female



### 0 ( Control) F CF7

- 66 4.224 31 Female



### 0 (Control) F CF8

- 66

-- -- -- 

### 0 (Control) F CF9

- 66

-- -- -- 

### 0 (Control) F CF10

- 66

-- -- -- 

### 0 (Control) G CG1

- 66 3.831 31 Female



### 0 (Control) G CG2

- 66 3.774 30 Male



### 0 (Control) G CG3

- 66 4.012 31 I ndeterminate



### 0 (Contr ol) G CG4

- 66 4.101 31 Female



### 0 (Control) G CG5

- 66 3.455 29 Female



### 0 (Control) G CG6

- 66 3.248 30 Female



### 0 (Control) G CG7

- 66 5.591 35 Female



### 0 (Control) G CG8

- 66 2.370 27 Female



### 0 (Control) G CG9

- 66

-- -- -- 

### 0 (Control) G CG10

- 66

-- -- -- 

### 0 (Control) H CH1

- 66 3.312 29 Male



### 0 (Control) H CH2

- 66 3.574 31 Male



### 0 (Control) H CH3

- 66 3.711 30 Male



### 0 (Control) H CH4

- 66 2.932 29 Female



### 0 (Control) H CH5

- 66 4.987 33 Female



### 0 (Control) H CH6

- 66 3.607 29 Female



### 0 (Control) H CH7

- 66 4.164 31 Female



### 0 (Cont rol) H CH8

- 66

-- -- -- 

### 0 (Control) H CH9

- 66

-- -- --



### ‘

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

.

### ABC Study No. 66086

Page 203 of 261 Appendix I -1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull

(continued)

Treatment

(μg/L) Replicate Juvenile

Code Weig ht (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex



### Control H CH10

- 66

-- -- --

4.97 A L1A1

- 66 5.936 35 Female

4.97 A L1A2

- 66 2.836 27 Indeterminate

4.97 A L1A3

- 66 4.133 32 Female

4.97 A L1A4

- 66 4.756 33 Female

4.97 A L1A5

- 66 4.522 33 Male

4.97 A L1A6

- 66 4.051 32 Female

4.97 A L1A7

- 66

-- -- --

4.97 A L1A8

- 66

-- -- --

4.97 A L1A9

- 66

-- -- --

4.97 A L1A10

- 66

-- -- --

4.97 B L1B1

- 66 3.922 31 Female

4.97 B L1B2

- 66 4.153 31 Male

4.97 B L1B3

- 66 4.327 33 Female

4.97 B L1B4

- 66

-- -- --

4.97 B L1B5

- 66

-- -- --

4.97 B L1B6

- 66

-- -- --

4.97 B L1B7

- 66

-- -- --

4.97 B L1B8

- 66

-- -- --

4.97 B L1B9

- 66

-- -- --

4.97 B L1B10

- 66

-- -- --

4.97 C L1C1

- 66 4.728 33 Female

4.97 C L1C2

- 66 3.790 31 Male

4.97 C L1C3

- 66 5.706 35 Female

4.97 C L1C4

- 66 3.502 31 Femal e

4.97 C L1C5

- 66 3.603 30 Female

4.97 C L1C6

- 66 3.600 30 Male

4.97 C L1C7

- 66 2.325 27 Male

4.97 C L1C8

- 66 1.937 25 Male

4.97 C L1C9

- 66 4.140 32 Male

4.97 C L1C10

- 66

-- -- --

4.97 D L1D1

- 66 5.251 34 Female

4.97 D L1D2

- 66 5.422 34 Male

4.97 D L1D3

- 6 6 3.722 30 Female

4.97 D L1D4

- 66 2.483 28 Female

4.97 D L1D5

- 66 3.263 29 Male

4.97 D L1D6

- 66 3.062 30 Male

4.97 D L1D7

- 66

-- -- --

4.97 D L1D8

- 66

-- -- --



### ‘

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

.

### ABC Study No. 66086

Page 204 of 261 Appendix I -1. Replicate Weight, Len gth, and Phenotypic Sex Data at NF Stage 66 Cull

(continued)

Treatment

(μg/L) Replicate Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex

4.97 D L1D9

- 66

-- -- --

4.97 D L1D10

- 66

-- -- --

10.3 A L2A1

- 66 2.391 24 Female

10.3 A L2A2

- 66 3.6 23 31 Female

10.3 A L2A3

- 66 3.247 29 Female

10.3 A L2A4

- 66 4.45 6 32 Male

10.3 A L2A5

- 66 3.841 30 Female

10.3 A L2A6

- 66

-- -- --

10.3 A L2A7

- 66

-- -- --

10.3 A L2A8

- 66

-- -- --

10.3 A L2A9

- 66

-- -- --

10.3 A L2A10

- 66

-- -- --

10.3 B L2B1

- 66 2.891 2 8 Female

10.3 B L2B2

- 66 4.952 34 Male

10.3 B L2B3

- 66 3.594 31 Female

10.3 B L2B4

- 66 2.465 28 Female

10.3 B L2B5

- 66

-- -- --

10.3 B L2B6

- 66

-- -- --

10.3 B L2B7

- 66

-- -- --

10.3 B L2B8

- 66

-- -- --

10.3 B L2B9

- 66

-- -- --

10.3 B L2B10

- 66

-- -- --

1 0.3 C L2C1

- 66 4.636 32 Female

10.3 C L2C2

- 66 1.434 23 Female

10.3 C L2C3

- 66 5.909 35 Female

10.3 C L2C4

- 66 2.877 29 Female

10.3 C L2C5

- 66 4.139 37 Female

10.3 C L2C6

- 66 3.511 31 Female

10.3 C L2C7

- 66 1.674 29 Male

10.3 C L2C8

- 66

-- -- --

10.3 C L2C 9

- 66

-- -- --

10.3 C L2C10

- 66

-- -- --

10.3 D L2D1

- 66 1.029 21 Female

10.3 D L2D2

- 66 4.449 32 Male

10.3 D L2D3

- 66 4.873 34 Male

10.3 D L2D4

- 66 3.695 32 Male

10.3 D L2D5

- 66 3.949 31 Male

10.3 D L2D6

- 66 2.037 26 Female

10.3 D L2D7

- 66 3.705 31 Male

1 0.3 D L2D8

- 66 2.956 28 Female



### ‘

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

.

### ABC Study No. 66086

Page 205 of 261 Appendix I -1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull

(continued)

Treatment

(μg/L) Replicate Juvenile

Code Weight (g) Snout

- to

- Vent

Len gth (mm) Phenotypic Sex

10.3 D L2D9

- 66

-- -- --

10.3 D L2D10

- 66

-- -- --

19.5 A L3A1

- 66 4.102 33 Female

19.5 A L3A2

- 66 3.184 30 Male

19.5 A L3A3

- 66 5.172 35 Male

19.5 A L3A4

- 66 4.020 32 Female

19.5 A L3A5

- 66 3.918 31 Female

19.5 A L3A6

- 66 4.104 32 Male

19.5 A L3A7

- 66 3.244 30 Male

19.5 A L3A8

- 66 2.583 27 Male

19.5 A L3A9

- 66 2.243 26 Female

19.5 A L3A10

- 66

-- -- --

19.5 B L3B1

- 66 4.074 33 Male

19.5 B L3B2

- 66 4.525 32 Female

19.5 B L3B3

- 66 4.479 33 Male

19.5 B L3B4

- 66 6.279 36 Female

19.5 B L 3B5

- 66 2.902 28 Male

19.5 B L3B6

- 66 3.375 30 Female

19.5 B L3B7

- 66 4.588 32 Female

19.5 B L3B8

- 66 4.815 35 Male

19.5 B L3B9

- 66 1.630 24 Male

19.5 B L3B10

- 66

-- -- --

19.5 C L3C1

- 66 5.394 35 Male

19.5 C L3C2

- 66 4.531 31 Female

19.5 C L3C3

- 66 6.042 3 3 Female

19.5 C L3C4

- 66 1.862 24 Male

19.5 C L3C5

- 66 1.107 22 Female

19.5 C L3C6

- 66 3.691 29 Female

19.5 C L3C7

- 66 1.088 20 Male

19.5 C L3C8

- 66 2.092 24 Male

19.5 C L3C9

- 66 3.716 30 Male

19.5 C L3C10

- 66

-- -- --

19.5 D L3D1

- 66 3.676 30 Female

19.5 D L3D2

- 66 4.297 32 Female

19.5 D L3D3

- 66 5.333 34 Female

19.5 D L3D4

- 66 3.681 29 Male

19.5 D L3D5

- 66 2.775 27 Male

19.5 D L3D6

- 66 2.265 26 Male

19.5 D L3D7

- 66

-- -- --

19.5 D L3D8

- 66

-- -- --



### ‘

-- ’ denotes a juvenile was not available for this samp le

.

### ABC Study No. 66086

Page 206 of 261 Appendix I -1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull

(continued)

Treatment

(μg/L) Replicate Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex

19.5 D L3D9

- 66

-- -- --

19.5 D L3D10

- 66

-- -- --

38.0 A L4A 1

- 66 3.122 30 Female

38.0 A L4A2

- 66 3.494 29 Male

38.0 A L4A3

- 66 2.903 28 Male

38.0 A L4A4

- 66 3.659 30 Female

38.0 A L4A5

- 66 5.013 32 Male

38.0 A L4A6

- 66 1.786 23 Male

38.0 A L4A7

- 66 4.305 32 Female

38.0 A L4A8

- 66 4.010 31 Male

38.0 A L4A9

- 66 3.117 29 Female

38.0 A L4A10

- 66

-- -- --

38.0 B L4B1

- 66 4.934 32 Female

38.0 B L4B2

- 66 3.944 29 Male

38.0 B L4B3

- 66 4.785 33 Female

38.0 B L4B4

- 66 4.189 31 Female

38.0 B L4B5

- 66 3.484 30 Female

38.0 B L4B6

- 66 3.880 31 Male

38.0 B L4B7

- 66

-- -- --

38.0 B L4B8

- 66

-- -- --

38.0 B L4B9

- 66

-- -- --

38.0 B L4B10

- 66

-- -- --

38.0 C L4C1

- 66 5.118 34 Male

38.0 C L4C2

- 66 2.920 29 Female

38.0 C L4C3

- 66 1.725 24 Female

38.0 C L4C4

- 66 3.153 30 Male

38.0 C L4C5

- 66 4.329 31 Female

38.0 C L4C6

- 66 4.031 31 Femal e

38.0 C L4C7

- 66 3.670 29 Female

38.0 C L4C8

- 66 4.515 32 Female

38.0 C L4C9

- 66 2.928 29 Female

38.0 C L4C10

- 66

-- -- --

38.0 D L4D1

- 66 3.398 29 Female

38.0 D L4D2

- 66 2.433 26 Male

38.0 D L4D3

- 66 4.041 30 Female

38.0 D L4D4

- 66 3.484 30 Female

38.0 D L4D5

- 66 2.993 29 Male

38.0 D L4D6

- 66 3.722 30 Male

38.0 D L4D7

- 66 1.445 23 Male



### ‘

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

.

### ABC Study No. 66086

Page 207 of 261 Appendix I -1. Replicate Weight, Length, and Phenotypic Sex Data at NF Stage 66 Cull

(continued)

Treatment

(μg/L ) Replicate Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex

38.0 D L4D8

- 66 3.760 29 Female

38.0 D L4D9

- 66

-- -- --

38.0 D L4D10

- 66

-- -- --



### ‘

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

.

### ABC Study No. 66086

Page 208 of 261 APPENDIX J – TERMINATION DATA

– GROWTH AND GENDER

### ABC Study No. 66086

Page 209 of 261 Appendix J-1. Replicate Weight, Length, and Sex Data at Termination

Treatment

(μg/L) R EP Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex G enotypic Sex



### 0 (Control) A CA1

- T 8.369 40 M M

0 (Control) A CA2

- T 10.216 40 F F

0 (Control) A CA3

- T 8.302 39 M M

0 (Control) A CA4

- T 6.430 36 M M

0 (Control) A CA5

- T 14.852 46 F F

0 (Control) A CA6

- T 12.185 45 F F

0 (Control) A CA7

- T 12.724 46 M M

0 (Control) A CA8

- T 7.641 38 F F

0 (Control) A C A9

- T 10.427 41 M M

0 (Control) A CA10

- T 8.250 37 M M

0 (Control) B CB1

- T 8.112 39 M M

0 (Control) B CB2

- T 10.430 42 F F

0 (Control) B CB3

- T 15.727 51 F F

0 (Control) B CB4

- T 6.800 36 M M

0 (Control) B CB5

- T 10.295 42 F F

0 (Control) B CB6

- T 14.757 4 4 M M

0 (Control) B CB7

- T 7.918 40 M M

0 (Control) B CB8

- T 9.079 40 M M

0 (Control) B CB9

- T 10.543 42 F F

0 (Control) B CB10

- T 13.247 47 F F

0 (Control) C CC1

- T 9.904 41 M M

0 (Control) C CC2

- T 9.811 40 M M

0 (Control) C CC3

- T 9.579 41 M M

0 (Contr ol) C CC4

- T 12.010 45 F F

0 (Control) C CC5

- T 16.785 48 M M

0 (Control) C CC6

- T 8.968 40 F F

0 (Control) C CC7

- T 5.727 36 F F

0 (Control) C CC8

- T 7.786 37 F F

0 (Control) C CC9

- T 10.468 41 M M

0 (Control) C CC10

- T

-- -- -- -- 0 (Control) D CD1

- T 11. 268 42 F F

0 (Control) D CD2

- T 11.920 42 F F

0 (Control) D CD3

- T 5.464 35 M M

0 (Control) D CD4

- T 7.899 37 M M

0 (Control) D CD5

- T 11.617 44 M M

0 (Control) D CD6

- T 7.410 39 M M

0 (Control) D CD7

- T 12.015 45 M M

0 (Control) D CD8

- T 11.805 43 F F

0 (Control) D CD9

- T 11.789 42 M M

“

-- ” denotes a juve nile was not available for this sample.

### ABC Study No. 66086

Page 210 of 261 Appendix J -1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μg/L) REP Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypi c Sex G enotypic Sex



### 0 (Control) D CD10

- T

-- -- -- -- 0 (Control) E CE1

- T 8.297 40 F F

0 (Control) E CE2

- T 11.321 45 M M

0 (Control) E CE3

- T 14.420 47 M M

0 (Control) E CE4

- T 12.117 44 M M

0 (Control) E CE5

- T 6.819 40 M M

0 (Control) E CE6

- T 12.106 47 M M

0 (Control) E CE7

- T 6.290 38 M F

0 (Control) E CE8

- T 8.824 39 M M

0 (Control) E CE9

- T 8.036 37 F F

0 (Control) E CE10

- T 10.742 43 F F

0 (Control) F CF1

- T 8.240 38 F F

0 (Control) F CF2

- T 9.185 41 F F

0 (Control) F CF3

- T 10.020 41 M M

0 (Cont rol) F CF4

- T 18.080 50 F F

0 (Control) F CF5

- T 14.757 47 M M

0 (Control) F CF6

- T 5.412 35 F F

0 (Control) F CF7

- T 14.354 49 F F

0 (Control) F CF8

- T 12.040 46 M M

0 (Control) F CF9

- T 9.425 43 M M

0 (Control) F CF10

- T 10.380 42 F F

0 (Control) G CG1

- T 7.486 38 M M

0 (Control) G CG2

- T 8.760 41 F F

0 (Control) G CG3

- T 7.158 37 M M

0 (Control) G CG4

- T 15.598 47 F F

0 (Control) G CG5

- T 9.373 41 M M

0 (Control) G CG6

- T 13.189 46 M F

0 (Control) G CG7

- T 11.110 43 F F

0 (Control) G CG8

- T 8.870 38 M F

0 (Control) G CG9

- T 12.122 43 M F

0 (Control) G CG10

- T 13.357 47 F F

0 (Control) H CH1

- T 13.053 48 M M

0 (Control) H CH2

- T 8.177 41 M M

0 (Control) H CH3

- T 9.542 42 F F

0 (Control) H CH4

- T 11.280 44 M M

0 (Control) H CH5

- T 10.628 44 F F

0 (Control) H CH6

- T 11.007 42 M M

0 (Control) H CH7

- T 14.608 47 F F

0 (Control) H CH8

- T 10.807 45 M M

“

-- ” denotes a juve nile was not available for this sample.

### ABC Study No. 66086

Page 211 of 261 Appendix J -1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μg/L) R EP Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex G enotypic Sex



### 0 (Control) H CH9

- T 8.495 39 M M

0 (Control) H CH10

- T 7.517 39 F F

4.97 A L1A1

- T 11.733 46 F F

4.97 A L1A2

- T 12.736 46 M M

4.97 A L1A3

- T 10.561 43 F M

4.97 A L1A4

- T 10.1 94 40 M M

4.97 A L1A5

- T 7.088 38 F F

4.97 A L1A6

- T 7.677 35 M M

4.97 A L1A7

- T 16.235 49 F F

4.97 A L1A8

- T 9.815 41 F F

4.97 A L1A9

- T 9.865 40 M M

4.97 A L1A10

- T 7.881 38 F F

4.97 B L1B1

- T 7.763 40 F F

4.97 B L1B2

- T 18.255 50 F F

4.97 B L1B3

- T 11.1 19 42 M M

4.97 B L1B4

- T 12.503 46 M M

4.97 B L1B5

- T 7.117 36 M M

4.97 B L1B6

- T 13.321 47 F F

4.97 B L1B7

- T 12.234 45 F F

4.97 B L1B8

- T 12.515 46 M M

4.97 B L1B9

- T 7.827 39 F F

4.97 B L1B10

- T 4.262 31 M F

4.97 C L1C1

- T 11.386 43 M M

4.97 C L1C2

- T 1 6.404 50 F M

4.97 C L1C3

- T 11.801 45 M M

4.97 C L1C4

- T 9.136 40 F M

4.97 C L1C5

- T 11.392 43 F M

4.97 C L1C6

- T 11.827 42 M F

4.97 C L1C7

- T 9.459 38 F F

4.97 C L1C8

- T 5.973 35 M M

4.97 C L1C9

- T 12.992 48 F F

4.97 C L1C10

- T 14.448 49 F F

4.97 D L1D1

- T 7.404 39 M F

4.97 D L1D2

- T 10.226 42 F M

4.97 D L1D3

- T 10.794 41 M M

4.97 D L1D4

- T 11.822 45 F M

4.97 D L1D5

- T 9.087 40 M M

4.97 D L1D6

- T 12.459 46 F F

4.97 D L1D7 -T 12.368 43 M M

### ABC Study No. 66086

Page 212 of 261 Appendix J -1. Replicate Weight, Length, and Sex Data at Terminatio n (continued)

Treatment

(μg/L) REP Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex G enotypic Sex



### 4.97 D L1D8

- T 9.078 40 F M

4.97 D L1D9

- T 11.091 41 M M

4.97 D L1D10

- T

-- -- -- --

10.3 A L2A1

- T 9.106 40 F F

10.3 A L2A2

- T 9.127 41 M M

10.3 A L2A3

- T 5.848 35 M M

10.3 A L2A4

- T 17.443 51 M M

10.3 A L2A5

- T 11.278 43 M F

10.3 A L2A6

- T 9.096 39 M M

10.3 A L2A7

- T 12.782 45 M F

10.3 A L2A8

- T 14.696 47 F M

10.3 A L2A9

- T 10.092 43 M M

10.3 A L2A10

- T 11.354 46 M M

10.3 B L2B1

- T 10.951 4 3 M M

10.3 B L2B2

- T 8.388 43 F F

10.3 B L2B3

- T 9.853 43 F F

10.3 B L2B4

- T 12.262 47 M M

10.3 B L2B5

- T 13. T 7 45 M M

10.3 B L2B6

- T 6.624 37 F M

10.3 B L2B7

- T 10.513 43 F M

10.3 B L2B8

- T 10.370 43 M M

10.3 B L2B9

- T 7.502 39 M F

10.3 B L2B10

- T 7.792 3 8 M M

10.3 C L2C1

- T 13.906 46 F F

10.3 C L2C2

- T 9.497 40 M F

10.3 C L2C3

- T 14.139 57 F M

10.3 C L2C4

- T 11.173 44 M M

10.3 C L2C5

- T 7.121 39 M M

10.3 C L2C6

- T 10.769 45 M M

10.3 C L2C7

- T 10.954 47 F M

10.3 C L2C8

- T 11.449 44 M F

10.3 C L2C9

- T 8.443 42 M F

10.3 C L2C10

- T 6.585 39 F F

10.3 D L2D1

- T 12.535 50 F M

10.3 D L2D2

- T 10.945 41 M M

10.3 D L2D3

- T 12.673 47 F F

10.3 D L2D4

- T 9.825 41 F M

10.3 D L2D5

- T 8.601 41 F F

10.3 D L2D6

- T 7.026 40 F F

“

-- ” denotes a juve nile was not available for this sample.

### ABC Study No. 66086

Page 213 of 261 Appendix J -1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μg/L) R EP Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex G enotypic Sex



### 10.3 D L2D7

- T 9.989 42 F F

10.3 D L2D8

- T 9.975 42 F F

10 .3 D L2D9

- T 10.318 42 F F

10.3 D L2D10

- T 10.181 42 M M

19.5 A L3A1

- T 10.841 41 F M

19.5 A L3A2

- T 3.779 32 F M

19.5 A L3A3

- T 4.115 31 F M

19.5 A L3A4

- T 11.123 43 M F

19.5 A L3A5

- T 9.906 44 M F

19.5 A L3A6

- T 12.187 46 M M

19.5 A L3A7

- T 12.967 42 M F

19.5 A L3A8

- T 9.545 43 F F

19.5 A L3A9

- T 7.236 38 F F

19.5 A L3A10

- T 11.781 47 F F

19.5 B L3B1

- T 13.369 48 F F

19.5 B L3B2

- T 7.435 41 F M

19.5 B L3B3

- T 6.286 39 M F

19.5 B L3B4

- T 14.964 47 M F

19.5 B L3B5

- T 9.696 41 M F

19.5 B L3B6

- T 9.339 42 M M

19.5 B L3B7

- T 5.438 37 F F

19.5 B L3B8

- T 13.995 49 M M

19.5 B L3B9

- T 3.271 31 F M

19.5 B L3B10

- T 11.995 46 M M

19.5 C L3C1

- T 2.082 38 F F

19.5 C L3C2

- T 7.530 38 M M

19.5 C L3C3

- T 15.295 52 M M

19.5 C L3C4

- T 7.804 40 M F

19.5 C L3C5

- T 10.270 41 F M

19.5 C L3C6

- T 14.036 48 F F

19.5 C L3C7

- T 14.363 50 M F

19.5 C L3C8

- T 8.691 41 M M

19.5 C L3C9

- T 9.788 42 M F

19.5 C L3C10

- T 12.231 46 M F

19.5 D L3D1

- T 12.929 45 F F

19.5 D L3D2

- T 13.544 49 F F

19.5 D L3D3

- T 9.972 44 M M

19.5 D L3D4

- T 10.472 43 M M

19.5 D L3D5

- T 12.265 43 F F

### ABC Study No. 66086

Page 214 of 261 Appendix J -1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μg/L) R EP Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex G enotypic Sex



### 19.5 D L3D6 -T 8.619 41 M M

19.5 D L3D 7

- T 12.841 47 F F

19.5 D L3D8

- T 7.525 40 M M

19.5 D L3D9

- T 12.391 47 M M

19.5 D L3D10

- T 11.365 45 F F

38.0 A L4A1

- T 10.222 40 M M

38.0 A L4A2

- T 15.497 48 F F

38.0 A L4A3

- T 19.538 55 F F

38.0 A L4A4

- T 8.486 42 M M

38.0 A L4A5

- T 9.025 41 M M

38.0 A L4A6

- T 7.493 42 M M

38.0 A L4A7

- T 14.821 48 M M

38.0 A L4A8

- T 10.758 43 F F

38.0 A L4A9

- T 6.041 37 M M

38.0 A L4A10

- T 6.449 37 F F

38.0 B L4B1

- T 10.147 41 M M

38.0 B L4B2

- T 9.870 42 M M

38.0 B L4B3

- T 14.339 46 M M

38.0 B L4B4

- T 14.331 48 F F

38.0 B L4B5

- T 12.778 46 M M

38.0 B L4B6

- T 6.179 42 F F

38.0 B L4B7

- T 11.442 45 F F

38.0 B L4B8

- T 8.545 41 F F

38.0 B L4B9

- T 12.476 44 M M

38.0 B L4B10

- T 5.477 31 M M

38.0 C L4C1

- T 8.232 38 F F

38.0 C L4C2

- T 6.759 36 M M

38.0 C L4C3

- T 11.379 44 M M

38.0 C L4C4

- T 10.723 42 M M

38.0 C L4C5

- T 13.129 48 M M

38.0 C L4C6

- T 6.736 38 F F

38.0 C L4C7

- T 19.080 50 M M

38.0 C L4C8

- T 11.828 45 M M

38.0 C L4C9

- T 6.916 35 F M

38.0 C L4C10

- T 6.906 37 F F

38.0 D L4D1

- T 9.463 43 F M

38.0 D L4D2

- T 10.567 44 M M

38 .0 D L4D3

- T 16.987 51 F F

38.0 D L4D4

- T 13.163 47 F F

### ABC Study No. 66086

Page 215 of 261 Appendix J -1. Replicate Weight, Length, and Sex Data at Termination (continued)

Treatment

(μg/L) R EP Juvenile

Code Weight (g) Snout

- to

- Vent

Length (mm) Phenotypic Sex G enotypic Sex



### 38.0 D L4D5

- T 1 0.850 44 F F

38.0 D L4D6

- T 9.537 37 M M

38.0 D L4D7

- T 11.891 42 F F

38.0 D L4D8

- T 7.040 38 M M

38.0 D L4D9

- T 8.596 42 M M

38.0 D L4D10

- T 13.680 48 F F

### ABC Study No. 66086

Page 216 of 261 APPENDIX K – TERMINATION DATA

– NUPTIAL PAD AND LIVE R WEIGHT

### ABC Study No. 66086

Page 217 of 261 Appendix K-1. Replicate Nuptial Pads and Liver Weight Data

Juvenile

Code Phenotypic

Sex G enotypic

Sex Nuptial Pads

(Present/Absent) Liver Cassette

Tare Weight

(g) Cassette +

Liver Weight

(g) Calculated

Liver Weight

(g)

CA1 -T M 

### M absent 3.392 3.8291 0.4371

CA2

- T F 

### F absent 3.3972 3.9102 0.5130

CA3 -T M 

### M absent 3.4099 4.0069 0.5970

CA4

- T M 

### M absent 3.3738 3.6852 0.3114

CA5

- T F 

### F present 3.401 0 4.3996 0.9986

CA6 -T F 

### F absent 3.3904 4.1986 0.8082

CA7

- T M 

### M present 3.4009 4.1249 0.7240

CA8 -T F 

### F absent 3.3963 3.8039 0.4076

CA9

- T M 

### M absent 3.3954 3.9459 0.5505

CA10

- T M 

### M absent 3.4019 3.8365 0.4346

CB1 -T M 

### M absent 3.4028 3.7974 0.3946

CB2

- T F 

### F absent 3.4051 4.0654 0. 66 03

CB3 -T F 

### F present 3.3836 4.2982 0.9146

CB4

- T M 

### M absent 3.3947 3.7300 0.3353

CB5

- T F 

### F absent 3.3986 3.9408 0.5422

CB6 -T M 

### M present 3.3989 4.4616 1.0627

CB7

- T M 

### M absent 3.3905 3.8233 0.4328

CB8 -T M 

### M absent 3.3956 3.9236 0.5280

CB9

- T F 

### F absent 3.3837 3.8952 0.5115

CB10

- T F 

### F present 3.3898 4.1645 0.7747

CC1 -T M 

### M absent 3.3940 3.9602 0. 5 66 2

CC2

- T M 

### M absent 3.4015 4.0012 0.5997

CC3 -T M 

### M present 3.3946 3.9041 0.5095

CC4

- T F 

### F absent 3.3995 4.3122 0.9127

CC5

- T M 

### M present 3.3907 4.4649 1.0742

CC6 -T F 

### F absent 3.3979 3.6305 0.2326

CC7

- T F 

### F absent 3.3940 4. 04 66 0.6526

CC8 -T F 

### F absent 3.4069 3.7947 0.3878

CC9

- T M 

### M absent 3.3838 3.9949 0.6111

CC10

- T

-- 

### -- -- -- -- --

CD1 -T F 

### F absent 3.4077 4.1896 0.7819

CD2

- T F 

### F absent 3.4013 4.2831 0.8818

CD3 -T M 

### M absent 3.4009 3.6417 0.2408

CD4

- T M 

### M absent 3.3972 3.9021 0.50 49

CD5

- T M 

### M absent 3.4000 4.2181 0.8181

CD6 -T M 

### M absent 3.4015 3.7779 0.3764

CD7

- T M 

### M absent 3.4014 4.2463 0.8449

CD8 -T F 

### F absent 3.3981 4.1062 0.7081

“

-- ” denotes a juve nile was not available for this sample.

### ABC Study No. 66086

Page 218 of 261 Appendix K-1. Replicate Nuptial Pad s and Liver Weight Data (continued)

Juvenile

Code Phenotypic

Sex G enotypic

Sex Nuptial Pads

(Present/Absent) Liver Cassette

Tare Weight

(g) Cassette +

Liver Weight

(g) Calculated

Liver Weight

(g)

CD9 -T M M absent 3.3998 4.1195 0.7197

CD10

- T

-- -- -- -- -- --

CE1 -T F 

### F absent 3.3971 3.8453 0.4482

CE2

- T M 

### M absent 3.4020 4.1695 0.7675

CE3

- T M 

### M present 3.3997 4.2736 0.8739

CE4 -T M 

### M absent 3.3733 4.2494 0.8761

CE5

- T M 

### M absent 3.4052 3.7816 0.3764

CE6 -T M 

### M absent 3.3917 4.2030 0.8113

CE7

- T M 

### F abs ent 3.3890 3.7088 0.3198

CE8

- T M 

### M absent 3.4015 3.9461 0.5446

CE9 -T F 

### F present 3.4097 3.8522 0.4425

CE10

- T F 

### F absent 3.3860 3.9249 0.5389

CF1 -T F 

### F absent 3.3854 3.8024 0.4170

CF2

- T F 

### F absent 3.3883 3.9478 0.5595

CF3

- T M 

### M present 3.3896 3.9454 0 .5558

CF4 -T F 

### F present 3.4001 4.5907 1.1906

CF5

- T M 

### M present 3.4090 4.2431 0.8341

CF6 -T F 

### F absent 3.3959 3.6573 0.2614

CF7

- T F 

### F present 3.4005 4.1791 0.7786

CF8

- T M 

### M present 3.4007 4.0775 0.6768

CF9 -T M 

### M absent 3.4009 3.9170 0.5161

CF10

- T F 

### F absent 3.4071 3.9838 0.5767

CG1 -T M 

### M absent 3.3485 3.7334 0.3849

CG2

- T F 

### F absent 3.3805 3.9223 0.5418

CG3

- T M 

### M absent 3.3954 3.7771 0.3817

CG4 -T F 

### F absent 3.3762 4.4591 1.0829

CG5

- T M 

### M absent 3.3782 3.9813 0.6031

CG6 -T M 

### F absent 3.3647 4.0931 0 .7284

CG7

- T F 

### F absent 3.3507 4.0327 0.6820

CG8

- T M 

### F absent 3.3449 3.8750 0.5301

CG9 -T M 

### F absent 3.3684 3.9886 0.6202

CG10

- T F 

### F absent 3.3492 4.1987 0.8495

CH1 -T M 

### M present 3.3814 4.0755 0.6941

CH2

- T M 

### M absent 3.3578 3.7730 0.4152

CH3

- T F 

### F abs ent 3.3800 3.9235 0.5435

CH4 -T M 

### M absent 3.3813 4.0287 0.6474

CH5

- T F 

### F absent 3.3645 3.9271 0.5626

CH6 -T M 

### M absent 3.3787 3.9833 0.6046

“

-- ” denotes a juve nile was not available for this sample.

### ABC Study No. 66086

Page 219 of 261 Appendix K-1. Replicate Nuptial Pads and Liver Weigh t Data (continued)

Juvenile

Code Phenotypic

Sex G enotypic

Sex Nuptial Pads

(Present/Absent) Liver Cassette

Tare Weight

(g) Cassette +

Liver Weight

(g) Calculated

Liver Weight

(g)

CH7 -T F 

### F present 3.3517 4.1361 0.7844

CH8 -T M 

### M absent 3.3505 3.8742 0.5 237

CH9 -T M 

### M absent 3.3673 3.9230 0.5557

CH10 -T F 

### F absent 3.3784 3.7718 0.3934

L1A1 -T F 

### F absent 3.3811 4.1898 0.8087

L1A2 -T M 

### M present 3.3761 4.0765 0.7004

L1A3 -T F 

### M present 3.3619 3.9902 0.6283

L1A4 -T M 

### M absent 3.3663 3.9858 0.6195

L1A5 -T F 

### F absent 3.3802 3.7608 0.3806

L1A6 -T M 

### M absent 3.3736 3.8356 0.4620

L1A7 -T F 

### F present 3.3457 4.3554 1.0097

L1A8 -T F 

### F absent 3.3644 3.8580 0.4936

L1A9 -T M 

### M absent 3.3674 4.0677 0.7003

L1A10 -T F 

### F absent 3.3682 3.8246 0.4564

L1B1 -T F 

### F absent 3.3690 3.8148 0.4458

L1B2 -T F 

### F present 3.3677 4.6705 1.3028

L1B3 -T M 

### M absent 3.3649 3.9960 0.6311

L1B4 -T M 

### M absent 3.3780 4.0267 0.6487

L1B5 -T M 

### M absent 3.3710 3.7808 0.4098

L1B6 -T F 

### F present 3.3676 4.1925 0.8249

L1B7 -T F 

### F present 3.3784 4.1232 0.744 8

L1B8 -T M 

### M absent 3.3673 4.0699 0.7026

L1B9 -T F 

### F absent 3.3662 3.8625 0.4963

L1B10 -T M 

### F present 3.3663 3.5730 0.2067

L1C1 -T M 

### M absent 3.3746 3.9639 0.5893

L1C2 -T F 

### M present 3.3684 4.3675 0.9991

L1C3 -T M 

### M present 3.3716 4.0283 0.6567

L1C4 -T F 

### M absent 3.3711 3.9570 0.5859

L1C5 -T F 

### M absent 3.3659 4.0204 0.6545

L1C6 -T M 

### F absent 3.3522 3.9878 0.6356

L1C7 -T F 

### F absent 3.3825 3.9272 0.5447

L1C8 -T M 

### M absent 3.3480 3.6558 0.3078

L1C9 -T F 

### F present 3.3765 4.1191 0.7426

L1C10 -T F 

### F present 3.34 87 4.2130 0.8643

L1D1 -T M 

### F absent 3.3675 3.8115 0.4440

L1D2 -T F 

### M present 3.3462 3.8721 0.5259

L1D3 -T M 

### M absent 3.3795 3.9676 0.5881

L1D4 -T F 

### M present 3.3625 4.0079 0.6454

L1D5 -T M 

### M absent 3.3510 3.7779 0.4269

### ABC Study No. 66086

Page 220 of 261 Appendix K-1. Replicate Nuptial Pad s and Liver Weight Data (continued)

Juvenile

Code Phenotypic

Sex G enotypic

Sex Nuptial Pads

(Present/Absent) Liver Cassette

Tare Weight

(g) Cassette +

Liver Weight

(g) Calculated

Liver Weight

(g)

L1D6 -T F 

### F present 3.3421 4.0248 0.6827

L1D7

- T M 

### M absen t 3.3637 4.1433 0.7796

L1D8 -T F 

### M absent 3.3664 3.9023 0.5359

L1D9

- T M 

### M absent 3.3457 3.9176 0.5719

L1D10

- T

-- 

### -- -- -- -- --

L2A1 -T F 

### F absent 3.3452 3.9344 0.5892

L2A2

- T M 

### M absent 3.3510 3.7650 0.4140

L2A3 -T M 

### M absent 3.3653 3.5798 0.2145

L2A4

- T M 

### M present 3.3668 4.3473 0.9805

L2A5

- T M 

### F present 3.3580 3.9712 0.6132

L2A6 -T M 

### M present 3.3460 3.8352 0.4892

L2A7

- T M 

### F present 3.3658 4.0871 0.7213

L2A8 -T F 

### M present 3.3580 4.2606 0.9026

L2A9

- T M 

### M present 3.3747 3.9487 0.5740

L2A10

- T M 

### M pre sent 3.3816 3.9908 0.6092

L2B1 -T M 

### M absent 3.3771 4.0713 0.6942

L2B2

- T F 

### F absent 3.3678 4.0185 0.6507

L2B3 -T F 

### F absent 3.3476 4.0093 0.6617

L2B4

- T M 

### M present 3.3778 4.2723 0.8945

L2B5

- T M 

### M absent 3.3657 4.3242 0.9585

L2B6 -T F 

### M absent 3.3651 3.7 719 0.4068

L2B7

- T F 

### M present 3.3660 3.9636 0.5976

L2B8 -T M 

### M present 3.3728 3.9576 0.5848

L2B9

- T M 

### F absent 3.3381 3.7940 0.4559

L2B10

- T M 

### M absent 3.3688 3.8967 0.5279

L2C1 -T F 

### F present 3.3467 4.1495 0.8028

L2C2

- T M 

### F present 3.3642 3.8974 0.5332

L2C3 -T F 

### M present 3.3690 4.2301 0.8611

L2C4

- T M 

### M absent 3.3781 4.0818 0.7037

L2C5

- T M 

### M absent 3.3442 3.7635 0.4193

L2C6 -T M 

### M present 3.3804 4.0129 0.6325

L2C7

- T F 

### M absent 3.3793 3.9450 0.5657

L2C8 -T M 

### F present 3.3677 4.0833 0.7156

L2C9

- T M 

### F a bsent 3.3705 3.7853 0.4148

L2C10

- T F 

### F absent 3.3501 3.7669 0.4168

L2D1 -T F 

### M present 3.3499 4.2718 0.9219

L2D2

- T M 

### M present 3.3519 4.0089 0.6570

L2D3 -T F 

### F present 3.3787 4.1412 0.7625

“

-- ” denotes a juve nile was not available for this sample.

### ABC Study No. 66086

Page 221 of 261 App endix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Juvenile

Code Phenotypic

Sex G enotypic

Sex Nuptial Pads

(Present/Absent) Liver Cassette

Tare Weight

(g) Cassette +

Liver Weight

(g) Calculated

Liver Weight

(g)

L2D4 -T F 

### M present 3.3691 3.9021 0.5330

L2D5 -T F 

### F absent 3.3668 3.8514 0.4846

L2D6 -T F 

### F absent 3.3447 3.7092 0.3645

L2D7 -T F 

### F absent 3.3680 3.9748 0.6068

L2D8 -T F 

### F absent 3.3765 4.0434 0.6669

L2D9 -T F 

### F absent 3.3710 4.0218 0.6508

L2D10 -T M 

### M absent 3.3739 3.9708 0.5969

L3A1 -T F 

### M absent 3.3849 4.1330 0.7481

L3A2 -T F 

### M absent 3.3703 3.5589 0.1886

L3A3 -T F 

### M absent 3.3650 3.5206 0.1556

L3A4 -T M 

### F absent 3.3662 4.0354 0.6692

L3A5 -T M 

### F absent 3.3465 3.9212 0.5747

L3A6 -T M 

### M absent 3.3468 4.1513 0.8045

L3A7 -T M 

### F abse nt 3.3787 4.2451 0.8664

L3A8 -T F 

### F absent 3.3758 4.0568 0.6810

L3A9 -T F 

### F absent 3.3512 3.7285 0.3773

L3A10 -T F 

### F absent 3.3829 4.2208 0.8379

L3B1 -T F 

### F present 3.3806 4.2384 0.8578

L3B2 -T F 

### M absent 3.3512 3.8535 0.5023

L3B3 -T M 

### F absent 3.3781 3.75 75 0.3794

L3B4 -T M 

### F present 3.3643 4.2884 0.9241

L3B5 -T M 

### F absent 3.3662 4.1090 0.7428

L3B6 -T M 

### M present 3.3794 3.9416 0.5622

L3B7 -T F 

### F absent 3.3712 3.7038 0.3326

L3B8 -T M 

### M absent 3.3612 4.2330 0.8718

L3B9 -T F 

### M absent 3.3736 3.5332 0.1596

L3B 10 -T M 

### M present 3.3692 4.1765 0.8073

L3C1 -T F 

### F NA 3.3841 3.4455 0.0614

L3C2 -T M 

### M absent 3.3706 3.7837 0.4131

L3C3 -T M 

### M present 3.3503 4.1797 0.8294

L3C4 -T M 

### F present 3.3568 3.7405 0.3837

L3C5 -T F 

### M absent 3.3509 3.9547 0.6038

L3C6 -T F 

### F present 3.3769 4.2026 0.8257

L3C7 -T M 

### F present 3.3676 4.2239 0.8563

L3C8 -T M 

### M absent 3.3626 3.8788 0.5162

L3C9 -T M 

### F absent 3.3424 4.0362 0.6938

L3C10 -T M 

### F absent 3.3665 4.0599 0.6934

L3D1 -T F 

### F absent 3.3669 4.2438 0.8769

L3D2 -T F 

### F present 3.3797 4.0986 0.7189

### ABC Study No. 66086

Page 222 of 261 Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Juvenile

Code Phenotypic

Sex G enotypic

Sex Nuptial Pads

(Present/Absent) Liver Cassette

Tare Weight

(g) Cassette +

Liver Weight

(g) Calculated

Liver Weight

(g)

L3D3 -T M 

### M ab sent 3.3499 3.9540 0.6041

L3D4 -T M 

### M present 3.3793 4.0085 0.6292

L3D5 -T F 

### F absent 3.3649 4.1129 0.7480

L3D6 -T M 

### M absent 3.3621 3.8835 0.5214

L3D7 -T F 

### F present 3.3789 3.9810 0.6021

L3D8 -T M 

### M absent 3.3606 3.9015 0.5409

L3D9 -T M 

### M present 3.3836 4 .1328 0.7492

L3D10 -T F 

### F absent 3.3384 4.0007 0.6623

L4A1 -T M 

### M absent 3.3710 3.9629 0.5919

L4A2 -T F 

### F present 3.3495 4.2880 0.9385

L4A3 -T F 

### F present 3.3414 4.5050 1.1636

L4A4 -T M 

### M absent 3.3709 3.8591 0.4882

L4A5 -T M 

### M absent 3.3533 3.9469 0.5936

L4A6 -T M 

### M absent 3.3669 3.7741 0.4072

L4A7 -T M 

### M present 3.3456 4.2056 0.8600

L4A8 -T F 

### F absent 3.3853 4.0539 0.6686

L4A9 -T M 

### M absent 3.3634 3.6160 0.2526

L4A10 -T F 

### F absent 3.3709 3.6733 0.3024

L4B1 -T M 

### M present 3.3819 3.9686 0.5867

L4B2 -T M 

### M a bsent 3.3770 3.9426 0.5656

L4B3 -T M 

### M present 3.3653 4.2644 0.8991

L4B4 -T F 

### F present 3.3535 4.2113 0.8578

L4B5 -T M 

### M present 3.3678 4.1542 0.7864

L4B6 -T F 

### F absent 3.3506 3.6840 0.3334

L4B7 -T F 

### F absent 3.3538 4.0102 0.6564

L4B8 -T F 

### F present 3.3705 3.7412 0.3707

L4B9 -T M 

### M present 3.3536 4.1148 0.7612

L4B10 -T M 

### M absent 3.3687 3.6622 0.2935

L4C1 -T F 

### F absent 3.3692 3.8264 0.4572

L4C2 -T M 

### M absent 3.3694 3.7519 0.3825

L4C3 -T M 

### M absent 3.3619 4.0489 0.6870

L4C4 -T M 

### M absent 3.3662 4.0275 0.6613

L4C5 -T M 

### M absent 3.3701 4.3592 0.9891

L4C6 -T F 

### F absent 3.3684 3.7574 0.3890

L4C7 -T M 

### M present 3.3511 4.5865 1.2354

L4C8 -T M 

### M present 3.3763 4.1720 0.7957

L4C9 -T F 

### M absent 3.3746 3.7426 0.3680

L4C10 -T F 

### F absent 3.3702 3.7528 0.3826

L4D1 -T F 

### M absent 3.3811 3.8079 0.4268

### ABC Study No. 66086

Page 223 of 261 Appendix K-1. Replicate Nuptial Pads and Liver Weight Data (continued)

Juvenile

Code Phenotypic

Sex G enotypic

Sex Nuptial Pads

(Present/Absent) Liver Cassette

Tare Weight

(g) Cassette +

Liver Weight

(g) Calculated

Liver Weight

(g)

L4D2 -T M 

### M absent 3.3481 4.0127 0.6646

L4D3 -T F 

### F present 3.3732 4.3717 0.9985

L4D4 -T F 

### F present 3.3792 4.0975 0.7183

L4D5 -T F 

### F absent 3.3710 4.0187 0.6477

L4D6 -T M 

### M absent 3.3466 3.9003 0.5537

L4D7 -T F 

### F present 3.3823 4.0349 0.6526

L4D8 -T M 

### M absent 3.3822 3.7380 0.3558

L4D9 -T M 

### M absent 3.3812 3.7980 0.4168

L4D10 -T F 

### F present 3.3730 4.1125 0.7395

### ABC Study No. 66086

Page 224 of 261 APPENDIX L – WATER QUALITY DATA

### ABC Study No. 66086

Page 225 of 261 Table L-1. Test Solution Temperature Measured in Each Replicate Test Chamber

Temperature °C

Control

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

0 21.2 21.0 21.1 21.1 21.1 20.9 20.9 20.9

6 20.9 20.7 20.8 20.8 20.9 20.7 20.8 20. 8

8 21.0 20.9 20.9 20.9 20.9 20.7 20.9 20.9

12 21.1 20.9 21.1 21.1 21.1 20.8 2 1.0 21.0

15 21.4 21.1 21.3 21.3 21.2 21.1 21.1 21.2

19 21.0 20.8 20.9 20.8 20.9 20.7 20.8 20.8

21 21.2 21.1 21.3 21.2 21.3 21.2 21.1 21.2

27 20.8 20.6 20.8 20.8 20.9 20.6 20.6 20.6

29 20.8 20.5 20.8 20.8 20.9 20.7 20.6 20.7

32 21.1 20.9 21.1 21.2 21. 2 20.9 20.9 20.9

34 20.8 20.6 20.8 20.8 20.8 20.6 20.6 20.7

36 20.9 20.6 20.9 20.8 20.9 20.7 20.7 20.7

39 20.9 20.8 20.9 21.0 21.0 20.9 20.7 20.7

41 20.8 20.6 21.1 21.1 21.0 21.0 21.1 21.1

43 21.2 20.9 21.5 21.5 21.5 20.9 20.9 20.9

46 21.6 20.7 20.9 21.0 21.5 20.8 20.6 20.7

50 20.6 20.6 20.7 20.8 20.8 20.7 20.5 20.5

64 21.6 21.5 21.6 21.6 21.8 21.5 21.3 21.4

67 20.7 20.1 20.8 20.3 20.6 20.4 20.1 20.1

71 21.1 21.0 21.2 21.0 21.2 21.0 20.9 21.0

75 21.3 21.2 21.2 21.2 21.3 21.3 21.1 21.1

78 21.4 21 .5 21.4 21.1 21.4 21.3 21.4 21.4

82 21.6 21.5 21.5 21.4 21.4 21.7 21.6 21.4

85 20.4 20.5 21.1 21.5 21.1 20.8 20.7 20.6

88 20.7 20.7 21.1 21.0 21.0 20.7 20.7 20.7

92 20.7 20.6 21.1 21.0 21.0 20.8 20.7 20.6

97 20.8 20.8 21.0 20.7 21.0 21.0 20.9 20.9

99 20.4 20.4 20.5 20.6 20.8 20.6 20.6 20.6

102 20.3 20.7 20.8 20.6 20.7 20.5 20.4 20.3

106 20.7 20.6 20.6 20.6 20.4 20.5 20.7 20.7

111 21.1 21.3 21.3 21.3 21.2 21.0 21.0 21.0

112 20.9

-- 20.7 20.8

-- -- -- 20.7

113

-- 20.9

-- -- 20.8 20.7 20.8

--

‘

-- ’ indicates measurement not collected

.

### ABC Study No. 66086

Page 226 of 261 Table L-1. Test Solution Temperature Measured in Each Replicate Test Chamber (continued)

Temperature °C

Level 1 ( 4.97 μg/L ) Level 2 ( 10.3 μg/L )

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

0 20.9 20.9 20.9 21.0 21.0 21.0 20.9 21.0

6 20.7 20.8 20.8 20.8 20.7 20.7 20.6 20.6

8 20.6 20.9 21.0 20.9 20.8 20.8 20.7 20.9

12 20.7 21.1 21.1 21.1 20.9 21.0 20.8 21.0

15 20.9 21.5 21.2 21.3 21.1 21.2 21.0 21.1

19 20.5 20.8 20.9 20.8 20.7 20.7 20.6 20.8

21 20. 9 21.3 21.4 21.2 21.2 21.2 21.0 21.1

27 20.4 20.8 20.2 20.8 20.6 20.8 20.6 20.7

29 20.4 20.8 20.7 20.8 20.6 20.7 20.5 20.5

32 20.9 21.3 21.0 21.1 21.0 21.1 20.9 21.0

34 20.4 21.1 20.7 20.8 20.6 20.7 20.5 20.6

36 20.4 21.1 20.8 20.8 20.7 20.7 20.5 20.6

39 20.6 20.9 20.9 20.9 20.8 20.8 20.6 20.7

41 20.8 21.2 21.2 21.2 21.0 21.0 20.6 20.9

43 20.7 21.4 21.2 21.5 20.9 21.5 21.0 21.4

46 20.8 20.7 20.9 20.9 20.8 20.8 20.6 20.9

50 20.4 20.7 20.8 20.7 20.7 20.7 20.4 20.5

64 21.2 21.7 21.5 21.6 21.4 21.4 2 1.4 21.7

67 20.4 20.5 20.6 20.5 20.5 20.4 20.2 20.5

71 21.1 21.3 21.3 20.5 21.2 21.1 20.9 21.1

75 21.0 21.2 21.2 21.3 21.5 21.2 21.1 21.4

78 21.4 21.5 21.1 21.3 21.3 21.2 21.7 21.4

82 21.4 21.5 21.2 21.5 21.4 21.5 21.5 21.7

85 20.3 20.9 20.8 21.0 20. 9 20.9 20.6 20.7

88 20.4 20.8 20.7 21.0 20.9 21.0 20.7 20.8

92 20.5 20.9 20.7 21.1 20.9 20.7 20.8 21.2

97 20.8 21.1 20.8 21.1 20.9 21.0 20.8 20.9

99 20.4 20.7 20.4 20.6 20.4 20.5 20.4 20.5

102 20.4 20.9 20.4 20.8 20.5 20.6 20.6 20.4

106 20.5 20.9 20. 5 20.8 20.6 20.6 20.5 20.7

111 20.8 21.5 21.2 21.4 21.3 21.1 21.0 21.1

112 20.5

-- -- 20.8

-- -- 20.6 20.9

113

-- 20.9 20.7

-- 20.7 20.7

-- --

‘

-- ’ indicates measurement not collected

.

### ABC Study No. 66086

Page 227 of 261 Table L-1. Test Solution Temperature Measured in Each Replicate Te st Chamber (continued)

Temperature °C

Level 3 ( 19.5 μg/L ) Level 4 ( 38.0 μg/L )

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

0 21.0 21.0 21.0 21.0 21.0 21.0 21.0 21.0

6 20.8 20.8 20.8 20.8 20.8 20.8 20.8 20.8

8 20.9 21.0 21.0 20.8 21.0 2 0.9 20.8 20.9

12 21.1 21.1 21.1 21.1 21.0 21.1 21.0 20.9

15 21.3 21.3 21.4 21.1 21.6 21.2 21.2 21.3

19 20.8 20.9 20.8 20.7 20.8 20.8 20.7 20.9

21 21.3 21.4 21.2 21.2 21.2 21.2 21.1 21.3

27 20.9 21.0 20.8 20.7 20.9 20.7 20.7 20.7

29 20.9 20.8 20.9 20. 8 21.0 20.7 20.7 20.7

32 21.2 21.2 21.2 21.0 21.3 21.0 21.1 21.1

34 20.8 20.9 20.9 20.6 21.3 20.6 20.6 20.7

36 20.9 21.0 20.9 20.7 21.3 20.7 20.6 20.8

39 20.9 21.0 20.9 20.9 21.1 20.6 20.7 20.9

41 21.2 21.2 21.1 21.1 21.1 19.9 20.7 21.0

43 21.6 21.5 21.0 21.4 20.9 20.9 20.9 20.9

46 20.9 20.9 20.8 20.8 20.8 20.8 20.8 20.9

50 20.8 20.8 20.7 20.7 20.7 20.7 20.7 20.8

64 21.8 21.9 21.8 21.7 21.6 21.7 21.5 21.7

67 20.5 20.6 20.6 20.5 20.4 20.5 20.3 20.5

71 21.3 21.2 21.1 21.0 21.1 21.0 21.0 21.1

75 21 .4 21.3 21.2 21.2 21.1 21.2 21.1 21.3

78 21.6 21.4 21.5 21.6 21.2 21.5 21.2 21.1

82 21.8 21.4 21.7 21.6 21.3 21.4 21.1 21.4

85 21.0 20.9 21.0 21.1 20.7 20.9 20.7 20.8

88 21.0 21.1 21.0 21.0 19.9 21.0 21.1 20.8

92 21.2 21.2 20.9 21.0 20.6 20.8 21.0 20. 6

97 21.0 21.0 20.9 20.9 20.8 21.1 21.2 20.8

99 20.8 20.9 20.7 20.8 20.1 20.8 20.8 20.5

102 20.6 20.8 20.7 20.9 20.5 20.8 20.8 20.3

106 20.7 20.7 20.7 20.8 20.8 20.6 20.7 20.4

111 21.3 21.3 21.4 21.3 21.3 21.1 21.2 21.1

112 20.9

-- 21.0

-- -- -- 20.9

--

113

-- 20.6

-- 20.9 21.0 20.9

-- --

‘

-- ’ indicates measurement not collected

.

### ABC Study No. 66086

Page 228 of 261 Table L-2. Test Solution Dissolved Oxygen Concentrations Measured in Each Replicate Test

Chamber

Dissolved Oxygen (mg/L)

Control

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

0 8.61 8.67 8.66 8.69 8.65 8.66 8.70 8.70

6 5.59 5.92 6.21 5.57 6.41 6.31 6.13 6.03

8 6.11 6.81 6.77 6.93 7.16 6.82 6.81 6.71

12 5.97 6.63 6.64 6.77 6.60 6.75 6.73 6.50

15 6.29 6.34 6.41 6.38 5.53 6.35 6.23 6.24

19 6.59 6.31 6.46 6.04 6.60 6.20 6.30 6.35

21 5.68 5.48 5.96 5.19 5.70 5.26 5.47 5.48

27 5.87 5.23 5.32 5.26 5.11 5.31 5.25 5.35

29 6.17 5.81 6.20 6.02 5.75 6.04 6.38 5.89

32 5.26 4.89 5.30 5.17 4.89 5.09 4.87 4.90

34 4.94 4.63 5.23 4.93 4.75 4.99 4.83 4.77

36 5.10 4 .82 5.21 4.92 4.69 4.74 4.81 5.01

39 5.41 5.43 6.02 5.98 5.17 5.86 5.83 5.82

41 4.01 5.13 5.80 6.30 5.14 5.85 5.67 6.14

43 5.06 5.73 6.69 6.75 6.32 6.06 5.89 6.14

46 5.61 5.53 6.02 5.61 5.25 5.64 5.54 5.95

50 4.87 5.73 5.95 5.64 5.71 6.11 5.91 5.82

5 3 6.35 5.99 6.95 6.56 6.43 6.89 7.32 6.96

57 6.69 6.96 7.40 7.09 7.05 7.17 7.59 7.32

60 4.18 6.03 5.79 5.35 5.11 5.81 5.68 5.44

64 5.83 6.54 6.46 6.44 6.10 5.53 6.97 6.60

67 6.73 5.71 7.71 6.08 6.17 6.75 6.67 6.49

71 7.21 7.31 7.35 7.27 7.13 7.39 7.41 7.22

75 6.42 6.44 6.46 6.48 6.35 6.43 6.28 6.28

78 6.02 6.23 6.22 8.22 6.34 6.25 6.43 6.40

82 6.63 6.46 7.32 6.70 7.06 6.70 6.93 6.43

85 7.95 6.62 7.35 7.32 7.15 7.08 7.24 7.40

88 6.97 7.05 7.34 7.11 7.03 6.83 6.96 7.05

92 7.39 7.00 7.13 7.66 7.14 6 .95 7.04 6.85

97 6.44 6.87 6.39 6.67 6.73 6.70 6.56 6.55

99 6.48 6.48 6.90 6.59 6.58 6.59 6.62 6.64

102 6.76 6.65 7.29 6.87 6.99 6.95 6.75 6.69

106 6.89 6.76 7.19 6.87 7.13 7.16 7.13 6.78

111 6.75 6.78 7.20 7.25 6.78 6.92 6.83 6.68

112 7.90

-- 6.91 7 .12

-- -- -- 6.93

113

-- 6.74

-- -- 6.72 6.98 7.09

--

Note: 100% dissolved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21

, and 22°C, respectively.

‘

-- ’ indicates measurement not collected

.

### ABC Study No. 66086

Page 229 of 261 Table L-2. Test Solution Dissolved Oxygen Conce ntrations Measured in Each Replicate Test

Chamber (continued)

Dissolved Oxygen (mg/L)

Level 1 ( 4.97 μg/L) Level 2 ( 10.3 μg/L)

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

0 8.72 8.71 8.69 8.66 8.69 8.71 8.69 8.69

6 5.90 6.39 6.32 6.57 5 .92 6.09 5.73 5.85

8 7.09 6.82 6.92 7.16 7.15 6.92 6.09 7.15

12 6.70 6.37 6.54 6.99 6.56 6.21 5.64 6.79

15 6.33 6.45 6.07 6.02 6.34 6.34 6.57 6.42

19 6.38 6.57 6.21 6.63 6.29 6.60 7.02 6.33

21 5.63 5.90 5.43 5.67 5.53 5.96 6.44 5.41

27 5.64 5.64 5.17 5.21 5.61 5.39 6.28 5.25

29 6.31 6.57 6.01 5.79 6.57 6.25 6.57 5.96

32 5.42 5.48 4.80 5.09 5.35 5.64 5.71 4.89

34 5.43 5.51 4.71 5.06 5.16 5.50 5.57 5.00

36 5.09 5.43 4.37 4.88 4.72 5.31 5.31 4.71

39 5.96 6.34 5.51 5.92 5.66 6.17 5.50 5.56

41 5.15 6 .40 5.60 5.03 5.55 5.60 4.55 4.78

43 5.73 7.16 6.04 6.34 6.15 6.45 5.27 6.02

46 6.13 6.11 5.52 5.36 5.79 5.75 4.96 5.59

50 5.88 5.95 5.30 5.61 5.57 5.56 5.82 5.92

53 6.46 6.98 6.75 6.84 6.18 6.45 6.66 6.96

57 7.34 7.46 7.41 7.28 7.16 7.39 7.32 7.57

6 0 5.36 5.37 5.47 4.33 5.50 5.66 5.21 5.42

64 6.29 6.98 6.85 6.77 6.51 6.58 6.69 6.85

67 6.79 6.83 6.81 6.62 6.87 6.89 6.55 6.62

71 7.35 7.23 7.45 6.56 7.11 7.10 6.85 7.36

75 6.25 5.84 6.40 6.81 6.19 5.53 6.34 6.40

78 6.32 6.20 6.22 6.03 6.35 5.53 6.04 6.31

82 6.74 7.89 6.44 5.76 5.91 6.60 6.36 6.82

85 6.84 6.78 6.91 7.32 7.13 7.16 6.77 6.44

88 6.67 6.71 7.21 6.92 6.79 6.93 6.95 6.94

92 6.79 6.81 7.01 6.79 6.89 6.90 6.87 7.05

97 6.57 6.68 6.62 6.33 6.71 6.74 6.80 6.86

99 6.83 6.61 6.67 6.53 6.45 6 .75 6.67 6.63

102 6.75 6.58 6.97 7.09 6.90 6.90 6.87 6.98

106 7.09 6.87 6.66 6.89 6.75 7.06 7.05 6.84

111 6.79 6.76 6.83 6.77 6.68 6.92 6.40 6.50

112 6.94

-- -- 6.97

-- -- 6.61 6.88

113

-- 6.71 6.66

-- 6.71 6.83

-- --

Note: 100% dissolved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21

, and 22°C, respectively.

‘

-- ’ indicates measurement not collected

.

### ABC Study No. 66086

Page 230 of 261 Table L-2. Test Solution Dissolved Oxygen Concentrations Measured in Each Replicate Test

Chamber (continued)

Dissolved Oxygen (mg/L)

Level 3 ( 19.5 μg/L) Level 4 ( 38.0 μg/L)

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

0 8.69 8.67 8.68 8.69 8.67 8.66 8.64 8.64

6 6.11 6.08 6.23 6.68 5.61 6.35 6.10 5.68

8 6.97 6.73 7.08 7.21 6.86 7.28 7.19 6.78

12 5.97 5.80 6.36 6.70 6. 99 5.58 6.74 6.78

15 6.43 6.37 6.19 6.02 6.06 6.32 6.06 5.81

19 6.82 7.01 6.31 6.56 6.39 6.73 6.42 5.53

21 5.96 6.41 5.48 5.64 5.58 5.71 5.40 5.34

27 5.42 5.72 5.20 5.33 5.74 5.34 5.62 4.87

29 5.60 5.91 5.93 5.88 5.66 5.99 5.74 5.56

32 5.04 5.35 4.96 5.31 4.66 5.20 5.06 4.76

34 4.94 4.97 4.92 5.21 4.77 4.94 4.59 4.57

36 4.91 4.90 4.88 5.00 4.53 5.00 4.61 4.38

39 5.32 5.56 5.55 6.16 5.15 5.64 5.37 5.03

41 4.89 4.31 5.76 5.44 5.73 5.09 4.70 4.74

43 6.18 5.23 5.59 6.61 5.34 5.71 5.56 5.40

46 5.52 4 .79 5.28 6.01 5.60 5.69 5.60 5.31

50 5.63 5.41 5.06 5.90 5.46 5.40 5.63 5.10

53 6.52 6.64 6.53 6.75 6.08 6.82 6.85 6.21

57 7.21 6.76 6.80 7.18 6.71 7.46 5.45 7.39

60 5.37 4.84 5.15 5.55 4.96 5.39 4.97 4.92

64 6.54 6.64 6.53 6.39 6.40 7.15 6.21 6.32

6 7 6.59 6.18 6.32 7.02 5.98 6.78 6.21 6.36

71 7.04 6.84 7.03 7.48 6.86 7.13 6.91 7.22

75 6.19 6.22 6.40 6.34 6.20 6.30 6.28 6.28

78 6.63 6.34 6.17 6.17 6.30 6.38 6.12 6.15

82 6.28 6.34 6.32 5.78 6.33 5.92 5.84 6.31

85 6.68 6.78 6.86 7.11 6.22 6.78 6.20 6.58

88 7.10 7.11 6.85 7.19 4.64 7.22 7.21 7.03

92 7.05 7.39 6.68 6.79 6.84 6.69 6.83 6.55

97 6.91 6.53 6.77 6.18 6.52 6.65 6.33 6.54

99 6.59 6.66 6.53 6.86 4.72 6.78 6.76 6.54

102 7.08 7.10 6.77 6.95 6.76 6.79 6.95 6.43

106 7.09 6.91 6.78 6.96 6.64 6.85 6.83 6.54

111 6.95 6.57 6.72 6.87 6.48 6.68 6.66 6.41

112 7.08

-- 6.98

-- -- -- 6.92 6.49

113

-- 6.74

-- 6.83 6.43 6.92

-- --

Note: 100% dissolved oxygen saturation is 8.7, 8.5 and 8.4 at temperatures of 20, 21

, and 22°C, respectively.

‘

-- ’ indi cates measurement not collected

.

### ABC Study No. 66086

Page 231 of 261 Table L-3. Test Solution pH Values Measured in Each Replicate Test Chamber

pH

Control

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

0 8.32 8.32 8.33 8.30 8.31 8.31 8.28 8.27

6 8.07 8.05 8.08 8.01 8.11 8.0 9 8.06 8.03

8 7.86 7.95 7.95 7.96 8.02 8.00 7.97 7.98

12 8.03 8.06 8.10 8.09 8.09 8.11 8.06 8.09

15 8.15 8.13 8.16 8.14 8.17 8.18 8.15 8.14

19 8.12 8.04 8.14 8.05 8.28 8.11 8.06 8.03

21 8.05 8.00 8.06 7.95 8.01 8.00 7.98 8.00

27 8.25 8.09 8.11 8.05 8 .04 8.04 8.03 8.03

29 8.25 8.14 8.19 8.14 8.13 8.14 8.15 8.11

32 7.89 7.87 7.97 7.93 7.90 7.93 7.89 7.89

36 8.46 8.27 8.14 8.07 8.03 7.99 7.98 7.97

41 7.91 7.95 8.03 8.10 7.90 8.04 8.03 8.07

43 7.82 7.94 8.18 8.21 8.12 8.04 8.00 8.02

46 8.01 8.08 8.1 3 8.05 8.01 8.08 8.04 8.08

50 7.99 8.15 8.19 8.12 8.15 8.22 8.18 8.17

53 8.07 8.02 8.20 8.14 8.14 8.19 8.23 8.19

57 8.13 8.15 8.24 8.20 8.21 8.12 8.07 8.19

60 7.76 7.92 7.91 7.84 7.80 7.85 7.86 7.82

64 7.85 7.90 7.93 7.90 7.88 7.88 7.93 7.95

67 7.47 7.59 7.93 7.71 7.70 7.81 7.77 7.77

71 8.08 8.13 8.14 8.10 8.11 8.14 8.13 8.11

75 7.70 7.98 8.02 7.94 7.96 7.99 7.96 7.96

78 8.06 7.90 7.95 7.86 7.86 7.87 7.82 7.80

82 8.12 8.04 8.05 7.99 8.01 8.04 7.99 7.95

85 7.90 7.85 8.04 8.09 7.97 7.93 7.91 7.91

88 7.66 7.68 7.90 7.78 7.77 7.75 7.73 7.75

92 7.83 7.88 8.04 7.91 7.90 7.95 7.95 7.91

97 7.63 7.68 7.79 7.72 7.73 7.78 7.74 7.74

99 7.60 7.70 7.81 7.77 7.79 7.83 7.79 7.80

102 7.67 7.68 7.78 7.66 7.67 7.66 7.63 7.63

106 7.84 7.81 7.92 7.75 7.85 7.83 7 .85 7.79

111 7.76 7.85 7.93 7.90 7.86 7.75 7.76 7.76

112 7.77

-- 7.80 7.77

-- -- -- 7.70

113

-- 7.84

-- -- 7.92 7.93 7.86

--

‘

-- ’ indicates measurement not collected

.

### ABC Study No. 66086

Page 232 of 261 Table L-3. Test Solution pH Values Measured in Each Replicate Test Chamber (continued)

pH

Level 1 ( 4.97 μg/L) Level 2 ( 10.3 μg/L)

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

0 8.26 8.26 8.24 8.25 8.22 8.23 8.22 8.21

6 8.00 8.07 8.08 8.07 8.02 8.06 7.99 7.99

8 8.02 7.98 7.98 8.00 8.01 8.00 7.92 8.00

12 8.09 8.08 8.0 7 8.15 8.07 8.05 7.98 8.10

15 8.14 8.13 8.13 8.11 8.17 8.15 8.20 8.18

19 8.11 8.06 8.06 7.99 8.10 8.04 8.06 8.01

21 8.01 8.03 7.99 8.01 8.02 8.04 8.11 8.00

27 8.06 8.07 8.04 8.01 8.05 8.08 8.14 8.01

29 8.13 8.15 8.09 8.06 8.13 8.11 8.14 8.08

32 7.93 7.99 7.91 7.83 7.94 7.98 7.98 7.93

36 7.98 7.99 7.94 7.94 7.93 7.94 7.96 7.91

41 7.98 8.13 8.02 7.97 8.00 8.02 7.92 7.85

43 8.00 8.24 8.11 8.08 8.04 8.11 8.01 8.06

46 8.09 8.12 8.05 8.04 8.07 8.13 7.95 8.02

50 8.19 8.20 8.10 8.15 8.14 8.11 8.12 8.16

53 8.13 8.21 8.19 8.20 8.14 8.16 8.17 8.23

57 8.23 8.21 8.20 8.20 8.14 8.25 8.27 8.28

60 7.84 7.83 7.84 7.74 7.87 7.89 7.82 7.85

64 7.87 8.09 7.98 7.98 7.95 7.96 7.95 7.97

67 7.83 7.86 7.80 7.79 7.82 7.84 7.81 7.77

71 8.17 8.14 8.17 7.96 8.10 8.08 8.0 7 8.14

75 7.97 7.95 8.01 7.96 7.97 7.96 8.00 8.00

78 7.84 7.86 7.92 7.86 7.85 7.83 7.90 7.89

82 7.94 7.94 7.99 7.90 7.98 7.94 7.95 8.00

85 7.83 7.92 7.97 7.87 7.89 7.87 7.87 7.79

88 7.67 7.69 7.89 7.75 7.74 7.74 7.72 7.74

92 7.86 7.90 7.98 7.89 7.92 7.93 7.86 7.97

97 7.73 7.78 7.76 7.75 7.73 7.73 7.76 7.74

99 7.79 7.76 7.80 7.77 7.80 7.81 7.82 7.80

102 7.59 7.69 7.66 7.69 7.68 7.62 7.64 7.63

106 7.79 7.76 7.71 7.85 7.73 7.78 7.75 7.75

111 7.81 7.80 7.81 7.77 7.74 7.80 7.66 7.70

112 7.75

-- -- 7. 76

-- -- 7.62 7.78

113

-- 7.79 7.81

-- 7.79 7.84

-- --

‘

-- ’ indicates measurement not collected

.

### ABC Study No. 66086

Page 233 of 261 Table L-3. Test Solution pH Values Measured in Each Replicate Test Chamber (continued)

pH

Level 3 ( 19.5 μg/L) Level 4 ( 38.0 μg/L)

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

0 8.21 8.22 8.20 8.23 8.24 8.23 8.23 8.22

6 8.01 8.02 8.04 8.07 7.98 8.06 8.05 8.01

8 7.97 7.98 8.00 8.00 7.96 8.01 7.99 7.92

12 8.00 7.99 8.08 8.06 8.13 7.96 8.10 8.12

15 8.16 8.20 8.15 8.12 8.13 8.15 8.13 8.13

19 8.06 8.06 8.21 8.01 7.96 7.97 7.98 7.99

21 8.04 8.11 8.00 7.99 8.01 8.01 7.98 7.99

27 8.04 8.07 8.00 7.99 7.97 8.01 7.99 7.96

29 8.07 8.08 8.06 8.04 8.02 8.06 8.03 8.02

32 7.92 7.95 7.91 7.93 7.86 7.92 7.90 7.87

36 7.90 7.90 7.88 7.89 8.46 8.27 8. 14 8.07

41 7.88 7.86 7.99 7.98 7.98 7.95 7.88 7.84

43 8.00 7.96 7.97 8.12 7.96 7.98 7.94 7.92

46 8.01 7.91 7.97 8.03 8.02 8.06 8.01 7.96

50 8.13 8.10 8.04 8.12 8.10 8.10 8.12 8.05

53 8.16 8.18 8.16 8.19 8.14 8.21 8.21 8.15

57 8.24 8.20 8.20 8.21 8.21 8.30 7.97 8.17

60 7.78 7.77 7.82 7.81 7.72 7.84 7.73 7.76

64 7.93 7.98 7.93 7.90 7.86 8.05 7.85 7.86

67 7.77 7.75 7.71 7.77 7.68 7.77 7.70 7.71

71 8.09 8.05 8.06 8.17 7.99 8.10 8.03 8.06

75 7.99 7.99 8.00 7.99 7.98 7.99 7.93 7.97

78 7.91 7.94 7.79 7 .85 7.83 7.88 7.82 7.84

82 7.98 7.95 7.97 8.01 7.93 7.98 7.87 7.91

85 7.91 7.89 7.85 7.92 7.86 7.91 7.78 7.80

88 7.80 7.83 7.72 7.80 7.33 7.81 7.80 7.84

92 7.97 8.03 7.87 7.85 7.86 7.87 7.91 7.83

97 7.74 7.79 7.76 7.77 7.73 7.74 7.78 7.77

99 7.81 7.9 1 7.76 7.87 7.53 7.80 7.89 7.82

102 7.69 7.72 7.63 7.67 7.61 7.64 7.68 7.61

106 7.90 7.84 7.71 7.79 7.72 7.77 7.75 7.69

111 8.03 7.86 7.94 7.79 7.70 7.78 7.77 7.75

112 7.81

-- 7.76

-- -- -- 7.75 7.65

113

-- 7.83

-- 7.88 7.75 7.90

-- --

‘

-- ’ indicates measurement not collected

.

### ABC Study No. 66086

Page 234 of 261 APPENDIX M – PLASMA VITELLOGENIN DATA

### ABC Study No. 66086

Page 235 of 261 Appendix M-1. Vitellogenin Data for NF Stage 62 Pooled Plasma

Treatment

(μg/L) REP Sample ID Vtg Conc.

( m n /mL) Plate # min.

detection max.

detection

Co ntrol A CA

- 180 0 3 28,442 2,318,572

B CB

- 180 0 3 28,442 2,318,572

C CC

- 90 8999.685 11 17,999 1,111,687

D CD

- 90 8999.685 11 17,999 1,111,687

E CE

- 90 9156.555 12 18,313 1,166,195

F CF

- 90 9156.555 12 18,313 1,166,195

G CG

- 180 16495.74 4 32,991 2,3 05,985

H CH

- 180 16495.74 4 32,991 2,305,985

4.97 A L1A

- 180 16495.74 4 32,991 2,305,985

B L1B

- 180 16495.74 4 32,991 2,305,985

C L1C

- 180 0 3 28,442 2,318,572

D L1D

- 180 0 3 28,442 2,318,572

10.3 A L2A

- 90 8999.685 11 17,999 1,111,687

B L2B

- 180 0 3 28,442 2,318,572

C L2C

- 180 16495.74 4 32,991 2,305,985

D L2D

- 180 0 3 28,442 2,318,572

19.5 A L3A

- 180 0 3 28,442 2,318,572

B L3B

- 180 16495.74 4 32,991 2,305,985

C L3C

- 180 0 3 28,442 2,318,572

D L3D

- 180 16495.74 4 32,991 2, 305,985

38.0 A L4A

- 180 16495.74 4 32,991 2,305,985

B L4B

- 180 0 3 28,442 2,318,572

C L4C

- 180 16495.74 4 32,991 2,305,985

D L4D

- 90 9156.555 12 18,313 1,166,195

### ABC Study No. 66086

Page 236 of 261 Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination , Based on Phenotypic Sex

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

Control A CA1

- T 1

- 90 1 9227.7 1

- R1 18455.4 1196909

A CA3

- T 3

- 90 3 9227.7 1

- R1 18455.4 1196909

A CA4

- T 4

- 180 4 171 44.91 7 34289.82 2215452

A CA9

- T 9

- 180 9 0 3 28442.16 2318572

A CA10

- T 10

- 180 10 0 3 28442.16 2318572

B CB1

- T 11

- 90 11 8999.685 11 17999.37 1111687

B CB4

- T 14

- 90 14 0 2

- R1 15544.53 1079139

B CB6

- T 16

- 180 16 50113.582 7 34289.82 2215452

B CB7

- T 17

- 180 17 17144.91 7 34289.82 2215452

B CB8

- T 18

- 180 18 64891.308 7 34289.82 2215452

C CC1

- T 21

- 180 21 16495.74 4 32991.48 2305985

C CC2

- T 22

- 90 22 8999.685 11 17999.37 1111687

C CC3

- T 23

- 90 23 9227.7 1

- R1 18455.4 1196909

C CC5

- T 25

- 90 25 9227.7 1

- R1 18455.4 1196909

C CC9

- T 29

- 180 29 14221.08 3 28442.16 2318572

D CD3

- T 33

- 180 33 0 8 38758.5 2745362

D CD4

- T 34

- 90 34 9156.555 12 18313.11 1166195

D CD5

- T 35

- 90 35 0 2

- R1 15544.53 1079139

D CD6

- T 36

- 90 36 0 2

- R1 15544.53 1079139

D CD7

- T 37

- 90 37 0 2

- R1 15544.53 1079139

D CD9

- T 39

- 90 39 19905.85 11 17999.37 1111687

### ABC Study No. 66086

Page 237 of 261 Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

Control E CE2

- T 42

- 180 42 16495.74 4 32991.48 2305984.5

E CE3

- T 43

- 180 43 16495.74 4 32991.48 2305984.5

E CE4

- T 44

- 180 44 16495.74 4 32991.48 2305984.5

E CE5

- T 45

- 180 45 17144.91 7 34289.82 2215452.06

E CE6

- T 46

- 180 46 17144.91 7 34289.82 2215452.06

E CE7

- T 47

- 180 47 17144.91 7 34289.82 2215452.06

E CE8

- T 48

- 180 48 0 8 38758.5 2745361.98

F CF3

- T 53

- 90 53 9227.7 1

- R1 18455.4 1196909.01

F CF5

- T 55

- 90 55 9156.555 12 18313.11 1166195.25

F CF8

- T 5 8

- 180 58 0 3 28442.16 2318571.9

F CF9

- T 59

- 90 59 38540.873 12 18313.11 1166195.25

G CG1

- T 61

- 90 61 8999.685 11 17999.37 1111687.29

G CG3

- T 63

- 90 63 0 2

- R1 15544.53 1079138.7

G CG5

- T 65

- 90 65 42828.694 2

- R1 15544.53 1079138.7

G CG6

- T 66

- 180 66 193 79.25 8 38758.5 2745361.98

G CG8

- T 68

- 180 68 0 8 38758.5 2745361.98

G CG9

- T 69

- 90 69 8999.685 11 17999.37 1111687.29

H CH1

- T 71

- 180 71 16495.74 4 32991.48 2305984.5

H CH2

- T 72

- 180 72 16495.74 4 32991.48 2305984.5

H CH4

- T 74

- 90 74 9156.555 12 1831 3.11 1166195.25

H CH6

- T 76

- 90 76 8999.685 11 17999.37 1111687.29

H CH8

- T 78

- 90 78 9156.555 12 18313.11 1166195.25

H CH9

- T 79

- 180 79 0 3 28442.16 2318571.9

### ABC Study No. 66086

Page 238 of 261 Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination , Based on Phenotypic S ex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

4.97 A L1A2

- T 82

- 90 82 8999.685 11 17999.37 1111687.29

A L1A4

- T 84

- 90 84 9227.7 1

- R1 18455.4 1196909.01

A L1A6

- T 86

- 180 86 17144.91 7 34289.82 2215452.06

A L1A9

- T 89

- 180 89 0 8 38758.5 2745361.98

B L1B3

- T 93

- 90 93 9227.7 1

- R1 18455.4 1196909.01

B L1B4

- T 94

- 90 94 9227.7 1

- R1 18455.4 1196909.01

B L1B5

- T 95

- 90 95 8999.685 11 17999.37 1111687.29

B L1B8

- T 98

- 180 98 0 3 2 8442.16 2318571.9

B L1B10

- T 100

- 180 100 17144.91 7 34289.82 2215452.06

C L1C1

- T 101

- 180 101 36151.126 7 34289.82 2215452.06

C L1C3

- T 103

- 90 103 0 2

- R1 15544.53 1079138.7

C L1C6

- T 106

- 180 106 16495.74 4 32991.48 2305984.5

C L1C8

- T 108

- 180 108 1649 5.74 4 32991.48 2305984.5

D L1D1

- T 111

- 180 111 0 8 38758.5 2745361.98

D L1D3

- T 113

- 90 113 0 2

- R1 15544.53 1079138.7

D L1D5

- T 115

- 90 115 9156.555 12 18313.11 1166195.25

D L1D7

- T 117

- 180 117 0 3 28442.16 2318571.9

D L1D9

- T 119

- 180 119 0 3 28442.16 2318571.9

### ABC Study No. 66086

Page 239 of 261 Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

10.3 A L2A2

- T 122

- 90 122 8999.685 11 17999.37 1111687.29

A L2A3

- T 123

- 90 123 9227.7 1

- R1 18455.4 1196909.01

A L2A4

- T 124

- 90 124 9227.7 1

- R1 18455.4 1196909.01

A L2A5

- T 125

- 90 125 168474.727 1

- R1 18455.4 1196909.01

A L2A6

- T 126

- 90 126 8999.685 11 17999.37 1111687.29

A L2A7

- T 127

- 180 127 16495.74 4 32991.48 2305984.5

A L2A9

- T 129

- 180 129 16495.74 4 32991.48 2305984.5

A L2A10

- T 130

- 90 130 8999.685 11 17999.37 1111687.29

B L2B1

- T 131

- 90 131 9156.555 12 18313.11 1166195.25

B L2B4

- T 134

- 90 134 9227.7 1

- R1 18455.4 11 96909.01

B L2B5

- T 135

- 90 135 9156.555 12 18313.11 1166195.25

B L2B8

- T 138

- 180 138 0 3 28442.16 2318571.9

B L2B9

- T 139

- 180 139 17144.91 7 34289.82 2215452.06

B L2B10

- T 140

- 180 140 17144.91 7 34289.82 2215452.06

C L2C2

- T 142

- 90 142 9156.555 12 1831 3.11 1166195.25

C L2C4

- T 144

- 90 144 0 2

- R1 15544.53 1079138.7

C L2C5

- T 145

- 90 145 0 2

- R1 15544.53 1079138.7

C L2C6

- T 146

- 90 146 0 2

- R1 15544.53 1079138.7

C L2C8

- T 148

- 180 148 0 8 38758.5 2745361.98

C L2C9

- T 149

- 180 149 0 8 38758.5 2745361.98

D L2D2

- T 152

- 180 152 0 3 28442.16 2318571.9

D L2D10

- T 160

- 180 160 17144.91 7 34289.82 2215452.06

### ABC Study No. 66086

Page 240 of 261 Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

19.5 A L3A4

- T 164

- 90 164 9227.7 1

- R1 18455.4 1196909.01

A L3A5

- T 165

- 90 165 9156.555 12 18313.11 1166195.25

A L3A6

- T 166

- 90 166 9156.555 12 18313.11 1166195.25

B L3B3

- T 173

- 90 173 0 2

- R1 15544.53 1079138.7

B L3B4

- T 174

- 90 174 0 2

- R1 15544.53 1079138.7

B L3B5

- T 175

- 180 175 17144.91 7 34289.82 2215452.06

B L3B6

- T 176

- 180 176 17144.91 7 34289.82 2215452.06

B L3B8

- T 178

- 90 178 8999.685 11 17999.37 1111687.29

B L3B10

- T 180

- 90 180 9 156.555 12 18313.11 1166195.25

C L3C2

- T 182

- 90 182 9227.7 1

- R1 18455.4 1196909.01

C L3C3

- T 183

- 90 183 9227.7 1

- R1 18455.4 1196909.01

C L3C4

- T 184

- 180 184 17144.91 7 34289.82 2215452.06

C L3C7

- T 187

- 180 187 0 8 38758.5 2745361.98

C L3C8

- T 188

- 180 188 0 8 38758.5 2745361.98

C L3C9

- T 189

- 180 189 0 8 38758.5 2745361.98

C L3C10

- T 190

- 90 190 8999.685 11 17999.37 1111687.29

D L3D3

- T 193

- 90 193 0 2

- R1 15544.53 1079138.7

D L3D4

- T 194

- 90 194 0 2

- R1 15544.53 1079138.7

D L3D6

- T 196

- 180 196 0 8 38758 .5 2745361.98

D L3D8

- T 198

- 90 198 8999.685 11 17999.37 1111687.29

D L3D9

- T 199

- 90 199 8999.685 11 17999.37 1111687.29

### ABC Study No. 66086

Page 241 of 261 Appendix M-2. Vitellogenin Data for Juvenile Males at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP F rog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

38.0 A L4A1

- T 201

- 180 201 16495.74 4 32991.48 2305984.5

A L4A4

- T 204

- 180 204 17144.91 7 34289.82 2215452.06

A L4A5

- T 205

- 180 205 17144.91 7 34289.82 2215452.06

A L4A6

- T 206

- 90 206 9156.555 12 18313.11 1166195.25

A L4A7

- T 207

- 90 207 8999.685 11 17999.37 1111687.29

A L4A9

- T 209

- 90 209 0 2

- R1 15544.53 1079138.7

B L4B1

- T 211

- 180 211 0 8 38758.5 2745361.98

B L4B2

- T 212

- 180 212 19379.25 8 38758.5 2745361.98

B L4B3

- T 213

- 180 213 19379.25 8 38758.5 2745361.98

B L4B5

- T 215

- 90 215 9156.555 12 18313.11 1166195.25

B L4B9

- T 219

- 180 219 0 8 38758.5 2745361.98

B L4B10

- T 220

- 180 220 19379.25 8 38758.5 2745361.98

C L4C2

- T 222

- 90 222 27180.931 12 18313.11 116619 5.25

C L4C3

- T 223

- 90 223 9156.555 12 18313.11 1166195.25

C L4C4

- T 224

- 90 224 9156.555 12 18313.11 1166195.25

C L4C5

- T 225

- 90 225 9227.7 1

- R1 18455.4 1196909.01

C L4C7

- T 227

- 90 227 29156.71 1

- R1 18455.4 1196909.01

C L4C8

- T 228

- 180 228 0 8 38758.5 2745361.98

D L4D2

- T 232

- 180 232 74942.043 4 32991.48 2305984.5

D L4D6

- T 236

- 180 236 17144.91 7 34289.82 2215452.06

D L4D8

- T 238

- 90 238 9227.7 1

- R1 18455.4 1196909.01

D L4D9

- T 239

- 90 239 9227.7 1

- R1 18455.4 1196909.01

### ABC Study No. 66086

Page 242 of 261 Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination , Based on Phenotypic Sex

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

Control A CA2

- T 2

- 90 2 9227.7 1

- R1 18455.4 1196909.01

A CA5

- T 5

- 18 0 5 17144.91 7 34289.82 2215452.06

A CA6

- T 6

- 180 6 17144.91 7 34289.82 2215452.06

A CA8

- T 8

- 180 8 0 3 28442.16 2318571.9

B CB2

- T 12

- 90 12 8999.685 11 17999.37 1111687.29

B CB3

- T 13

- 90 13 7772.265 2

- R1 15544.53 1079138.7

B CB5

- T 15

- 90 15 0 2

- R1 15 544.53 1079138.7

B CB9

- T 19

- 180 19 40189.923 4 32991.48 2305984.5

B CB10

- T 20

- 180 20 16495.74 4 32991.48 2305984.5

C CC4

- T 24

- 90 24 9227.7 1

- R1 18455.4 1196909.01

C CC6

- T 26

- 90 26 8999.685 11 17999.37 1111687.29

C CC7

- T 27

- 180 27 0 3 28442.16 231 8571.9

C CC8

- T 28

- 180 28 0 3 28442.16 2318571.9

D CD1

- T 31

- 180 31 0 8 38758.5 2745361.98

D CD2

- T 32

- 180 32 0 8 38758.5 2745361.98

D CD8

- T 38

- 90 38 9156.555 12 18313.11 1166195.25

### ABC Study No. 66086

Page 243 of 261 Appendix M-3. Vitellogenin Data for Juvenile Females at Study Term ination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

Control E CE1

- T 41

- 90 41 8999.685 11 17999.37 1111687.29

E CE9

- T 49

- 180 49 0 8 38758.5 274536 1.98

E CE10

- T 50

- 180 50 0 8 38758.5 2745361.98

F CF1

- T 51

- 90 51 9227.7 1

- R1 18455.4 1196909.01

F CF2

- T 52

- 90 52 9227.7 1

- R1 18455.4 1196909.01

F CF4

- T 54

- 90 54 8999.685 11 17999.37 1111687.29

F CF6

- T 56

- 180 56 0 3 28442.16 2318571.9

F CF7

- T 57

- 180 57 0 3 28442.16 2318571.9

F CF10

- T 60

- 90 60 9156.555 12 18313.11 1166195.25

G CG2

- T 62

- 90 62 8999.685 11 17999.37 1111687.29

G CG4

- T 64

- 90 64 0 2

- R1 15544.53 1079138.7

G CG7

- T 67

- 180 67 0 8 38758.5 2745361.98

G CG10

- T 70

- 180 70 16495.74 4 329 91.48 2305984.5

H CH3

- T 73

- 90 73 9156.555 12 18313.11 1166195.25

H CH5

- T 75

- 90 75 8999.685 11 17999.37 1111687.29

H CH7

- T 77

- 90 77 8999.685 11 17999.37 1111687.29

H CH10

- T 80

- 180 80 14221.08 3 28442.16 2318571.9

### ABC Study No. 66086

Page 244 of 261 Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

4.97 A L1A1

- T 81

- 180 81 0 3 28442.16 2318571.9

A L1A3

- T 83

- 9 0 83 9227.7 1

- R1 18455.4 1196909.01

A L1A5

- T 85

- 90 85 9227.7 1

- R1 18455.4 1196909.01

A L1A7

- T 87

- 180 87 17144.91 7 34289.82 2215452.06

A L1A8

- T 88

- 180 88 17144.91 7 34289.82 2215452.06

A L1A10

- T 90

- 180 90 0 8 38758.5 2745361.98

B L1B1

- T 91

- 180 91 0 8 38758.5 2745361.98

B L1B2

- T 92

- 90 92 9227.7 1

- R1 18455.4 1196909.01

B L1B6

- T 96

- 90 96 8999.685 11 17999.37 1111687.29

B L1B7

- T 97

- 180 97 0 3 28442.16 2318571.9

B L1B9

- T 99

- 180 99 0 3 28442.16 2318571.9

C L1C2

- T 102

- 180 102 17144.91 7 34289.8 2 2215452.06

C L1C4

- T 104

- 90 104 0 2

- R1 15544.53 1079138.7

C L1C5

- T 105

- 90 105 0 2

- R1 15544.53 1079138.7

C L1C7

- T 107

- 180 107 16495.74 4 32991.48 2305984.5

C L1C9

- T 109

- 180 109 0 8 38758.5 2745361.98

C L1C10

- T 110

- 180 110 0 8 38758.5 2745361.98

D L1D2

- T 112

- 90 112 7772.265 2

- R1 15544.53 1079138.7

D L1D4

- T 114

- 90 114 0 2

- R1 15544.53 1079138.7

D L1D6

- T 116

- 90 116 9156.555 12 18313.11 1166195.25

D L1D8

- T 118

- 180 118 0 3 28442.16 2318571.9

### ABC Study No. 66086

Page 245 of 261 Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

10.3 A L2A1

- T 121

- 90 121 9156.555 12 18313.11 1166195.25

A L2A8

- T 128

- 180 128 45364.625 4 32991.48 2305984.5

B L2B2

- T 132

- 90 132 9227.7 1

- R1 18455.4 1196909.01

B L2B3

- T 133

- 90 133 9227.7 1

- R1 18455.4 1196909.01

B L2B6

- T 136

- 180 136 0 3 28442.16 2318571.9

B L2B7

- T 137

- 180 137 0 3 28442.16 2318571.9

C L2C1

- T 141

- 180 141 1714 4.91 7 34289.82 2215452.06

C L2C3

- T 143

- 90 143 40739.093 11 17999.37 1111687.29

C L2C7

- T 147

- 180 147 0 8 38758.5 2745361.98

C L2C10

- T 150

- 90 150 8999.685 11 17999.37 1111687.29

D L2D1

- T 151

- 180 151 0 3 28442.16 2318571.9

D L2D3

- T 153

- 180 153 0 3 28442.16 2318571.9

D L2D4

- T 154

- 90 154 8999.685 11 17999.37 1111687.29

D L2D5

- T 155

- 90 155 9156.555 12 18313.11 1166195.25

D L2D6

- T 156

- 90 156 0 2

- R1 15544.53 1079138.7

D L2D7

- T 157

- 90 157 0 2

- R1 15544.53 1079138.7

D L2D8

- T 158

- 90 158 0 2

- R1 1554 4.53 1079138.7

D L2D9

- T 159

- 180 159 17144.91 7 34289.82 2215452.06

### ABC Study No. 66086

Page 246 of 261 Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /m L) Plate # min.

detection max.

detection

19.5 A L3A1

- T 161

- 180 161 17144.91 7 34289.82 2215452.06

A L3A2

- T 162

- 90 162 9227.7 1

- R1 18455.4 1196909.01

A L3A3

- T 163

- 90 163 9227.7 1

- R1 18455.4 1196909.01

A L3A8

- T 168

- 180 168 16495.74 4 32991.48 2305984 .5

A L3A9

- T 169

- 180 169 16495.74 4 32991.48 2305984.5

A L3A10

- T 170

- 90 170 8999.685 11 17999.37 1111687.29

B L3B1

- T 171

- 90 171 8999.685 11 17999.37 1111687.29

B L3B2

- T 172

- 90 172 0 2

- R1 15544.53 1079138.7

B L3B7

- T 177

- 180 177 17144.91 7 34289.82 2215452.06

B L3B9

- T 179

- 90 179 9156.555 12 18313.11 1166195.25

C L3C1

- T 181

- 90 181 9227.7 1

- R1 18455.4 1196909.01

C L3C5

- T 185

- 180 185 17144.91 7 34289.82 2215452.06

C L3C6

- T 186

- 180 186 17144.91 7 34289.82 2215452.06

D L3D1

- T 191

- 90 191 9156.555 12 18313.11 1166195.25

D L3D2

- T 192

- 90 192 0 2

- R1 15544.53 1079138.7

D L3D5

- T 195

- 180 195 19379.25 8 38758.5 2745361.98

D L3D7

- T 197

- 180 197 0 8 38758.5 2745361.98

D L3D10

- T 200

- 180 200 77088.478 4 32991.48 2305984.5

### ABC Study No. 66086

Page 247 of 261 Appendix M-3. Vitellogenin Data for Juvenile Females at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Vtg Conc.

( n g /mL) Plate # min.

detection max.

detection

38.0 A L4A2

- T 202

- 180 202 16495.74 4 32991.48 2305984.5

A L4A3

- T 203

- 180 203 17144.91 7 34289.82 2215452.06

A L4A8

- T 208

- 90 208 0 2

- R1 15544.53 1079138.7

A L4A10

- T 210

- 90 210 0 2

- R1 15544.53 1079138.7

B L4B4

- T 214

- 90 214 9156.555 12 18313.11 1166195.25

B L4B6

- T 216

- 90 216 45506.302 2

- R1 15544.53 1079 138.7

B L4B7

- T 217

- 90 217 0 2

- R1 15544.53 1079138.7

B L4B8

- T 218

- 90 218 0 2

- R1 15544.53 1079138.7

C L4C1

- T 221

- 180 221 0 8 38758.5 2745361.98

C L4C6

- T 226

- 90 226 9227.7 1

- R1 18455.4 1196909.01

C L4C9

- T 229

- 180 229 0 8 38758.5 2745361.98

C L4C10

- T 230

- 180 230 0 8 38758.5 2745361.98

D L4D1

- T 231

- 180 231 16495.74 4 32991.48 2305984.5

D L4D3

- T 233

- 180 233 16495.74 4 32991.48 2305984.5

D L4D4

- T 234

- 180 234 107361.268 7 34289.82 2215452.06

D L4D5

- T 235

- 180 235 17144.91 7 34289.82 2215452.06

D L4D7

- T 237

- 90 237 32859.473 12 18313.11 1166195.25

D L4D10

- T 240

- 90 240 9227.7 1

- R1 18455.4 1196909.01

### ABC Study No. 66086

Page 248 of 261 APPENDIX N – P LASMA T4 DATA

### ABC Study No. 66086

Page 249 of 261 Appendix N-1. T4 Data for NF Stage 62 Pooled Plasma

Treatment

(μg/L) REP Sample ID T 4 Conc.

(ng/mL) Plate # min.

detection max.

detection

Control A CA

- 30 303.46 3 16 7,963

B CB

- 30 204.6 3 16 7,963

C CC

- 30 173.95 7

- R1 15 7,665

D CD

- 30 109.91 7

- R1 15 7,665

E CE

- 30 241.35 8

- R1 12 6,961

F CF

- 30 320.03 8

- R1 12 6,961

G CG

- 30 182.4 8 4 13 7,496

H CH

- 30 283.01 4 13 7,496

4.97 A L1A

- 30 149.9 4 13 7,496

B L1B

- 30 234.93 4 13 7,496

C L1C

- 30 445.28 3 16 7,963

D L1D

- 30 303.38 3 16 7,963

10.3 A L2A

- 30 333.14 7

- R1 15 7,665

B L2B

- 30 208.8 3 16 7,963

C L2C

- 30 122 .9 4 13 7,496

D L2D

- 30 145.12 3 16 7,963

4

19.5 A L3A

- 30 314.03 3 16 7,963

B L3B

- 30 185.12 4 13 7,496

C L3C

- 30 505.01 3 16 7,963

D L3D

- 30 117.37 4 13 7,496

38.0 A L4A

- 30 222.3 4 13 7,496

B L4B

- 30 410.92 3 16 7,963

C L4C

- 30 266 .77 4 13 7,496

D L4D

- 30 246.56 8

- R1 12 6,961

### ABC Study No. 66086

Page 250 of 261 Appendix N-2. T4 Data for Juvenile Males at Study Termination , Based on Phenotypic Sex

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

det ection

Control 

### A CA1

- T 1

- 30 1 1 65.89 15.3 4228.5



### A CA3

- T 3

- 30 3 1 35.39 15.3 4228.5



### A CA4

- T 4

- 30 4 5 68.5 17.1 6648.3



### A CA7

- T 7

- 30 7 7

- R1 169.3 15 7665.3



### A CA9

- T 9

- 30 9 3 70.01 15.9 7963.2



### A CA10

- T 10

- 30 10 3 242.23 15.9 7963.2



### B CB1

- T 11

- 30 11 7

- R1 24.79 15 7665.3



### B CB4

- T 14

- 30 14 2 25.67 18.6 9006.9



### B CB6

- T 16

- 30 16 5 66.38 17.1 6648.3



### B CB7

- T 17

- 30 17 5 29.63 17.1 6648.3



### B CB8

- T 18

- 30 18 5 26.25 17.1 6648.3



### C CC1

- T 21

- 30 21 4 70.45 13.2 7496.1



### C CC2

- T 22

- 30 22 7

- R1 67.02 15 766 5.3



### C CC3

- T 23

- 30 23 1 46.53 15.3 4228.5



### C CC5

- T 25

- 30 25 1 172.67 15.3 4228.5



### C CC9

- T 29

- 30 29 3 58.77 15.9 7963.2



### D CD3

- T 33

- 30 33 6 & 6

- R1 25.36 13.8 8004



### D CD4

- T 34

- 30 34 8

- R1 44.84 12 6960.6



### D CD5

- T 35

- 30 35 2 49.65 18.6 9006.9



### D CD6

- T 3 6

- 30 36 2 23.88 18.6 9006.9



### D CD7

- T 37

- 30 37 2 37.32 18.6 9006.9



### D CD9

- T 39

- 30 39 7

- R1 99.43 15 7665.3

### ABC Study No. 66086

Page 251 of 261 Appendix N-2. T4 Data for Juvenile Males at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

detection

Control E CE2

- T 42

- 30 42 4 34.77 13.2 7496.1

E CE3

- T 43

- 30 43 4 76.21 13.2 7496.1

E CE4

- T 44

- 30 44 4 59.93 13.2 7496.1

E CE5

- T 45

- 30 45 5 31.45 17.1 6648.3

E CE6

- T 46

- 30 46 5 38.91 1 7.1 6648.3

E CE7

- T 47

- 30 47 5 41.36 17.1 6648.3

E CE8

- T 48

- 30 48 6 & 6

- R1 16.545 13.8 8004

F CF3

- T 53

- 30 53 1 24.95 15.3 4228.5

F CF5

- T 55

- 30 55 8

- R1 78.15 12 6960.6

F CF8

- T 58

- 30 58 3 86.42 15.9 7963.2

F CF9

- T 59

- 30 59 8

- R1 57.08 12 6960.6

G CG1

- T 61

- 30 61 7

- R1 30.11 15 7665.3

G CG3

- T 63

- 30 63 2 43.32 18.6 9006.9

G CG5

- T 65

- 30 65 2 91.87 18.6 9006.9

G CG6

- T 66

- 15 66 9 264.94 16.65 3474.15

G CG8

- T 68

- 30 68 6 & 6

- R1 129.085 13.8 8004

G CG9

- T 69

- 30 69 7

- R1 85.45 15 7665.3

H CH1

- T 71

- 30 71 4 101.97 13.2 7496.1

H CH2

- T 72

- 30 72 4 42.32 13.2 7496.1

H CH4

- T 74

- 30 74 8

- R1 59.77 12 6960.6

H CH6

- T 76

- 30 76 7

- R1 20.35 15 7665.3

H CH8

- T 78

- 30 78 8

- R1 62.57 12 6960.6

H CH9

- T 79

- 30 79 3 45.85 15.9 7963.2

### ABC Study No. 66086

Page 252 of 261 Appendix N-2. T4 Data for Juv enile Males at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

detection

4.97 A L1A2

- T 82

- 30 82 7

- R1 185.63 15 7665.3

A L1A4

- T 84

- 30 84 1 82.9 15.3 4228.5

A L1A6

- T 86

- 30 86 5 31.78 17.1 6648.3

A L1A9

- T 89

- 30 89 6 & 6

- R1 20.47 13.8 8004

B L1B3

- T 93

- 30 93 1 52.1 15.3 4228.5

B L1B4

- T 94

- 30 94 1 25.57 15.3 4228.5

B L1B5

- T 95

- 30 95 7

- R1 47.06 15 7665.3

B L1B8

- T 98

- 30 98 3 58.44 15.9 7963.2

B L1B10

- T 100

- 30 100 5 8.55 17.1 6648.3

C L1C1

- T 101

- 30 101 5 30.29 17.1 6648.3

C L1C3

- T 103

- 30 103 2 26.08 18.6 9006.9

C L1C6

- T 106

- 30 106 4 97.24 13.2 7496.1

C L1C8

- T 108

- 30 108 4 69.73 13.2 7496.1

D L1D1

- T 111

- 15 111 9 56.49 16.65 3474.15

D L1D3

- T 113

- 30 113 2 78.36 18.6 9006.9

D L1D5

- T 115

- 30 115 8

- R1 42.31 12 6960.6

D L1D7

- T 117

- 30 117 3 165.72 15.9 7963.2

D L1D9

- T 119

- 30 119 3 28.14 15.9 7963.2

### ABC Study No. 66086

Page 253 of 261 Appendix N-2. T4 Data for Juvenile Males at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

detection

10.3 A L2A2

- T 122

- 30 122 7

- R1 31.76 15 7665.3

A L2A3

- T 123

- 30 123 1 27.29 15.3 4228.5

A L2A4

- T 124

- 30 124 1 58.71 15.3 4228.5

A L2A5

- T 125

- 30 125 1 132.74 15.3 4228.5

A L2A6

- T 126

- 30 126 7

- R1 24.79 15 7665.3

A L2A7

- T 127

- 30 127 4 39.29 13.2 7496.1

A L2A9

- T 129

- 30 129 4 50.09 13.2 7496.1

A L2A10

- T 130

- 30 130 7

- R1 54.72 15 7665.3

B L2B1

- T 131

- 30 131 8

- R1 42.86 12 6960. 6

B L2B4

- T 134

- 30 134 1 76.3 15.3 4228.5

B L2B5

- T 135

- 30 135 8

- R1 94.1 12 6960.6

B L2B8

- T 138

- 30 138 3 128.22 15.9 7963.2

B L2B9

- T 139

- 30 139 5 19.55 17.1 6648.3

B L2B10

- T 140

- 30 140 5 55.06 17.1 6648.3

C L2C2

- T 142

- 30 142 8

- R1 58.3 12 6960.6

C L2C4

- T 144

- 30 144 2 63.12 18.6 9006.9

C L2C5

- T 145

- 30 145 2 48.52 18.6 9006.9

C L2C6

- T 146

- 30 146 2 103.53 18.6 9006.9

C L2C8

- T 148

- 30 148 6 & 6

- R1 29.37 13.8 8004

C L2C9

- T 149

- 15 149 9 79.97 16.65 3474.15

D L2D2

- T 152

- 30 152 3 125.32 15.9 796 3.2

D L2D10

- T 160

- 30 160 5 29.81 17.1 6648.3

### ABC Study No. 66086

Page 254 of 261 Appendix N-2. T4 Data for Juvenile Males at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

detect ion

19.5 A L3A4

- T 164

- 30 164 1 54.03 15.3 4228.5

A L3A5

- T 165

- 30 165 8

- R1 37.3 12 6960.6

A L3A6

- T 166

- 30 166 8

- R1 48.21 12 6960.6

A L3A7

- T 167

- 30 167 4 56.04 13.2 7496.1

B L3B3

- T 173

- 30 173 2 19.8 18.6 9006.9

B L3B4

- T 174

- 30 174 2 226.14 18.6 9 006.9

B L3B5

- T 175

- 30 175 5 20.68 17.1 6648.3

B L3B6

- T 176

- 30 176 5 76.35 17.1 6648.3

B L3B8

- T 178

- 30 178 7

- R1 47.88 15 7665.3

B L3B10

- T 180

- 30 180 8

- R1 199.9 12 6960.6

C L3C2

- T 182

- 30 182 1 23.94 15.3 4228.5

C L3C3

- T 183

- 30 183 1 94.87 15.3 42 28.5

C L3C4

- T 184

- 30 184 5 40.97 17.1 6648.3

C L3C7

- T 187

- 30 187 6 & 6

- R1 102.745 13.8 8004

C L3C8

- T 188

- 30 188 6 & 6

- R1 30.19 13.8 8004

C L3C9

- T 189

- 30 189 6 & 6

- R1 42.23 13.8 8004

C L3C10

- T 190

- 30 190 7

- R1 49.9 15 7665.3

D L3D3

- T 193

- 15 193 9 25 16.65 3474.15

D L3D4

- T 194

- 30 194 2 63.87 18.6 9006.9

D L3D6

- T 196

- 15 196 9 157.31 16.65 3474.15

D L3D8

- T 198

- 30 198 7

- R1 38.93 15 7665.3

D L3D9

- T 199

- 30 199 7

- R1 108.09 15 7665.3

### ABC Study No. 66086

Page 255 of 261 Appendix N-2. T4 Data for Juvenile Males at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

detection

38.0 A L4A1

- T 201

- 30 201 4 63.43 13.2 7496.1

A L4A4

- T 204

- 15 204 9 8.325 16.65 3474.15

A L4A5

- T 205

- 30 205 5 46.06 17.1 6648.3

A L4A6

- T 206

- 30 206 8

- R1 47.57 12 6960.6

A L4A7

- T 207

- 30 207 8

- R1 35.87 12 6960.6

A L4A9

- T 209

- 30 209 2 41.76 18.6 9006.9

B L4B1

- T 211

- 30 211 6 & 6

- R1 156.54 13.8 8004

B L4B2

- T 212

- 15 212 9 88.12 16.65 3474.15

B L4B3

- T 213

- 30 213 6 & 6

- R1 34.295 13.8 8004

B L4B5

- T 215

- 30 215 8

- R1 49.92 12 6960.6

B L4B9

- T 219

- 15 219 9 78.38 16.65 3474.15

B L4B10

- T 220

- 15 220 9 8.325 16.65 3474.15

C L4C2

- T 222

- 30 222 8

- R1 43.81 12 6960.6

C L4C3

- T 223

- 30 223 8

- R1 83.18 12 6960.6

C L4C4

- T 224

- 30 224 8

- R1 90.75 12 6960.6

C L4C5

- T 225

- 30 225 1 47.14 15.3 4228.5

C L4C7

- T 227

- 30 227 1 50.88 15.3 4228.5

C L4C8

- T 228

- 30 228 6 & 6

- R1 16.56 13.8 8004

D L4D2

- T 232

- 15 232 9 47.7 16.65 3474.15

D L4D6

- T 236

- 30 236 9 132.76 33.3 6948 .3

D L4D8

- T 238

- 15 238 9 8.325 16.65 3474.15

D L4D9

- T 239

- 30 239 1 36.36 15.3 4228.5

### ABC Study No. 66086

Page 256 of 261 Appendix N-3. T4 Data for Juvenile Females at Study Termination , Based on Phenotypic Sex

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

detection

Control A CA2

- T 2

- 30 2 1 58.99 15.3 4228.5

A CA5

- T 5

- 30 5 5 66.81 17.1 6648.3

A CA6

- T 6

- 30 6 5 48.16 17.1 6648.3

A CA8

- T 8

- 30 8 9 147.02 33.3 6948.3

B CB2

- T 12

- 30 12 7

- R1 58.42 15 7665.3

B CB3

- T 13

- 30 13 2 126.17 18.6 9006.9

B CB5

- T 15

- 30 15 2 50.23 18.6 9006.9

B CB9

- T 19

- 30 19 9 289.28 33.3 6948.3

B CB10

- T 20

- 30 20 4 99.13 13.2 7496.1

C CC4

- T 24

- 30 24 1 69.26 15.3 4228.5

C CC6

- T 26

- 30 26 7

- R1 46.9 15 7665.3

C CC7

- T 27

- 30 27 3 34.39 15.9 7963.2

C CC8

- T 28

- 30 28 3 116.12 15.9 7963.2

D CD1

- T 31

- 30 31 9 93.38 33.3 6948.3

D CD2

- T 32

- 15 32 9 110.57 16.65 3474.15

D CD8

- T 38

- 30 38 8

- R1 45.88 12 6960.6

### ABC Study No. 66086

Page 257 of 261 Appendix N-3. T4 Data for Juvenile Females at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

detection

Control E CE1

- T 41

- 30 41 7

- R1 42.2 15 7665.3

E CE9

- T 49

- 15 49 9 38.94 16.65 3474.15

E CE10

- T 50

- 30 50 6 & 6

- R1 70.01 13.8 8004

F CF1

- T 51

- 30 51 1 44.18 15.3 4228.5

F CF2

- T 52

- 30 52 1 43.02 15.3 4228.5

F CF4

- T 54

- 30 54 7

- R1 140.62 15 7665.3

F CF6

- T 56

- 30 56 3 46.73 15.9 7963.2

F CF7

- T 57

- 30 57 3 80.16 15.9 7963.2

F CF10

- T 60

- 30 60 8

- R1 46.6 12 6960.6

G CG2

- T 62

- 30 62 7

- R1 35.19 15 7665.3

G CG4

- T 64

- 30 64 2 72.39 18.6 9006.9

G CG7

- T 67

- 15 67 9 90.73 16.65 3474.15

G CG10

- T 70

- 30 70 4 68.47 13.2 7496.1

H CH3

- T 73

- 30 73 8

- R1 41.86 12 6960.6

H CH5

- T 75

- 30 75 7

- R1 34.85 15 7665.3

H CH7

- T 77

- 30 77 7

- R1 25.8 15 7665.3

H CH10

- T 80

- 30 80 3 50.76 15.9 7963.2

### ABC Study No. 66086

Page 258 of 261 Appendix N-3. T4 Data for Juvenile Females at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

detection

4.97 A L1A1

- T 81

- 30 81 3 111.96 15.9 7963.2

A L1A3

- T 83

- 30 83 1 32.01 15.3 4228.5

A L1A5

- T 85

- 30 85 1 38.04 15.3 4228.5

A L1A7

- T 87

- 30 87 5 40.11 17.1 6648.3

A L1A8

- T 88

- 30 88 5 68.36 17.1 6648.3

A L1A10

- T 90

- 30 90 6 & 6

- R1 63.735 13.8 8004

B L1B1

- T 91

- 15 91 9 56.64 16.65 3474.15

B L1B2

- T 92

- 30 92 1 84.12 15.3 4228.5

B L1B6

- T 96

- 30 96 7

- R1 129.12 15 7665.3

B L1B7

- T 97

- 30 97 3 143.09 15.9 7963.2

B L1B9

- T 99

- 15 99 9 8.325 16.65 3474.15

C L1C2

- T 102

- 30 102 5 94.54 17.1 6648.3

C L1C4

- T 104

- 30 104 2 50.42 18.6 9006.9

C L1C5

- T 105

- 30 105 2 74.71 18.6 9006.9

C L1C7

- T 107

- 30 107 4 24.87 13.2 7496.1

C L1C9

- T 109

- 30 109 6 & 6

- R1 73.185 13.8 8004

C L1C10

- T 110

- 30 110 6 & 6

- R1 64.5 13.8 8004

D L1D2

- T 112

- 30 112 2 101.96 18.6 9006.9

D L1D4

- T 114

- 30 114 2 119.87 18.6 9006.9

D L1D6

- T 116

- 30 116 8

- R1 22.36 12 6960.6

D L1D8

- T 118

- 30 118 3 99 15.9 7963.2

### ABC Study No. 66086

Page 259 of 261 Appendix N-3. T4 Data for Juvenile Females at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

detection

10.3 A L2A1

- T 121

- 30 121 8

- R1 80.87 12 6960.6

A L2A8

- T 128

- 30 128 4 85.93 13.2 7496.1

B L2B2

- T 132

- 30 132 1 44.44 15.3 4228.5

B L2B3

- T 133

- 15 133 9 30.49 16.65 3474.15

B L2B6

- T 136

- 30 136 3 25.26 15.9 7963.2

B L2B7

- T 137

- 30 137 3 137.9 15.9 7963.2

C L2C1

- T 141

- 30 141 5 93.78 17.1 6648.3

C L2C3

- T 143

- 30 143 7

- R1 148.32 15 7665.3

C L2C7

- T 147

- 15 147 9 66.65 16.65 3474.15

C L2C10

- T 150

- 30 150 7

- R1 17.09 15 7665 .3

D L2D1

- T 151

- 30 151 3 231.11 15.9 7963.2

D L2D3

- T 153

- 30 153 3 88.86 15.9 7963.2

D L2D4

- T 154

- 30 154 7

- R1 28.97 15 7665.3

D L2D5

- T 155

- 30 155 8

- R1 6 12 6960.6

D L2D6

- T 156

- 30 156 2 22.91 18.6 9006.9

D L2D7

- T 157

- 30 157 2 80.08 18.6 9006.9

D L2D8

- T 158

- 30 158 2 88.16 18.6 9006.9

D L2D9

- T 159

- 30 159 5 57.71 17.1 6648.3

### ABC Study No. 66086

Page 260 of 261 Appendix N-3. T4 Data for Juvenile Females at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc

. (ng/mL) min.

detection max.

detection

19.5 A L3A1

- T 161

- 30 161 5 62.82 17.1 6648.3

A L3A2

- T 162

- 15 162 9 30.12 16.65 3474.15

A L3A3

- T 163

- 15 163 9 8.325 16.65 3474.15

A L3A8

- T 168

- 30 168 4 57.05 13.2 7496.1

A L3A9

- T 169

- 30 169 4 33.98 13.2 7496 .1

A L3A10

- T 170

- 30 170 7

- R1 28.04 15 7665.3

B L3B1

- T 171

- 30 171 7

- R1 95.73 15 7665.3

B L3B2

- T 172

- 30 172 2 42.59 18.6 9006.9

B L3B7

- T 177

- 30 177 5 18.47 17.1 6648.3

B L3B9

- T 179

- 30 179 8

- R1 6 12 6960.6

C L3C1

- T 181

- 15 181 9 8.325 16.65 3474.15

C L3C5

- T 185

- 30 185 5 35.01 17.1 6648.3

C L3C6

- T 186

- 30 186 5 70.7 17.1 6648.3

D L3D1

- T 191

- 30 191 8

- R1 45.7 12 6960.6

D L3D2

- T 192

- 30 192 2 69.3 18.6 9006.9

D L3D5

- T 195

- 30 195 6 & 6

- R1 45.625 13.8 8004

D L3D7

- T 197

- 15 197 9 97.94 16.65 3474. 15

D L3D10

- T 200

- 30 200 4 109 13.2 7496.1

### ABC Study No. 66086

Page 261 of 261 Appendix N-3. T4 Data for Juvenile Females at Study Termination , Based on Phenotypic Sex (continued)

Treatment

(μg/L) REP Frog Code Sample

ID Plasma Vial ID Plate # T4 Conc.

(ng/mL) min.

detection max.

detecti on

38.0 A L4A2

- T 202

- 30 202 4 38.24 13.2 7496.1

A L4A3

- T 203

- 30 203 5 183.11 17.1 6648.3

A L4A8

- T 208

- 30 208 2 39.81 18.6 9006.9

A L4A10

- T 210

- 30 210 2 63.67 18.6 9006.9

B L4B4

- T 214

- 30 214 8

- R1 73.37 12 6960.6

B L4B6

- T 216

- 30 216 2 134.75 18.6 9006.9

B L4B7

- T 217

- 30 217 2 47.59 18.6 9006.9

B L4B8

- T 218

- 30 218 9 44.81 33.3 6948.3

C L4C1

- T 221

- 15 221 9 42.06 16.65 3474.15

C L4C6

- T 226

- 30 226 1 47.34 15.3 4228.5

C L4C9

- T 229

- 30 229 6 & 6

- R1 32.43 13.8 8004

C L4C10

- T 230

- 15 230 9 145.98 16.65 3474.15

D L4D1

- T 231

- 30 231 4 72.78 13.2 7496.1

D L4D3

- T 233

- 30 233 4 34.65 13.2 7496.1

D L4D4

- T 234

- 15 234 9 24.16 16.65 3474.15

D L4D5

- T 235

- 30 235 5 77.26 17.1 6648.3

D L4D7

- T 237

- 30 237 8

- R1 41.08 12 6960.6

D L4D10

- T 240

- 30 240 9 100 .5 33.3 6948.3