# Page 1 of 52 ADDENDUM TO FINAL REPORT TITLE D

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

AUTHOR

Simon Dinehart, Ph.D.

STUDY COMPLETION DATE

28 December 2012

ADDENDUM COMPLETION DATE

09 April 2013

SPONSOR

Battelle

- Pacific Northwest Division

Marine Research Operations

1529 West Sequim Bay Road

Sequim, Washington 98382

PERFORMING LABORATOR IES

ABC Laboratories, Inc.

7200 E. ABC Lane

Columbia, Missouri 65202

United States

Battelle

- Pacific Northwest Division

Marine Research Oper ations

1529 West Sequim Bay Road

Sequim, Washington 98382

PROJECT IDENTIFICATION

ABC Study No. 66086

# ABC Study No. 66086

Page 3 of 52 STUDY PERSONNEL

Study Director

Simon Dinehart, Ph.D., Toxicologist

ABC Laboratories, Inc.

7200 E. ABC Lane

Columbia, Missouri 65202

Principal Investigator s

Statistics: Dr. Valerie Cullinan (Battelle

- Pacific Northwest Division)

# ABC Study No. 66086

Page 4 of 52 TABLE OF CONTENTS

Page

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

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

STUDY PERSONNEL ....................................................................................................................3

TABLE OF CONTENTS .................................................................................................................4

LIST OF TABLES ...........................................................................................................................4

LIST OF FIGURES .........................................................................................................................5

LIST OF APPENDICES ..................................................................................................................6

1.0 INTRODUCTION ...............................................................................................................7

2.0 STATISTICAL ANALYSES ..............................................................................................7

3.0 BIOLOGICAL RESULTS AND DISCUSSION .................................................................8

3.1 Growth at Termination: ............................................................................................8

3.2 Nuptial Pad Development at Termination: ............................................................11

3.3 Live r Weight at Termination: ................................................................................13

3.4 Genotypic/Phenotypic Sex Ratios..........................................................................15

3.5 Plasma Thyroxine (T4) Results .............................................................................15

4.0 CONCLUSIONS ................................................................................................................17

LIST OF TABLES

Page

Table 3.1.1. Mean (and standard devia tion) whole body weight and snout

- to

- vent length

at the termination of the Xenopus laevis 4- tert -octylphenol exposure. ...................9

Table 3.2.1. Mean (standard deviation) proportion of female and male frogs and total

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

4- tert -octylphenol exposure. ..................................................................................12

Table 3.2.2. Proportion of total frogs within each treatment having nuptial pads at the

termination of the Xenopus laev is 4

- tert -octylphenol exposure. ...........................13

Table 3.3.1. Mean (and standard deviation) liver weight:whole body weight for male and

female f rogs at the termination of the Xenopus laevis 4- tert -octylphenol

exposure. ................................................................................................................14

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

laevis 4- tert -octylphenol exposure. ........................................................................15

Table 3.5.1. Mean (and standard deviation) plasma thyroxine (T4) for frogs at the

termination of the Xenopus laevis 4- tert -octylphenol exposure. ...........................16

# ABC Study No. 66086

Page 5 of 52 Table 4.0.1. Calculated No -Observable- Effect Concentration and Lowest

- Observable

Effect Concentration based on mean measured 4

- tert -octylphenol

concentrations for parameter measured for Xenopus laevis exposed to 4

- tert

octylphenol. ............................................................................................................17

LIST OF FIGURES

Page

Figure 3.1.1. Boxplot of whole body weight for male frogs at the termination of the

Xenopus laevis 4- tert

- octylphenol exposure. The box represen ts the

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

median. Gender is based on genotypic sex. ............................................................9

Figure 3.1.2. Boxplot of whole body weight for female frogs at the termination of the

Xenopus laevis 4- tert

- octylphenol exposure. The box represents the

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

median. Gender is based on genotypic sex. ..........................................................10

Figure 3.1.3. 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. ..........................................................10

Figure 3.1.4. 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 horizo ntal line is the

median. Gender is based on genotypic sex. ..........................................................11

Figure 3.2.1. 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. ..........................................12

Figure 3.2.2. Boxplot of the proportion of female frogs having nuptial pads at the

termination of the Xenopus laevis 4- tert

- octylphenol exposure. The box

represent s the interquartile range, the circle is the mean, and the horizontal

line is the median. Gender is based on genotypic sex. ..........................................13

Figure 3.3.1. 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. ..........................................................14

Figure 3.3.2. Boxplot of liver weight:whole body weight for female frogs at the

termination of the Xenopus laevis 4- tert

- octylphenol exposure. The box

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

line is the median. Gender is based on genotypic sex. ..........................................15

# ABC Study No. 66086

Page 6 of 52 Figure 3.5.1. Boxplot of plasma thyroxine concentration for male frogs at the termination

of the Xenopus laevis 4- tert -oc tylphenol 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. ........................16

Figure 3.5.2. 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 ci rcle is the mean, and the horizontal

line is the median. Gender is based on genotypic sex. ..........................................17

LIST OF APPENDICES

Page

APPENDIX A

– AMENDED STATISTICAL REPORT .............................................................18

APPENDIX B

– TERMINATION DATA

– GROWTH AND GENDER ....................................42

# ABC Study No. 66086

Page 7 of 52 1.0 INTRODUCTION

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

Disruptor Screening Program (EDSP)

. To support this program, U.S. EPA has contracted with

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

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

standardizing, and validating an ecotoxicological test for identifying and characterizing

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

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

for obtain ing technical support for the Larval Amphibian Growth and Development Assay

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

endocrine disrupting in vertebrates.

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

use in examining adverse responses in amphibians exposed to vertebrate

- relevant endocrine

disrupting compounds. The chemical 4- tert -octylphenol is a known endocrine

- disruptor and was

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

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

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

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

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

Th is addendum to the final report issued December 19, 2012 was prepare d to incorporate data

changes to 43 genotypic sex determinations for 43 frogs sampled at test termination. These data

changes reflect the g enetic analyses completed by U.S. EPA in Duluth, Minnesota , after

December 19, 2012. The specific statistical analy ses that were affected by these data changes

are whole body weight, snout

- to

- vent length, nuptial pad presence/absence, liver weight:body

weight ratios, plasma thyroxine levels, and genetic sex ratios . Statistical methods utilized and

the final report sections impacted by these data changes are discussed in this addendum.

2.0 STATISTICAL ANALYSES

Parametric assumptions were tested using the residuals from a one

- way analysis of variance

(ANOVA) of the treatment means of the raw variable or following a tran sformation. 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

tra nsformed 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 n ormality and

homogeneity of within class variances were met, otherwise the step

- down Jonckheere

- Terpstra

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

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

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

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

- Wallis (K

- W) test followed by a

step

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

# ABC Study No. 66086

Page 8 of 52 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 Jonckheere

Terpstra nonparametric test of ordered alternatives was run, however, because this test does not

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

treatments (n=4), the LOEC/ NOEC was 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 o f within class variances were met. When the ANOVA (a comparison among all

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

between the control and treatment doses. The K

- W test was used when assumptions of normality

a nd 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 LO EC/NOEC. No

identified outliers were removed from the analyses. A binomial probability was calculated on

the incidence of nuptial pads with the expected proportion of nuptial pad occurrence calculated

from the mean of the control replicates. Significance was based on α = 0.05. Descriptive

statistics included the mean, standard deviation (StDev), and the coefficient of variation (CV) of

replicates within treatments. The mean and standard deviation of the treatment

- related responses

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

detected by the K

- W test or the K

- W multiple comparisons test were between median values.

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

- T

and CE7

- T 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. Statistics were done and graphs prepared in Minitab v16, except for the ana lytical

chemistry data and nuptial pad binomial probability, which were analyzed in Microsoft Excel.

The statistical report from Battelle is presented in Appendix A

.

3.0 BIOLOGICAL RESULTS AND DISCUSSION

3.1 G rowth at Termination:

The whole body (wet) weight, snout

- to

- vent length, phenotypic sex, and corrected genotypic sex

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

fr ogs measured at the termination of the 4

- tert

- octylphenol exposure was 10. 1 g in the control

treatment and ranged from 10.0 to 10.8 g in the 4

- tert

- octylphenol treatments ( Table 3.1.1 ;

Figure 3. 1.1 ). Mean whole body (wet) weight of female frogs measured at the termination of the

4- tert -octylphenol exposure was 10.8 g in the control treatment and ranged from 9.16 to 11.4 g in

the 4

- tert

- octylphenol treatments ( Table 3.1.1 ; Figure 3.1.2 ). 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 measu red at the termination of the 4

- tert -octylphenol

exposure was 42 mm in the control treatment and ranged from 41 to 44 mm in the 4

- tert

octylphenol treatments ( Table 3.1.1 ; Figure 3.1.3 ). Signi ficant differences among treatments

were detected in snout- to

- vent length of male frogs at the termination of the exposure. However,

# ABC Study No. 66086

Page 9 of 52 the K- W comparisons to control test determined that there were no differences in mean values

between any 4

- tert -octylpheno l treatment and the control treatment. Mean 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 3.1.1 ;

Figure 3.1.4 ). 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.

Table 3. 1.1. 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 10.1 (0.93) 10.8 (1.33) 42 (1.7) 42 (1.9)

4.97 10.0 (0.35) 11.4 (0.82) 41 (0.4) 44 (1.0)

10.3 10.4 (0.40) 10.6 (1.40) 42 (0.7) 44 (2.5)

19.5 10.8 (0.74) 9.16 (2.36) 44 (0.5) 42 (3.3 )

38.0 10.2 (1.14) 10.9 (2.84) 42 (0.8) 43 (4.0)

p

- value 0.571 0.654 0.040 0.557

Test: K

- W K

- W K

- W K

- W

Note: Gender is based on genotypic sex.

38.00 19.50 10.30 4.97 0.00 11.5

11.0

10.5

10.0

9.5

9.0

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



## Figure 3. 1.1. Boxplot of whole body weight for male frogs at the ter mination 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.

# ABC Study No. 66086

Page 10 of 52 38.0 19.5 10.3 4.97 0.00 14

13

12

11

10

9

8

7

Mean Me asured D ose (μg/L )Whole Body Weight (g)



## Figure 3. 1.2. Boxp lot of whole body weight for female frogs at the termination of the Xenopus laevis

4- tert

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

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

38.0 19.5 10.3 4.97 0.00 44

43

42

41

40

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



## Figure 3. 1.3

. 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 horizonta l line is the median. Gender is based on genotypic sex.

# ABC Study No. 66086

Page 11 of 52 38.0 19.5 10.3 4.97 0.00 47

46

45

44

43

42

41

40

39

38

Mean Me asured Do se (μg/L )Snout-to-Vent Length (mm)



## Figure 3. 1.4. Boxplot of snout

- to

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

4- tert

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

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



# 3.2 Nuptial Pad Development at Termination:

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

Appendix B

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

nuptial pads at the termination of the exposure were 0.2 3 and 0. 20 , respectively. The mean

proportions of male and fe male frogs in the 4

- tert

- octylphenol treatments that had nuptial pads at

the termination of the exposure ranged from 0.2 1 to 0.5 0 and from 0. 29 to 0. 51 , respectively

( Table 3.2.1 ; Figure 3.2.1 ; Figure 3.2.2 ). 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 ( Tab le 3.2.2 ). 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 = 0.2 1 for males, 0. 20 for females; n = 8). The

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

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

concentration. The proportion of males in the 10.3

- μg/L 4

- tert

- octylphenol concentration having

nuptial pads was 0.5 2 , which was considered significantly greater than chance ( p < 0.001). The

proportions of females in the 4.97

- , 10.3

-, and 38.0

- μg/L 4

- tert

- octylphenol concentrations

having nuptial pads were 0.50, 0.41, and 0.50, respective ly, which were considered significantly

greater than chance ( p = 0.00 1, 0.016, and 0.002, respectively).

# ABC Study No. 66086

Page 12 of 52 Table 3. 2.1. Mean (standard deviation) proportion of female and male frogs and total number of frogs

having nuptial pads at the termination of the X enopus laevis 4- tert

- octylphenol exposure.

Mean Measured

Concentration

(μg/L) N Nuptial Pads

(proportion of

females) Nuptial Pads

(proportion of males) Total Number of Frogs

with Nuptial Pads

<MQL 78 0.23 (0.25) 0.20 (0.17) 16

4.97 39 0.21 (0.17) 0.51 (0 .23) 14

10.3 40 0.50 (0.18) 0.45 (0.18) 19

19.5 39 1 

## 0.33 (0.23) 0.29 (0.22) 12

38.0 40 0.28 (0.28) 0.45 (0.33) 15

p

- value

– 0.360 0.137

–

Test: – 1- way ANOVA 1- way ANOVA

–

1 

## One female in replicate C was missing forelimbs

Note: Gender is bas ed on genotypic sex.

38.00 19.50 10.30 4.97 0.00 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. 2.1. 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

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

# ABC Study No. 66086

Page 13 of 52 38.00 19.50 10.30 4.97 0.00 0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Mean Me asured D ose (μg/ L)Proportion of Females with Nuptial Pads



## Figure 3. 2.2. Boxplot of the proportion of female frogs having nuptial pads 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.

Table 3. 2.2. Proportion of total frogs within each treatment having nuptial pads at the termination of the

Xenopus laevis 4

- tert

- octylphenol exposure



## .

Mean Measured

Concentration

(μg/L) N Proportion of Total

Males with Nuptial

Pads Binomial

Probability Proportion of

Total Females

with Nuptial Pads Binomial

Probability

<MQL 78 0.20

– 0.21

–

4.97 39 0.21 0.545 0.50 0.001

10.3 40 0.52 0.001 0.41 0.016

19.5 39 1 

## 0.36 0.056 0.24 0.284

38.0 40 0.29 0.189 0.50 0.002

1 

## One female in replicate C was missing forelimbs

Note: Gender is based on genotypic sex.



# 3.3 Liver Weight at Termination:

The liver (w et) weight for each frog at termination is presented in Appendix B

. Mean liver

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

the 4

- tert

- octylphenol exposure were 0.058 for males and 0.060 for females. The ratio for males

ranged from 0.056 to 0.062 in the 4

- tert

- octylphenol treatments ( Table 3.3.1 ; Figure 3.3.1 ). The

ratio for females ranged from 0.056 to 0.063 in the 4

- tert

- octylphenol treatments ( Table 3.3.1 ;

Figure 3.3.2 ). 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.

# ABC Study No. 66086

Page 14 of 52 Table 3. 3.1. 

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

frogs at the termination of the Xenopus laevis 4- tert

- octylphenol exposure.

Mean Measured

Concentration (μg/L) Liver

Weight:Whole Body

Weight (Males) Liver

Weight:Whole Body

Weight (Females)

<MQL 0.058 (0.003) 0.060 (0.006)

4.97 0.056 (0.004) 0.059 (0.003)

10.3 0.058 (0.006) 0.063 (0.002)

19.5 0.062 (0.003) 0.056 (0.005)

38.0 0.058 (0.004) 0.056 (0.002)

p

- value 0.196 0.187

Test: K

- W K

- W

Note: Gender is based on genotypic sex.

38.00 19.50 10.30 4.97 0.00 0.066

0.064

0.062

0.060

0.058

0.056

0.054

0.052

0.050

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



## Figure 3. 3.1. 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.

# ABC Study No. 66086

Page 15 of 52 38.0 19.5 10.3 4.97 0.00 0.070

0.065

0.060

0.055

0.050

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



## Figure 3.3.2. Boxplot of liver weig ht:whole body weight for female frogs at the termination of the

Xenopus laevis 4- tert -octylphenol exposure. The box rep resents the interqua rtile range, the

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



# 3.4 Genotypic/Phenotypic Sex Ratios

Detailed results of genetic sexing of the juveni le frogs using PCR follo wed by gel separation are

presented in Appendix B . The phenotypic and genotypic sex ratios at the termination differed

within each dose ( Table 3.4.1 ). The genotypic sex determinati ons of five individuals differed

from their phenotypic determinations.

Table 3.4.1. Frog ph enotypic and genotypic 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 33 45 0

4.97 39 21 18 20 19 2

10.3 40 18 22 17 23 1

19.5 40 18 22 18 22 0

38.0 40 18 22 16 24 2

1 

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



# 3.5 Plasma Thyroxine (T4) Results

The mean plasma T4 concentrations for control-treatment male and female frogs at the

termination of the 4- tert -octylphenol exposure were 67.5 and 72.1 ng/mL, respectively

( Table 3.5.1 ; Figure 3.5.1 ). Plasma T4 concentrations for frogs in the 4- tert -octylphenol

# ABC Study No. 66086

Page 16 of 52 treatments ranged from 55.4 to 79.4 ng/mL for males and from 47.2 to 72.6 ng/mL for females

( Table 3.5.1 ; Figure 3.5.2 ). 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 B

.

Table 3.5.1. Mean (and st andard deviation) plasma thyroxine (T4) for frogs at the termination of the

Xenopus laevis 4- tert -octylphenol exposure.

Mean Measured

Concentration (μg/L) Male Plasma

Thyroxine (T4)

(ng/mL) Female Plasma

Thyroxine (T4)

(ng/mL)

<MQL 67.5 (28.9) 72.1 (26.8)

4.97 59.7 (16.3) 74.6 (12.1)

10.3 65.9 (10.5) 76.2 (12.0)

19.5 70.2 (22.8) 47.2 (17.6)

38.0 55.4 (12.1) 72.6 (11.6)

p -value 0.852 0.184

Test: 1-Way ANOVA K-W

Note: Gender is based on genotypic sex.

38.0 19.5 10.3 4.97 0.00 110

100

90

80

70

60

50

40

30

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



## Figure 3.5.1. Boxplot of plasma thyroxine concentration for male frogs at the termination of the

Xenopus laevis 4- tert -octylphenol exposure. The box repres ents 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.

# ABC Study No. 66086

Page 17 of 52 38.0 19.5 10.3 4.97 0.00 130

120

110

100

90

80

70

60

50

40

Mean Me asured D ose (μg/L )Thyroxine Concentration (ng/mL)



## Figure 3. 5.2. Boxplot of plasma thyroxin e 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.



# 4.0 CONCLUSIONS

The no-observable- effect concentration and lowest -observable- effect concentration were

calculated as mean measured concentrations of 38.0 μg/L 4

- tert

- octylphenol and >38.0 μg/L

4- tert

- octylphenol, respectively, for each Xenopus laevis parameter. ( Table 4.0.1 ). These NOEC

and LOEC values do not differ from those presented in the final report issues Dec 19, 2012.

Table 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

- Effect

Concentration (μg/L) Lowest

- Observable

Effect Concentration

(μg/L)

Frogs at Termination

Whole Bod y Weight (Male) 38.0 >38.0

Whole Body Weight (Female) 38.0 >38.0

Snout

- to

- Vent Length (Male) 1 

## 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

Nup tial 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

1 

# K- W test was significant ( p = 0.04 0), but there were no differences between any treatment

and the control ( p > 0.013).

# ABC Study No. 66086

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# ABC Study No. 66086

Page 42 of 52 APPENDIX B – TERMINATION DATA

– GROWTH AND GENDER

# ABC Study No. 66086

Page 43 of 52 ID Tank

(Rep) Conc

. Sample

(62, 66,

Ter m) Body

Weight

(g) Snout- Vent

Length

(mm) T4

(ng/mL) VTG

(ng/mL) Pheno

Sex

(M, F,

I) Battelle

Geno

Sex EPA

Duluth

Geno sex Corrected

Geno Sex Nuptial

pad

(Y, N) Liver

Cassette

Tare

Weight

(g) Liver +

Cassette

Weight

(g) Liver

Weight

(g) Note

CA1-T A CON Term 8.369 40 65.89 9227.7 M M M N 3.392 3.8291 0.4371 spinal curvature

CA2-T A CON Term 10.216 40 58.99 9227.7 F F F N 3.3972 3.9102 0.5130

CA3-T A CON Term 8.302 39 35.39 9227.7 M M M N 3.4099 4.0069 0.5970

CA4-T A CON Term 6.430 36 68.5 17144.91 M M M N 3.3738 3.6852 0.3114

CA5-T A CON Term 14.852 46 66.81 17144.91 F F F Y 3.401 4.3996 0.9986

CA6-T A CON Term 12.185 45 48.16 17144.91 F F F N 3.3904 4.1986 0.8082

CA7-T A CON Term 12.724 46 169.3 8999.685 M M M Y 3.4009 4.1249 0.724 0

CA8-T A CON Term 7.641 38 147.02 0 F F F N 3.3963 3.8039 0.4076

CA9-T A CON Term 10.427 41 70.01 0 M M M N 3.3954 3.9459 0.5505

CA10-T A CON Term 8.250 37 242.23 0 M M M N 3.4019 3.8365 0.4346

CB1 -T B CON Term 8.112 39 24.79 8999.685 M M M N 3.4028 3.7974 0.3946

CB2 -T B CON Term 10.430 42 58.42 8999.685 F F F N 3.4051 4.0654 0.6603

CB3 -T B CON Term 15.727 51 126.17 7772.265 F F F Y 3.3836 4.2982 0.9146

CB4 -T B CON Term 6.800 36 25.67 0 M M M M N 3.3947 3.7300 0.3353

CB5 -T B CON Term 10.295 42 50.23 0 F F F F N 3.3986 3.9408 0.5422

CB6 -T B CON Term 14.757 44 66.38 50113.582 M M M Y 3.3989 4.4616 1.0627

CB7 -T B CON Term 7.918 40 29.63 17144.91 M M M N 3.3905 3.8233 0.4328

CB8 -T B CON Term 9.079 40 26.25 64891.308 M M M N 3.3956 3.9236 0.5280 testes reduced in

size and darkly

pigmented

CB9 -T B CON Term 10.543 42 289.28 40189.923 F F F N 3.3837 3.8952 0.5115

CB10 -T B CON Term 13.247 47 99.13 16495.74 F F F Y 3.3898 4.1645 0.7747

CC1 -T C CON Term 9.904 41 70.45 16495.74 M M M N 3.3940 3.9602 0.5662

CC2 -T C CON Term 9.811 40 67.02 8999.685 M M M N 3.4015 4.0012 0.5997 abnormal

coloration

“ . ” denotes a juvenile was not available for this sampl e.

# ABC Study No. 66086

Page 44 of 52 ID Tank

(Rep) Conc

. Sample

(62, 66,

Term) Body

Weight

(g) Snout- Vent

Length

(mm) T4

(ng/mL) VTG

(ng/mL) Pheno

Sex

(M, F,

I) Battelle

Geno

Sex EPA

Duluth

Geno sex Corrected

Geno Sex Nuptial

pad

(Y, N) Liver

Cassette

Tare

Weight

(g) Liver +

Cassette

Weight

(g) Liver

Weight

(g) Note

CC3 -T C CON Term 9.579 41 46.53 9227.7 M M M Y 3.3946 3.9041 0.5095 Patchy white and

brown skin

pattern.

CC4 -T C CON Term 12.010 45 69.26 9227.7 F F F N 3.3995 4.3122 0.9127

CC5 -T C CON Term 16.785 48 172.67 9227.7 M M M Y 3.3907 4.4649 1.0742

CC6 -T C CON Term 8.968 40 46.9 8999.685 F F F N 3.3979 3.6305 0.2326

CC7 -T C CON Term 5.727 36 34.39 0 F F F N 3.3940 4.0466 0.6526

CC8 -T C CON Term 7.786 37 116.12 0 F F F N 3.4069 3.7947 0.3878 spinal curvature

CC9 -T C CON Term 10.468 41 58.77 14221.08 M M M N 3.3838 3.9949 0.6111

CC10 -T C CON Term

. . no sample no sample

. . . . . . .

CD1-T D CON Term 11.268 42 93.38 0 F F F N 3.4077 4.1896 0.7819

CD2-T D CON Term 11.920 42 110.57 0 F F F N 3.4013 4.2831 0.8818

CD3-T D CON Term 5.464 35 25.36 0 M M M N 3.4009 3.6417 0.2408

CD4-T D CON Term 7.899 37 44.84 9156.555 M M M N 3.3972 3.9021 0.5049

CD5-T D CON Term 11.617 44 49.65 0 M M M N 3.4000 4.2181 0.8181

CD6-T D CON Term 7.410 39 23.88 0 M M M N 3.4015 3.7779 0.3764

CD7-T D CON Term 12.015 45 37.32 0 M M M N 3.4014 4.2463 0.8449

CD8-T D CON Term 11.805 43 45.88 9156.555 F F F N 3.3981 4.1062 0.7081

CD9-T D CON Term 11.789 42 99.43 19905.85 M M M N 3.3998 4.1195 0.7197

CD10-T D CON Term

. . no sample no sample

. . . . . . .

CE1 -T E CON Term 8.297 40 42.2 8999 .685 F F F N 3.3971 3.8453 0.4482

CE2 -T E CON Term 11.321 45 34.77 16495.74 M M M N 3.4020 4.1695 0.7675

CE3 -T E CON Term 14.420 47 76.21 16495.74 M M M Y 3.3997 4.2736 0.8739

CE4 -T E CON Term 12.117 44 59.93 16495.74 M M M N 3.3733 4.2494 0.8761

CE5 -T E CON Term 6.819 40 31.45 17144.91 M M M N 3.4052 3.7816 0.3764

“ . ” denotes a juvenile was not available for this sampl e.

# ABC Study No. 66086

Page 45 of 52 ID Tank

(Rep) Conc

. Sample

(62, 66,

Term) Body

Weight

(g) Snout- Vent

Length

(mm) T4

(ng/mL) VTG

(ng/mL) Pheno

Sex

(M, F,

I) Battelle

Geno

Sex EPA

Duluth

Geno sex Corrected

Geno Sex Nuptial

pad

(Y, N) Liver

Cassette

Tare

Weight

(g) Liver +

Cassette

Weight

(g) Liver

Weight

(g) Note

CE6 -T E CON Term 12.106 47 38.91 17144.91 M M M N 3.3917 4.2030 0.8113

CE7 -T E CON Term 6.290 38 41.36 17144.91 M F M M N 3.3890 3.7088 0.3198

CE8 -T E CON Term 8.824 39 16.545 0 M M M N 3.4015 3.9461 0.5446

CE9 -T E CON Term 8.036 37 38.94 0 F F F Y 3.4097 3.8522 0.4425

CE10 -T E CON Term 10.742 43 70.01 0 F F F N 3.3860 3.9249 0.5389

CF1-T F CON Term 8.240 38 44.18 9227.7 F F F N 3.3854 3.8024 0.4170 spinal curvature

CF2-T F CON Term 9.185 41 43.02 9227.7 F F F N 3.3883 3.9478 0.5595

CF3-T F CON Term 10.020 41 24.95 9227.7 M M M Y 3.3896 3.9454 0.5558 darkly pigmented

test es

CF4-T F CON Term 18.080 50 140.62 8999.685 F F F Y 3.4001 4.5907 1.1906

CF5-T F CON Term 14.757 47 78.15 9156.555 M M M Y 3.4090 4.2431 0.8341

CF6-T F CON Term 5.412 35 46.73 0 F F F N 3.3959 3.6573 0.2614

CF7-T F CON Term 14.354 49 80.16 0 F F F Y 3.4005 4.1791 0.7786

CF8-T F CON Term 12.040 46 86.42 0 M M M Y 3.4007 4.0775 0.6768

CF9-T F CON Term 9.425 43 57.08 38540.873 M M M N 3.4009 3.9170 0.5161

CF10-T F CON Term 10.380 42 46.6 9156.555 F F F N 3.4071 3.9838 0.5767

CG1-T G CON Term 7.486 38 30.11 8999.685 M M M N 3.3485 3.7334 0.3849

CG2-T G CON Term 8.760 41 35.19 8999.685 F F F N 3.3805 3.9223 0.5418

CG3-T G CON Term 7.158 37 43.32 0 M M M N 3.3954 3.7771 0.3817

CG4-T G CON Term 15.598 47 72.39 0 F F F N 3.3762 4.4591 1.0829

CG5-T G CON Term 9.373 41 91.87 42828.694 M M M N 3.3782 3.9813 0.6031

CG6-T G CON Term 13.189 46 264.94 19379.25 M F M M N 3.3647 4.0931 0.7284

CG7-T G CON Term 11.110 43 90.73 0 F F F N 3.3507 4.0327 0.6820

CG8-T G CON Term 8.870 38 1 29.085 0 M F M M N 3.3449 3.8750 0.5301

CG9-T G CON Term 12.122 43 85.45 8999.685 M F M M N 3.3684 3.9886 0.6202

“ . ” denotes a juvenile was not available for this sampl e.

# ABC Study No. 66086

Page 46 of 52 ID Tank

(Rep) Conc

. Sample

(62, 66,

Term) Body

Weight

(g) Snout- Vent

Length

( mm) T4

(ng/mL) VTG

(ng/mL) Pheno

Sex

(M, F,

I) Battelle

Geno

Sex EPA

Duluth

Geno sex Corrected

Geno Sex Nuptial

pad

(Y, N) Liver

Cassette

Tare

Weight

(g) Liver +

Cassette

Weight

(g) Liver

Weight

(g) Note

CG10-T G CON Term 13.357 47 68.47 16495.74 F F F N 3.3492 4.1987 0.8495

CH1-T H CON Term 13.053 48 101.97 16495.74 M M M Y 3.3814 4.0755 0.6941

CH2-T H CON Term 8.177 41 42.32 16495.74 M M M N 3.3578 3.7730 0.4152

CH3-T H CON Term 9.542 42 41.86 9156.555 F F F N 3.3800 3.9235 0.5435

CH4-T H CON Term 11.280 44 59.77 9156.555 M M M N 3.3813 4.0287 0.6474

CH5-T H CON Term 10.628 44 34.85 8999.685 F F F N 3.3645 3.9271 0.5626

CH6-T H CON Term 11.007 42 20.35 8999.685 M M M N 3.3787 3.9833 0.6046

CH7-T H CON Term 14.608 47 25.8 8999.68 5 F F F Y 3.3517 4.1361 0.7844

CH8-T H CON Term 10.807 45 62.57 9156.555 M M M N 3.3505 3.8742 0.5237

CH9-T H CON Term 8.495 39 45.85 0 M M M N 3.3673 3.9230 0.5557

CH10-T H CON Term 7.517 39 50.76 14221.08 F F F N 3.3784 3.7718 0.3934

L1A1 -T A L1 Term 11.733 46 111.96 0 F F F N 3.3811 4.1898 0.8087

L1A2 -T A L1 Term 12.736 46 185.63 8999.685 M M M Y 3.3761 4.0765 0.7004

L1A3 -T A L1 Term 10.561 43 32.01 9227.7 F M M M Y 3.3619 3.9902 0.6283

L1A4 -T A L1 Term 10.194 40 82.9 9227.7 M M M N 3.3663 3.9858 0.6195

L1A5 -T A L1 Term 7.088 38 38.04 9227.7 F F F N 3.3802 3.7608 0.3806

L1A6 -T A L1 Term 7.677 35 31.78 17144.91 M M M N 3.3736 3.8356 0.4620

L1A7 -T A L1 Term 16.235 49 40.11 17144.91 F F F Y 3.3457 4.3554 1.0097

L1A8 -T A L1 Ter m 9.815 41 68.36 17144.91 F F F N 3.3644 3.8580 0.4936

L1A9 -T A L1 Term 9.865 40 20.47 0 M M M N 3.3674 4.0677 0.7003

L1A10 -T A L1 Term 7.881 38 63.735 0 F F F N 3.3682 3.8246 0.4564

L1B1 -T B L1 Term 7.763 40 56.64 0 F F F N 3.3690 3.8148 0.4458

L1B2 -T B L1 Term 18.255 50 84.12 9227.7 F F F Y 3.3677 4.6705 1.3028

L1B3 -T B L1 Term 11.119 42 52.1 9227.7 M M M N 3.3649 3.9960 0.6311

L1B4 -T B L1 Term 12.503 46 25.57 9227.7 M M M N 3.3780 4.0267 0.6487

“ . ” denotes a juvenile was not available for this sampl e.

# ABC Study No. 66086

Page 47 of 52 ID Tank

(Rep) Conc

. Sample

(62, 66,

Term) Body

Weight

(g) Snout- Vent

Length

(mm) T4

(ng/mL) VTG

(ng/mL) Pheno

Sex

(M, F,

I) Battelle

Geno

Sex EPA

Duluth

Geno sex Corrected

Geno Sex Nuptial

pad

(Y, N) Liver

Cassette

Tare

Weight

(g ) Liver +

Cassette

Weight

(g) Liver

Weight

(g) Note

L1B5 -T B L1 Term 7.117 36 47.06 8999.685 M M M N 3.3710 3.7808 0.4098

L1B6 -T B L1 Term 13.321 47 129.12 8999.685 F F F Y 3.3676 4.1925 0.8249

L1B7 -T B L1 Term 12.234 45 143.09 0 F F F Y 3.3784 4 .1232 0.7448

L1B8 -T B L1 Term 12.515 46 58.44 0 M M M N 3.3673 4.0699 0.7026

L1B9 -T B L1 Term 7.827 39 8.325 0 F F F N 3.3662 3.8625 0.4963

L1B10 -T B L1 Term 4.262 31 8.55 17144.91 M F M M Y 3.3663 3.5730 0.2067

L1C1 -T C L1 Term 11.386 43 30.29 3 6151.126 M M M N 3.3746 3.9639 0.5893

L1C2 -T C L1 Term 16.404 50 94.54 17144.91 F M F F Y 3.3684 4.3675 0.9991

L1C3 -T C L1 Term 11.801 45 26.08 0 M M M Y 3.3716 4.0283 0.6567

L1C4 -T C L1 Term 9.136 40 50.42 0 F M F F N 3.3711 3.9570 0.5859

L1C5 -T C L1 Term 11.392 43 74.71 0 F M F F N 3.3659 4.0204 0.6545

L1C6 -T C L1 Term 11.827 42 97.24 16495.74 M F M M N 3.3522 3.9878 0.6356 spinal curvature

L1C7 -T C L1 Term 9.459 38 24.87 16495.74 F F F N 3.3825 3.9272 0.5447 spinal curvature

L1C8 -T C L1 Te rm 5.973 35 69.73 16495.74 M M M N 3.3480 3.6558 0.3078

L1C9 -T C L1 Term 12.992 48 73.185 0 F F F Y 3.3765 4.1191 0.7426

L1C10 -T C L1 Term 14.448 49 64.5 0 F F F Y 3.3487 4.2130 0.8643

L1D1 -T D L1 Term 7.404 39 56.49 0 M F M M N 3.3675 3.8115 0.4440

L1D2 -T D L1 Term 10.226 42 101.96 7772.265 F M F F Y 3.3462 3.8721 0.5259

L1D3 -T D L1 Term 10.794 41 78.36 0 M M M N 3.3795 3.9676 0.5881

L1D4 -T D L1 Term 11.822 45 119.87 0 F M F F Y 3.3625 4.0079 0.6454

L1D5 -T D L1 Term 9.087 40 42.31 9156.55 5 M M M N 3.3510 3.7779 0.4269

L1D6 -T D L1 Term 12.459 46 22.36 9156.555 F F F Y 3.3421 4.0248 0.6827

L1D7 -T D L1 Term 12.368 43 165.72 0 M M M N 3.3637 4.1433 0.7796 spinal curvature

L1D8 -T D L1 Term 9.078 40 99 0 F M F F N 3.3664 3.9023 0.5359

“ . ” denotes a juvenile was not available for this sampl e.

# ABC Study No. 66086

Page 48 of 52 ID Tank

(Rep) Conc

. Sample

(62, 66,

Term) Body

Weight

(g) Snout- Vent

Length

(mm) T4

(ng/mL) VTG

(ng/mL) Pheno

Sex

(M, F,

I) Battelle

Geno

Sex EPA

Duluth

Geno sex Corrected

Geno Sex Nuptial

pad

(Y, N) Liver

Cassette

Tare

Weight

(g) Liver +

Cassette

Weight

(g) Liver

Weight

(g) Note

L1D9 -T D L1 Term 11.091 41 28.14 0 M M M N 3.3457 3.9176 0.5719 spinal curvature

L1D10 -T D L1 Term

. . no sample no sample

. . . . . . .

L2A1 -T A L2 Term 9.1 06 40 80.87 9156.555 F F F N 3.3452 3.9344 0.5892 spinal curvature

L2A2 -T A L2 Term 9.127 41 31.76 8999.685 M M M N 3.3510 3.7650 0.4140

L2A3 -T A L2 Term 5.848 35 27.29 9227.7 M M M N 3.3653 3.5798 0.2145

L2A4 -T A L2 Term 17.443 51 58.71 9227.7 M M M Y 3.3668 4.3473 0.9805

L2A5 -T A L2 Term 11.278 43 132.74 168474.727 M F M M Y 3.3580 3.9712 0.6132

L2A6 -T A L2 Term 9.096 39 24.79 8999.685 M M M Y 3.3460 3.8352 0.4892

L2A7 -T A L2 Term 12.782 45 39.29 16495.74 M F M M Y 3.3658 4.0871 0.7213

L2A8-T A L2 Term 14.696 47 85.93 45364.625 F M F F Y 3.3580 4.2606 0.9026

L2A9 -T A L2 Term 10.092 43 50.09 16495.74 M M M Y 3.3747 3.9487 0.5740

L2A10 -T A L2 Term 11.354 46 54.72 8999.685 M M M Y 3.3816 3.9908 0.6092

L2B1 -T B L2 Term 10.951 43 42.86 9156.555 M M M N 3.3771 4.0713 0.6942

L2B2 -T B L2 Term 8.388 43 44.44 9227.7 F F F N 3.3678 4.0185 0.6507

L2B3 -T B L2 Term 9.853 43 30.49 9227.7 F F F N 3.3476 4.0093 0.6617

L2B4 -T B L2 Term 12.262 47 76.3 9227.7 M M M Y 3.3778 4.2723 0.8945

L 2B5 -T B L2 Term 13.667 45 94.1 9156.555 M M M N 3.3657 4.3242 0.9585

L2B6 -T B L2 Term 6.624 37 25.26 0 F M F F N 3.3651 3.7719 0.4068

L2B7 -T B L2 Term 10.513 43 137.9 0 F M F F Y 3.3660 3.9636 0.5976

L2B8 -T B L2 Term 10.370 43 128.22 0 M M M Y 3.3728 3.9576 0.5848

L2B9 -T B L2 Term 7.502 39 19.55 17144.91 M F M M N 3.3381 3.7940 0.4559

L2B10 -T B L2 Term 7.792 38 55.06 17144.91 M M M N 3.3688 3.8967 0.5279

L2C1 -T C L2 Term 13.906 46 93.78 17144.91 F F F Y 3.3467 4.1495 0.8028

L2C2 -T C L2 Ter m 9.497 40 58.3 9156.555 M F M M Y 3.3642 3.8974 0.5332

L2C3 -T C L2 Term 14.139 57 148.32 40739.093 F M F F Y 3.3690 4.2301 0.8611

“ . ” denotes a juvenile was not available for this sampl e.

# ABC Study No. 66086

Page 49 of 52 ID Tank

(Rep) Conc

. Sample

(62, 66,

Term) Body

Weight

(g) Snout- Vent

Length

(mm) T4

(ng/mL) VTG

(ng/mL) Pheno

Sex

(M, F,

I) Battelle

Geno

Sex EPA

Duluth

Geno sex Corrected

Geno Sex Nuptial

pad

(Y, N) Liver

Cassette

Tare

Weight

(g) Liver +

Cassette

Weight

(g) Liver

Weight

(g) Note

L2C4 -T C L2 Term 11.173 44 63.12 0 M M M N 3.3781 4.0818 0.7037

L2C5 -T C L2 Term 7.121 39 48.52 0 M M M N 3.3442 3.7635 0.4193

L2C6 -T C L2 Term 10.769 45 103.53 0 M M M Y 3.3804 4.0129 0.6325

L2C7 -T C L2 Term 10.954 47 66.65 0 F M M M N 3.3793 3.9450 0.5657

L2C8 -T C L2 Te rm 11.449 44 29.37 0 M F M M Y 3.3677 4.0833 0.7156

L2C9 -T C L2 Term 8.443 42 79.97 0 M F M M N 3.3705 3.7853 0.4148

L2C10 -T C L2 Term 6.585 39 17.09 8999.685 F F F N 3.3501 3.7669 0.4168

L2D1 -T D L2 Term 12.535 50 231.11 0 F M F F Y 3.3499 4.2718 0 .9219

L2D2 -T D L2 Term 10.945 41 125.32 0 M M M Y 3.3519 4.0089 0.6570 spinal curvature

L2D3 -T D L2 Term 12.673 47 88.86 0 F F F Y 3.3787 4.1412 0.7625

L2D4 -T D L2 Term 9.825 41 28.97 8999.685 F M F F Y 3.3691 3.9021 0.5330

L2D5 -T D L2 Term 8.601 41 6 9156.555 F F F N 3.3668 3.8514 0.4846

L2D6 -T D L2 Term 7.026 40 22.91 0 F F F N 3.3447 3.7092 0.3645

L2D7 -T D L2 Term 9.989 42 80.08 0 F F F N 3.3680 3.9748 0.6068

L2D8 -T D L2 Term 9.975 42 88.16 0 F F F N 3.3765 4.0434 0.6669

L2D9 -T D L2 Term 10.318 42 57.71 17144.91 F F F N 3.3710 4.0218 0.6508

L2D10 -T D L2 Term 10.181 42 29.81 17144.91 M M M N 3.3739 3.9708 0.5969

L3A1 -T A L3 Term 10.841 41 62.82 17144.91 F M F F N 3.3849 4.1330 0.7481

L3A2 -T A L3 Term 3.779 32 30.12 9227.7 F M F F N 3.3703 3.5589 0.1886

L3A3 -T A L3 Term 4.115 31 8.325 9227.7 F M F F N 3.3650 3.5206 0.1556

L3A4 -T A L3 Term 11.123 43 54.03 9227.7 M F M M N 3.3662 4.0354 0.6692

L3A5 -T A L3 Term 9.906 44 37.3 9156.555 M F M M N 3.3465 3.9212 0.5747

L3A6 -T A L3 Term 12.187 46 48.21 9156.555 M M M N 3.3468 4.1513 0.8045

L3A7 -T A L3 Term 12.967 42 56.04 16495.74 M F M M N 3.3787 4.2451 0.8664

L3A8 -T A L3 Term 9.545 43 57.05 16495.74 F F F N 3.3758 4.0568 0.6810

“ . ” denotes a juvenile was not available f or this sampl e.

# ABC Study No. 66086

Page 50 of 52 ID Tank

(Rep) Conc

. Sample

(62, 66,

Term) Body

Weight

(g) Snout- Vent

Length

(mm) T4

(ng/mL) VTG

(ng/mL) Pheno

Sex

(M, F,

I) Battelle

Geno

Sex EPA

Duluth

Geno sex Corrected

Geno Sex Nuptial

pad

(Y, N) Liver

Cassette

Tare

Weight

(g) Li ver +

Cassette

Weight

(g) Liver

Weight

(g) Note

L3A9 -T A L3 Term 7.236 38 33.98 16495.74 F F F N 3.3512 3.7285 0.3773

L3A10 -T A L3 Term 11.781 47 28.04 8999.685 F F F N 3.3829 4.2208 0.8379

L3B1 -T B L3 Term 13.369 48 95.73 8999.685 F F F Y 3.3806 4.2384 0.8578

L3B2 -T B L3 Term 7.435 41 42.59 0 F M F F N 3.3512 3.8535 0.5023

L3B3 -T B L3 Term 6.286 39 19.8 0 M F M M N 3.3781 3.7575 0.3794

L3B4 -T B L3 Term 14.964 47 226.14 0 M F M M Y 3.3643 4.2884 0.9241

L3B5 -T B L3 Term 9.696 41 20.68 17144.91 M F M M N 3.3662 4.1090 0.7428

L3B6 -T B L3 Term 9.339 42 76.35 17144.91 M M M Y 3.3794 3.9416 0.5622

L3B7 -T B L3 Term 5.438 37 18.47 17144.91 F F F N 3.3712 3.7038 0.3326

L3B8 -T B L3 Term 13.995 49 47.88 8999.685 M M M N 3.3612 4.2330 0.8718

L3B9 -T B L3 Term 3.271 31 6 9156.555 F M F F N 3.3736 3.5332 0.1596

L3B10 -T B L3 Term 11.995 46 199.9 9156.555 M M M Y 3.3692 4.1765 0.8073

L3C1 -T C L3 Term 2.082 38 8.325 9227.7 F F F NA 3.3841 3.4455 0.0614 missing forelimbs

L3C2 -T C L3 Term 7.530 38 23.94 9227.7 M M M N 3.3706 3.7837 0.4131

L3C3 -T C L3 Term 15.295 52 94.87 9227.7 M M M Y 3.3503 4.1797 0.8294

L3C4 -T C L3 Term 7.804 40 40.97 17144.91 M F M M Y 3.3568 3.7405 0.3837

L3C5 -T C L3 Term 10.270 41 35.01 17144.91 F M F F N 3.3509 3.9547 0.6038

L3C6 -T C L3 Term 14.036 48 70.7 17144.91 F F F Y 3.3769 4.2026 0.8257

L3C7 -T C L3 Term 14.363 50 102.745 0 M F M M Y 3.3676 4.2239 0.8563

L3C8 -T C L3 Term 8.691 41 30.19 0 M M M N 3.3626 3.8788 0.5162

L3C9 -T C L3 Term 9.788 42 42.23 0 M F M M N 3.3424 4.0362 0.6938

L3C10 -T C L3 Term 12.231 46 49.9 8999.685 M F M M N 3.3665 4.0599 0.6934

L3D1 -T D L3 Term 12.929 45 45.7 9156.555 F F F N 3.3669 4.2438 0.8769

L3D2 -T D L3 Term 13.544 49 69.3 0 F F F Y 3.3797 4.0986 0.7189

L3D3 -T D L3 Term 9.972 44 25 0 M M M N 3.3499 3.9540 0.6041

“ . ” denotes a juvenile was not available for this sampl e.

# ABC Study No. 66086

Page 51 of 52 ID Tank

(Rep) Conc

. Sample

(62, 66,

Term) Body

Weight

(g) Snout- Vent

Length

(mm) T4

(ng/mL) VTG

(ng/mL) Pheno

Sex

(M, F,

I) Battelle

Geno

Sex EPA

Duluth

Geno sex Corrected

Geno Sex Nuptial

pad

(Y, N) Liver

Cassette

Tare

Weight

(g) Liver +

Cassette

Weight

(g) Liver

Weight

(g) Note

L3D4 -T D L3 Term 10.472 43 63.87 0 M M M Y 3.3793 4.0085 0.6292

L3D5 -T D L3 Term 12.265 43 45.625 19379.25 F F F N 3.3649 4.1129 0.7480

L3D6 -T D L3 Term 8.619 41 157.31 0 M M M N 3.3621 3.8835 0.5214 spinal curvature

L3D7 -T D L3 Term 12.841 47 97.94 0 F F F Y 3.3789 3.9810 0.6021

L3D8 -T D L3 Term 7.525 40 38.93 8999.685 M M M N 3.3606 3.9015 0.5409

L3D9 -T D L3 Term 12.391 47 108.09 8999.685 M M M Y 3.3836 4.1328 0.7492

L3D10 -T D L3 Term 11.365 45 109 77088.478 F F F N 3.3384 4.0007 0.6623

L4A1 -T A L4 Term 10.222 40 63.43 16495.74 M M M N 3.3710 3.9629 0.5919

L4A2 -T A L4 Term 15.497 48 38.2 4 16495.74 F F F Y 3.3495 4.2880 0.9385

L4A3 -T A L4 Term 19.538 55 183.11 17144.91 F F F Y 3.3414 4.5050 1.1636

L4A4 -T A L4 Term 8.486 42 8.325 17144.91 M M M N 3.3709 3.8591 0.4882

L4A5 -T A L4 Term 9.025 41 46.06 17144.91 M M M N 3.3533 3.9469 0 .5936

L4A6 -T A L4 Term 7.493 42 47.57 9156.555 M M M N 3.3669 3.7741 0.4072

L4A7 -T A L4 Term 14.821 48 35.87 8999.685 M M M Y 3.3456 4.2056 0.8600

L4A8 -T A L4 Term 10.758 43 39.81 0 F F F N 3.3853 4.0539 0.6686

L4A9 -T A L4 Term 6.041 37 41.76 0 M M M N 3.3634 3.6160 0.2526

L4A10 -T A L4 Term 6.449 37 63.67 0 F F F F N 3.3709 3.6733 0.3024

L4B1 -T B L4 Term 10.147 41 156.54 0 M M M Y 3.3819 3.9686 0.5867

L4B2 -T B L4 Term 9.870 42 88.12 19379.25 M M M N 3.3770 3.9426 0.5656

L4B3 -T B L4 Ter m 14.339 46 34.295 19379.25 M M M Y 3.3653 4.2644 0.8991

L4B4 -T B L4 Term 14.331 48 73.37 9156.555 F F F Y 3.3535 4.2113 0.8578

L4B5 -T B L4 Term 12.778 46 49.92 9156.555 M M M Y 3.3678 4.1542 0.7864

L4B6 -T B L4 Term 6.179 42 134.75 45506.302 F F F N 3.3506 3.6840 0.3334

L4B7 -T B L4 Term 11.442 45 47.59 0 F F F N 3.3538 4.0102 0.6564

L4B8 -T B L4 Term 8.545 41 44.81 0 F F F Y 3.3705 3.7412 0.3707

“ . ” denotes a juvenile was not available for this sampl e.

# ABC Study No. 66086

Page 52 of 52 ID Tank

(Rep) Conc

. Sample

(62, 66,

Term) Body

Weight

(g) Snout- Vent

Length

(mm) T4

(ng/mL) VTG

(ng/mL) Pheno

Sex

(M, F,

I) Battelle

Geno

Sex EPA

Duluth

Geno sex Corrected

Geno Sex Nuptial

pad

(Y, N) Liver

Cassette

Tare

Weight

(g) Liver +

Cassette

Weight

(g) Liver

Weight

(g) Note

L4B9 -T B L4 Term 12.476 44 78.38 0 M M M Y 3.3536 4.1148 0.7612

L4B10 -T B L4 Term 5.477 31 8.325 19379.25 M M M N 3.3687 3.6622 0.2935

L4C1 -T C L4 Term 8.232 38 42.06 0 F F F N 3.3692 3.8264 0.4572

L4C2 -T C L4 Term 6.759 36 43.81 27180.931 M M M N 3.3694 3.7519 0.3825

L4C3 -T C L4 Term 11.379 44 83.18 9156.555 M M M N 3.3619 4.0489 0.6870

L4C4 -T C L4 Term 10.723 42 90.75 9156.555 M M M N 3.3662 4.0275 0.6613

L4C5 -T C L4 Term 13.129 48 47.14 9227.7 M M M N 3.3701 4.3592 0.9891

L4C6 -T C L4 Term 6.736 38 47.34 9227.7 F F F N 3.3684 3.7574 0.3890

L4C7 -T C L4 Term 19.080 50 50.88 29156.71 M M M Y 3.3511 4.5865 1.2354

L4C8 -T C L4 Term 11.828 45 16.56 0 M M M Y 3.3763 4.1720 0.7957

L4C9 -T C L4 Term 6.916 35 32.43 0 F M M M N 3.3746 3.7426 0.3680

L4C10 -T C L4 Term 6.906 37 145.98 0 F F F N 3.3702 3.7528 0.3826

L4D1 -T D L4 Term 9.463 43 72.78 16495.74 F M M M N 3.3811 3.8079 0.4268

L4D2 -T D L4 Term 10.567 44 47.7 74942.043 M M M N 3.3481 4.0127 0.6646

L4D3 -T D L4 Term 16.987 51 3 4.65 16495.74 F F F Y 3.3732 4.3717 0.9985

L4D4 -T D L4 Term 13.163 47 24.16 107361.268 F F F Y 3.3792 4.0975 0.7183

L4D5 -T D L4 Term 10.850 44 77.26 17144.91 F F F N 3.3710 4.0187 0.6477

L4D6 -T D L4 Term 9.537 37 132.76 17144.91 M M M N 3.3466 3.9003 0.5537

L4D7 -T D L4 Term 11.891 42 41.08 32859.473 F F F Y 3.3823 4.0349 0.6526

L4D8 -T D L4 Term 7.040 38 8.325 9227.7 M M M N 3.3822 3.7380 0.3558

L4D9 -T D L4 Term 8.596 42 36.36 9227.7 M M M N 3.3812 3.7980 0.4168

L4D10 -T D L4 Term 13.680 48 100.5 9227.7 F F F Y 3.3730 4.1125 0.7395

“ . ” denotes a juvenile was not available for this sample.