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ADDENDUM TO FINAL REPORT TITLED 

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 LABORATORIES

ABC Laboratories, Inc. 

7200 E. ABC Lane 

Columbia, Missouri 65202 

United States

Battelle-Pacific Northwest Division 

Marine Research Operations 

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 Investigators

Statistics: Dr. Valerie Cullinan (Battelle-Pacific Northwest Division)
ABC Study No. 66086 

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TABLE OF CONTENTS

Page

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

STATEMENT OF GLP COMPLIANCE ........................................................................................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 Liver 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 deviation) 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 laevis 4-tert-octylphenol exposure. ...........................13

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. ................................................................................................................14

Table 3.4.1. Frog phenotypic and genotypic sex ratios 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
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Table 4.0.1. Calculated No-Observable-Effect Concentration and Lowest-ObservableEffect Concentration based on mean measured 4-tert-octylphenol 

concentrations for parameter measured for Xenopus laevis exposed to 4-tertoctylphenol. ............................................................................................................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 represents 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 horizontal 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 

represents 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
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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 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 circle 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
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1.0 INTRODUCTION

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

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

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

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

standardizing, and validating an ecotoxicological test for identifying and characterizing 

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

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

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

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

endocrine disrupting in vertebrates. 

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

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

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

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

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

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

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

This addendum to the final report issued December 19, 2012 was prepared to incorporate data 

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

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

December 19, 2012. The specific statistical analyses 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 transformation. Deviations 

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

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

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

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

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

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

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

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

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

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

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

step-down Dunn’s test was used. Statistical outliers were defined as those values outside the 
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bounds determined by Q1 – 1.5*IQD and Q3 + 1.5*IQD where Q1 and Q3 are the first and third 

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

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

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

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

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

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

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

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

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

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

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

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

identified outliers were removed from the analyses. 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 analytical 

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 Growth 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

frogs 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 measured 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-tertoctylphenol treatments (Table 3.1.1; Figure 3.1.3). Significant differences among treatments 

were detected in snout-to-vent length of male frogs at the termination of the exposure. However, 
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the K-W comparisons to control test determined that there were no differences in mean values 

between any 4-tert-octylphenol treatment and the control treatment. Mean snout-to-vent length 

of female frogs measured at the termination of the 4-tert-octylphenol exposure was 42 mm in the 

control treatment and ranged from 42 to 44 mm in the 4-tert-octylphenol treatments (Table 3.1.1; 

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-toVent Length (mm)Female Snout-toVent 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. 

0.00 4.97 10.30 19.50 38.00

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 termination of the Xenopus laevis

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

mean, and the horizontal line is the median. Gender is based on genotypic sex. 
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0.00 4.97 10.3 19.5 38.0

14

13

12

11

10

9

8

7

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 3.1.2. 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. 

0.00 4.97 10.3 19.5 38.0

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

the horizontal line is the median. Gender is based on genotypic sex. 
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0.00 4.97 10.3 19.5 38.0

47

46

45

44

43

42

41

40

39

38

Mean Measured Dose (μg/L)

Snout-to-Vent Length (mm)

Figure 3.1.4. 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 in 

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 0.23 and 0.20, respectively. The mean 

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

the termination of the exposure ranged from 0.21 to 0.50 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 (Table 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 = 0.21 for males, 0.20 for females; n = 8). The 

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

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

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

nuptial pads was 0.52, 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, respectively, which were considered significantly 

greater than chance (p = 0.001, 0.016, and 0.002, respectively). 
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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.

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 based on genotypic sex. 

0.00 4.97 10.30 19.50 38.00

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 

circle is the mean, and the horizontal line is the median. Gender is based on genotypic sex. 
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0.00 4.97 10.30 19.50 38.00

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

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

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

Table 3.2.2. 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 (wet) 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. 
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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. 

0.00 4.97 10.30 19.50 38.00

0.066

0.064

0.062

0.060

0.058

0.056

0.054

0.052

0.050

Mean Measured Dose (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. 
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0.00 4.97 10.3 19.5 38.0

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 weight:whole body weight for female frogs at the termination of the 

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

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

3.4 Genotypic/Phenotypic Sex Ratios 

Detailed results of genetic sexing of the juvenile frogs using PCR followed 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 determinations of five individuals differed 

from their phenotypic determinations. 

Table 3.4.1. Frog phenotypic and genotypic sex ratios at the termination of the Xenopus laevis 4-tertoctylphenol exposure. 

Mean Measured 

Concentration (μg/L) N 

Phenotype Genotype Number of 

Mismatches 1 Females Males Females Males 

<MQL 78 33 45 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 
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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 standard deviation) plasma thyroxine (T4) for frogs at the termination of the 

Xenopus laevis 4-tert-octylphenol exposure. 

Mean Measured 

Concentration (μg/L) 

Male Plasma 

Thyroxine (T4) 

(ng/mL) 

Female Plasma 

Thyroxine (T4) 

(ng/mL) 

<MQL 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.

0.00 4.97 10.3 19.5 38.0

110

100

90

80

70

60

50

40

30

Mean Measured Dose (μg/L)

Thyroxine Concentration (ng/mL)

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

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

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

is based on genotypic sex. 
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0.00 4.97 10.3 19.5 38.0

130

120

110

100

90

80

70

60

50

40

Mean Measured Dose (μg/L)

Thyroxine Concentration (ng/mL)

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 

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-octylphenol concentrations for parameter 

measured for Xenopus laevis exposed to 4-tert-octylphenol.

Parameter No-Observable-Effect 

Concentration (μg/L)

Lowest-ObservableEffect Concentration 

(μg/L)

Frogs at Termination

Whole Body 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

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

1

 K-W test was significant (p = 0.040), but there were no differences between any treatment 

and the control (p > 0.013).
ABC Study No. 66086 

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APPENDIX A – AMENDED STATISTICAL REPORT
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APPENDIX B – TERMINATION DATA – GROWTH AND GENDER
ABC Study No. 66086 

Page 43 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

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 0.7240 

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 sample. 
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 sample. 
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 

testes

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 38 129.085 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 sample. 
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 8999.685 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 Term 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 sample. 
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 36151.126 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 Term 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 9156.555 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 sample. 
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 9.106 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

L2B5-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 Term 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 sample. 
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 Term 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 for this sample. 
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) 

Liver + 

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 sample. 
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 38.24 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 Term 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 sample. 
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 34.65 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.