DOCUMENT AMENDMENT FORM

Fort Environmental Laboratories

Document or Study Title: Larval Amphibian Growth and Development Study (LAGDA)

Amendment Number: 07 Document ID Number: BPNL01-3 (00242)

Submitted By: Douglas J. Fort Date: 3/15/2013

Amendment Relating To: 

[ ] Protocol [ ] Study Plan [ ] QAPP [ ] QAMP [ ] SOP

[ X ] Other (describe): Final Report

Original Specifications: The following Compliance Statement, tables, and sections within the body of the final report:

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

This study was conducted in accordance with 40 CFR 160 at Fort Environmental Laboratories, Inc., Stillwater, Oklahoma using 

standard U.S. EPA procedures and is considered facility records under Fort Environmental Laboratories Standard Operating 

Procedures with the exception of: 1) histopathology will be reported separately, 2) the test article was not specifically characterized, 

and 3) a reserve sample of the test article was not retained.

Page 32, Section 9. Study Protocol Adherence

“There were six amendments to and one deviation from the Study Protocol...”

Additional pages/sections, tables, figures and appendices affected:

Page 10 Section 1.2. Results and Conclusions, Page 27 Section 5.5.3. Nuptial Pad Development, Page 28 Section 5.5.5. 

Biochemistry, Page 28 5.5.6. Gender Ratios, Page 29 Section 5.6. Concentration Measurement Endpoints, Page 29 Section 6.2. 

Assessment of the LAGDA Results, Page 30 Section 7. Conclusions and Assay Recommendations, Tables 13, 14, 15, 16, 17, and 

18, Figures 11, 12, 13, 14, 17, and 18, and Appendices B, C, and E.

Planned Change:

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

“... with the exception of: 1) histopathology will be reported separately, 2) the test article was not specifically characterized, 3) a 

reserve sample of the test article was not retained, and 4) re-analysis of selected hind foot samples for genotyping by USEPA 

(Duluth, MN).”

Page 32, Section 9. Study Protocol Adherence

“There were six amendments to and two deviations from the Study Protocol...”

Protocol deviation 02 form attached.

Additional pages/sections, tables, figures and appendices affected:

See attachments for updated content in the areas described above.

Reason for Change: Additional genotyping from re-analysis of samples for genotyping and VTG analysis was made available 

to confirm or correct original data. Genotyping (PCR) re-analysis was performed by USEPA (Duluth, MN). VTG re-analysis was 

performed by FEL. Samples for re-analysis were selected by USEPA based on inconsistencies between necropsy phenotype, 

histology phenotype, and genotype (PCR). Protocol deviation 02 was issued to address these changes.

Page 10, 1.2. RESULTS AND CONCLUSIONS

The proportion of male frogs for which the expression of nuptial pads could not be 

determined in the 53.0 μg/L tOP treatment at the conclusion of exposure was significantly 

different from the control (KW ANOVA, p=0.001). Only two females in the 4-tert-octylphenol 

treatments had nuptial pads; one each in the 8.93 and 29.3 μg/L tOP treatments. No statistical 

analyses were conducted on the proportion of females with nuptial pads. To further evaluate the 

KW 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 males. The proportions of 

males in the 8.93 and 29.3 μg/L tOP concentrations having nuptial pads were 1.0, which were 

considered significantly greater than chance (p = 0.009 and 0.048, respectively). A binomial 

probability was not calculated for females. Significant differences among treatments were 

detected in liver weight:whole body weight for male and female frogs at the termination of the 

exposure. However, in neither case could the individual differences between any treatment and 

the control be determined (p > 0.013). Plasma T4 concentrations in 10 weeks postmetamorphosis (wpm) juvenile frogs were not significantly different from the control (KW 

ANOVA, p=0.165). Plasma VTG concentrations in male frogs from the 8.93, 15.8, 29.3, and 

53.0 μg/L tOP treatments were significantly greater than that in the control treatment (KW 

ANOVA, p <0.001). VTG concentrations in female frogs from the 29.3 and 53.0 μg/L tOP 

treatment was significantly greater than that in the control treatment (KW ANOVA, p <0.001). 

The phenotypic and genotypic sex ratios were 51.3:48.7 male:female and 50.0:50.0 male:female, 

respectively, in the control. The ratios were similar in each of the tOP treatments and similar in 

each case to the control. NOEC values ranged from <8.93 (male plasma VTG at study 

completion) to 53.0 μg/L tOP. Results from the present study suggested that tOP exposure 

during sexual differentiation of X. laevis may alter plasma VTG levels in NF stage 62 larvae and 

both male and female frogs at study completion.

Page 27, 5.5.3. NUPTIAL PAD DEVELOPMENT

The mean proportion of male frogs in the control treatment that had nuptial pads at the 

termination of the tOP exposure was 0.72. No females from the control treatment had nuptial 

pads. The mean proportion of male frogs from the tOP treatments that had nuptial pads at the 

termination of the exposure ranged from 0.50 to 1.0 (Table 13; Figure 11). The proportion of 

male frogs expressing nuptial pads in tOP treated frogs at the conclusion of exposure was 

significantly different among treatments (KW ANOVA, P=0.036), however the multiple 

comparisons test did not detect differences between any of the treatments and the control (KW 

ANOVA, p>0.05). The proportion of male frogs for which expression of nuptial pads could not 

be determined was 0.11 for frogs from the control treatment and ranged from 0.10 to 0.81 for 

frogs from the tOP treatments (Table 13; Figure 12). The proportion of male frogs for which the 

expression of nuptial pads could not be determined in the 53.0 μg/L tOP treatment at the 

conclusion of exposure was significantly different from the control (KW ANOVA, p=0.001). 

Only two females in the 4-tert-octylphenol treatments had nuptial pads; one each in the 8.93 and

29.3 μg/L tOP treatments). No statistical analyses were conducted on the proportion of females 

with nuptial pads. 
In addition to the KW 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 

males (Table 14). The expected proportion of nuptial pad occurrence was calculated for males 

from the mean proportion from the control replicates (probability of nuptial pads occurring by 

chance = 0.71 for males; n = 8). The probability of obtaining the total number of occurrences 

out of 40 frogs (the number in each treatment) from chance alone was calculated independently 

for each exposure concentration. The proportions of males in the 8.93 and 29.3 μg/L tOP 

concentrations having nuptial pads were both 1.0, which was considered significantly greater 

than chance (p = 0.009 and 0.048, respectively). A binomial probability was not calculated for 

females.

Page 28, 5.5.4. LIVER WEIGHT

Mean liver weight (normalized to the whole body weight) of frogs measured for the 

control treatment at the termination of the tOP exposure were 0.072 for males and 0.075 for 

females. Normalized liver weight for males ranged from 0.064 to 0.071 in the tOP treatments 

(Table 15; Figure 13). Significant differences among treatments for male frogs were detected, 

however the individual differences between treatment and the control could not be determined 

(KW ANOVA, p=0.037). The normalized liver weight for females ranged from 0.068 to 0.075 

in the tOP treatments (Table 15; Figure 14). Significant differences among treatments for female 

frogs were detected, however the individual differences between treatment and the control could 

not be determined (KW ANOVA, p=0.048).

Page 28, 5.5.5. BIOCHEMISTRY

The plasma VTG concentrations for male and female frogs in the control treatment at 

study termination were 4,585 and 36,811 ng/mL, respectively. Plasma VTG concentrations in 

male frogs in the tOP treatments at termination of the exposure ranged from 7,348 to 32,974 

ng/mL (Table 16; Figure 17). Plasma VTG concentrations in male frogs from the 8.93, 15.8, 

29.3, and 53.0 μg/L tOP treatments were significantly greater than that in the control treatment 

(KW ANOVA, p <0.001). VTG concentrations in female frogs in the tOP treatments at

termination of the exposure ranged from 42,266 to 141,624 ng/mL (Table 16; Figure 18). VTG 

concentration in female frogs from the 29.3 and 53.0 μg/L tOP treatment were significantly 

greater than that in the control treatment (KW ANOVA, p <0.001). The mean plasma T4 

concentration in the control treatment at the termination of the tOP exposure was 5.27 ng/mL. 

Mean T4 concentrations for frogs in the 4-tert-octylphenol treatments at termination of the 

exposure ranged from 5.48 to 6.82 ng/mL (Table 16; Figure 16). Plasma T4 concentrations in 10 

wpm juveniles were not significantly different from the control (KW ANOVA, p=0.165).

Page 28, 5.5.6. GENDER RATIOS

The phenotypic and genotypic sex ratios at the termination are presented in Table 17. The 

phenotypic and genotypic sex ratios were 51.3:48.7 male:female and 50.0:50.0 male:female, 
respectively, in the control. The ratios were similar in each of the tOP treatments and similar in 

each case to the control.

Page 29, 5.6. CONCENTRATION MEASUREMENT ENDPOINTS

The No Observable Effect Concentration and Lowest Observable-Effect Concentration are 

reported in Table 18 for each measurement endpoint. NOEC values ranged from <8.93 to 53.0 

μg/L tOP.

Page 29, 6.2. ASSESSMENT OF THE LAGDA RESULTS

Little effect of tOP exposure was noted in terms of the time required to complete 

metamorphosis and general measures of growth, body and liver weights, and endpoints 

associated with sexual differentiation with the exception of plasma VTG. Increased plasma 

VTG in NF stage 62 larvae exposed to tOP and in genetically male and female juvenile frogs at 

the test conclusion was noted. 

Page 30, 7. CONCLUSIONS AND ASSAY RECOMMENDATIONS

Performance of the LAGDA study demonstrated the utility of the assay as a potential Tier 

2 ecotoxicological EDSP test. The general framework of the model and the endpoints measured 

are appropriate for measuring both thyroid and hypothalamic–pituitary–gonadal (HPG) axis 

activity in a lower, but representative vertebrate. Results from the present study suggested that 

tOP exposure at metamorphic climax and during sexual differentiation of X. laevis altered 

plasma VTG levels metamorphosing larvae and 10 week post-metamorphic frogs regardless of 

gender . Overall, results from the present study suggested that tOP may be a HPG-active test 

article. Several recommendations for improving the LAGDA model as it currently is presented 

are provide below:
Table 13 Effect of 4-tert-Octylphenol Exposure on the Proportion of Female Frogs, Male 

Frogs, and Total Number of Frogs Expressing Nuptial Pads in 10 Week Post-Metamorphic X. 

laevis (LAGDA)1

Treatment (μg 

a.i./L) 

[measured]2

Nuptial Pads 

as proportion 

of females

Nuptial Pads as 

proportion of 

males3

Total Number of 

Frogs with Nuptial 

Pads4

Nuptial Pads 

Not 

Determined5

0.00 [<3.1] 0 0.72 (0.16) 25 0.11 (0.16)

6.25 [8.93] 0.046

1.00 (–) 15 0.10 (0.20)

12.5 [15.8] 0 0.76 (0.25) 12 0.10 (0.13)

25.0 [29.3] 0.046

1.00 (–) 10 0.40 (0.27)

50.0 [53.0] 0 0.50 (0.58) 3 0.81 (0.13)

7

 

1 Represents mean with standard deviation cited in parenthesis.

2 Nominal concentration with mean measured concentration in brackets.

3

Significantly different among treatments based on KW ANOVA, p=0.036 (adjusted for ties); but the multiple 

comparisons test did not find differences between any individual treatment and the control.

4 Total number of frogs = 80 for control and 40 for each 4-tert-octylphenol dose.

5 The proportion of male frogs for which presence or absence of nuptial pads could not be determined. This is not 

an endpoint included in the LAGDA.

6 Only one female in each treatment had nuptial pads.

7 Median significantly greater than control, (KW ANOVA, p=0.001).

Table 14 Effect of 4-tert-Octylphenol Exposure on Proportion of Frogs within Each 

Treatment Having Nuptial Pads in 10 Week Post-Metamorphic X. laevis (LAGDA)

Treatment (μg 

a.i./L) [measured]8

Proportion of 

Females with 

Nuptial Pads

Binomial 

Probability

Proportion of 

Males with 

Nuptial Pads

Binomial 

Probability

0.00 [<3.1] 0 – 0.71 –

6.25 [8.93] 0.049– 1.00 0.009

12.5 [15.8] 0 – 0.71 0.647

25.0 [29.3] 0.049– 1.00 0.048

50.0 [53.0] 0 – 0.75 0.677

 

8 Nominal concentration with mean measured concentration in brackets.

9 Only one female in each treatment had nuptial pads.
Table 15 Effect of 4-tert-Octylphenol Exposure on Liver Weight:Whole Body Weight for 

Male and Female Frogs within Each Treatment in 10 Week Post-Metamorphic X. laevis

(LAGDA)

Treatment

(μg a.i./L)

[measured]1

Rep

Liver Weight:Whole Body Weight

Female Male

Replicate 

Mean

Treatment 

Mean2 SEM3 Replicate Mean

Treatment 

Mean4 SEM

0.00

[<3.1]

A 0.071

0.075 0.001

0.067

0.072 0.001

B 0.071 0.070

C 0.076 0.073

D 0.084 0.074

E 0.073 0.069

F 0.076 0.078

G 0.073 0.073

H 0.077 0.068

6.25

[8.93]

A 0.076

0.073 0.002

0.068

0.065 0.001 B 0.068 0.063

C 0.077 0.063

D 0.073 0.067

12.5

[15.8]

A 0.078

0.075 0.003

0.069

0.071 0.002 B 0.074 0.073

C 0.081 0.069

D 0.069 0.075

25.0

[29.3]

A 0.071

0.069 0.001

0.076

0.066 0.003 B 0.065 0.065

C 0.069 0.065

D 0.069 0.060

50.0

[53.0]

A 0.066

0.068 0.002

0.073

0.064 0.004 B 0.073 0.062

C 0.069 0.055

D 0.065 0.066

 

1 Nominal concentration with mean measured concentration in brackets.

2

Significant differences among treatments were detected in liver weight:whole body weight, however the individual 

individual differences between and treatment and the control could not be determined (p = 0.037).

3

Standard Error of the Mean

4

Significant differences among treatments were detected in liver weight:whole body weight, however the individual 

individual differences between and treatment and the control could not be determined (p = 0.048).
Table 16 Effect of 4-tert-Octylphenol Exposure on Plasma Vitellogenin (VTG) and Thyroxin (T4) Levels in 

10 Week Post-Metamorphic X. laevis (LAGDA)

Treatment

(μg a.i./L)

[measured]1

Rep

VTG T4 Female Male

Replicate 

Mean

(ng/mL)

Treatment 

Mean

(ng/mL)

SEM2 CV

(%)3

Replicate 

Mean

(ng/mL)

Treatment 

Mean

(ng/mL)

SEM CV

(%)

Replicate 

Mean

(ng/mL)

Treatment 

Mean

(ng/mL) 4

SEM CV

(%)

0.00

[<3.1]

A 39092

36811 2595 19.9

3431

4585 321 19.8

4.08

5.27 0.42 22.7

B 41120 3789 3.73

C 45884 3951 5.95

D 42933 4259 4.52

E 39941 5563 4.98

F 29721 5215 7.43

G 25194 4491 5.91

H 30607 5982 5.58

6.25

[8.93]

A 26771

42266 5283 25.0

7126

73486823 22.4

5.64

5.99 0.25 8.4 B 47524 6559 6.60

C 50110 9718 6.18

D 44658 5990 5.52

12.5

[15.8]

A 63260

55862 2477 8.9

11576

87246954 21.9

6.25

5.48 0.50 18.2 B 53758 7813 6.43

C 53686 7955 4.52

D 52743 7553 4.73

25.0

[29.3]

A 107822

9843454947 10.1

29172

2037163765 37.0

6.62

6.19 0.19 6.1 B 105170 24104 6.37

C 94255 13830 5.78

D 86487 14379 5.98

50.0

[53.0]

A 155892

14162454894 6.9

43491

3297464270 25.9

6.67

6.82 0.47 13.7 B 139921 35617 5.57

C 134330 23695 7.73

D 136353 29094 7.28

 

1 Nominal concentration with mean measured concentration in brackets.

2

Standard Error of the Mean

3 Coefficient of variation = (Standard deviation / mean)100.

4 Medians not significantly different from the control (KW ANOVA, p=0.165).

5 Median significantly greater than control, (KW-ANOVA multiple comparisons, p<0.001).

6 Medians significantly greater than control, (KW-ANOVA multiple comparisons, p<0.001).
Table 17 Effect of 4-tert-Octylphenol Exposure on Gender Ratios in 

10 Week Post-Metamorphic X. laevis (LAGDA)

Treatment 

(μg a.i./L) 

[measured]1

Rep. n

Phenotype Genotype

Phenotypic 

Male

Phenotypic 

Female

Phenotype 

Ratio (M:F)

Genotypic 

Male

Genotypic 

Female

Genotype 

Ratio (M:F)

0.00 

[<3.0]

A 10 3 7

51.3:48.7

3 7

50.0:50.0

B 10 7 3 7 3

C 10 3 7 3 7

D 10 8 2 8 2

E 10 7 3 7 3

F 10 4 6 3 7

G 10 4 6 4 6

H 10 5 5 5 5

6.25 [8.93]

A 10 5 5

42.5:57.5

4 6

40.0:60.0 B 10 3 7 3 7

C 10 4 6 4 6

D 10 5 5 5 5

12.5 [15.8]

A 10 3 7

37.5:62.5

6 4

50.0:50.0 B 10 4 6 5 5

C 10 7 3 8 2

D 10 1 9 1 9

25.0 [29.3]

A 10 4 6

40.0:60.0

4 6

37.5:62.5 B 10 5 5 5 5

C 10 2 8 2 8

D 10 5 5 4 6

50.0 [53.0]

A 10 4 6

45.0:55.0

4 6

47.5:52.5 B 10 8 2 8 2

C 10 3 7 4 6

D 10 3 7 3 7

 

1 Nominal concentration with mean measured concentration in brackets.
Table 18 No Observed Effect Concentration and Lowest Observed Effect Concentrations

for Xenopus laevis exposed to 4-tert-octylphenol in LAGDA.

Parameter No-Observable-Effect 

Concentration (μg/L)

Lowest-ObservableEffect Concentration 

(μg/L)

NF Stage 62

Time to Metamorphosis (Replicate Analysis) 53 >53

Time to Metamorphosis (Kaplan-Meier) 53 >53

Whole Body Weight 53 >53

Snout-to-Vent Length 53 >53

Plasma Thyroxine Level 53 >53

Plasma Vitellogenin Level 15.8 29.3

NF Stage 66 Cull

Whole Body Weight 53 >53

Snout-to-Vent Length 53 >53

Frogs at Termination

Whole Body Weight 53 >53

Liver Weight:Whole Body Weight (Male)153 >53

Liver Weight:Whole Body Weight (Female) 53 >53

Snout-to-Vent Length 53 >53

Nuptial Pad Development (Male)253 >53

Plasma Thyroxine Level 53 >53

Plasma Vitellogenin Level (Male) <8.93 8.93

Plasma Vitellogenin Level (Female) 15.8 29.3

 

1 K-W tests were significant for males (p = 0.048) and females (p = 0.037), but in neither case could the individual 

differences between the treatment and the control be determined (p > 0.013).

2 Only two females had nuptial pads.
Figure 11.

0.00 8.93 15.8 29.3 53.0

1.0

0.8

0.6

0.4

0.2

0.0

Mean Measured Dose (μg/L)

Proportion of Males with Nuptial Pads

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.

Figure 12.

0.00 8.93 15.8 29.3 53.0

1.0

0.8

0.6

0.4

0.2

0.0

Mean Measured Dose (μg/L)

Proportion with Nuptial Pads Not Determined

Boxplot of the proportion of male frogs for which nuptial pads could not be determined 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.
Figure 13.

0.00 8.93 15.8 29.3 53.0

0.080

0.075

0.070

0.065

0.060

0.055

Mean Measured Dose (μg/L)

Liver Weight:Whole Body Weight

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.

Figure 14.

0.00 8.93 15.8 29.3 53.0

0.085

0.080

0.075

0.070

0.065

Mean Measured Dose (μg/L)

Liver Weight:Whole Body Weight

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.
Figure 17.

0.00 8.93 15.8 29.3 53.0

40000

30000

20000

10000

0

Mean Measured Dose (μg/L)

Vitellogenin Concentration (ng/mL)

Boxplot of plasma vitellogenin 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, and the horizontal line is the median.

Figure 18.

0.00 8.93 15.8 29.3 53.0

160000

140000

120000

100000

80000

60000

40000

20000

Mean Measured Dose (μg/L)

Vitellogenin Concentration (ng/mL)

Boxplot of plasma vitellogenin 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.
APPENDIX B. PROTOCOL, PROTOCOL AMENDMENTS, AND PROTOCOL DEVIATION

– Additional Protocol Deviation, Deviation 02
APPENDIX C. STATISTICAL ANALYSES (BATTELLE PACIFIC NORTHWEST 

LABORATORY) – Amended Report
PNWD-4374

4-tert-octylphenol: Larval Amphibian Growth and 

Development Assay Using Xenopus laevis

Amended Final Report of

Statistical Analyses for Fort Environmental Study No. BPNL01-00242

Prepared for 

Fort Environmental Laboratories, Inc.

515 South Duncan Street

Stillwater, Oklahoma 74074

Prepared by

Roy K. Kropp and Valerie I. Cullinan

Battelle Pacific Northwest Division

1529 W. Sequim Bay Road

Sequim, Washington 98382

March 25, 2013
PNWD-4374

LEGAL NOTICE

This report was prepared by Battelle Memorial Institute (Battelle) as an account of sponsored research activities. Neither Client nor 

Battelle nor any person acting on behalf of either: 

MAKES ANY WARRANTY OR REPRESENTATION, EXPRESS OR IMPLIED with respect to the accuracy, completeness, 

or usefulness of the information contained in this report, or that use of any information, apparatus, process, or composition disclosed 

in this report may not infringe privately owned rights: or 

Assumes any liabilities with respect to the use of or for damages resulting from the use of any information, apparatus, process, or 

composition disclosed in this report.

Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, 

does not necessarily constitute or imply its endorsement, recommendation, or favoring by Battelle. The views and opinions of 

authors expressed herein do not necessarily state or reflect those of Battelle. 

Balfeile

'. Tic Busincss o/Innovation

\, PNWD-4374

Study Title: 4-tert-octylphenol: Larval Amphibian Growth and Development Assay UsingXenopus

laevis

The statistical report has been reviewed bV Quality Assurance. The following audit was conducted.

Type of Audit Dates Conducted

Audit of Data

Qualrty, September 5,2012

Statistical report

Audit of Data

Quality,

Revised

Statistical report

Audit of Data

Quality,

Amended

Statistical report

September 25,2A12 September 25,2012 October 3,2012

March 23,2013 March 25,2013 March 25,2013

The final amended statistical report has been reviewed and is an accurate representation of the analyses

conducted.

JS lt4ar 20 t3

Date Reported to

Battelle Principal

Investigator and

Management

September t2,20L2

Date Reported to the

Study Director and

Testing Facility

Management

September 25,2012
PNWD-4374

1

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 has been 

subcontracted to Fort Environmental Laboratories and will be used to obtain 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 4-tert-octylphenol, a known vertebrate-relevant 

endocrine-disrupting compound. This report presents the results of the statistical analyses conducted on 

measurements of development and growth, and secondary mechanistic biomarker responses that should 

be indicative of potential adverse effects on population relevant parameters and potential endocrine 

disrupter activity for Xenopus laevis. 

This amended statistical report is a revision of the final report that was issued December 19, 2012. The 

statistical analyses (and the report) were revised to incorporate data changes made to 19 genotypic sex 

determinations and revised vitellogenin determinations for termination stage frogs. The specific 

analyses that were affected are nuptial pad presence/absence, liver weight:body weight ratios, plasma 

vitellogenin levels, and genetic sex ratios. The reanalyses changed the lowest observed effects 

concentration (LOEC) and the no observed effects concentration (NOEC) values for female liver 

weight:body weight ratio and male plasma vitellogenin level.

Statistical Methods

Parametric assumptions were tested using the residuals from a one-way analysis of variance (ANOVA)

of the treatment means of the raw variable or following a transformation. Deviations from normality 

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

with Levene’s test (α=0.05). Count data was transformed to the square root, proportional data was 

transformed to the arcsine square root, and continuous data was transformed to the natural logarithm. If 

the responses were monotonic, the William's test of ordered alternatives was used to estimate the LOEC 

and the 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 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.

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 
PNWD-4374

2

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

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

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

within class variances were met. When the ANOVA (a comparison among all treatments) was 

significant, the Dunnett Method was used to compare the mean responses between the control and 

treatment doses. The K-W test was used when assumptions of normality and homogeneity of within 

class variances were not met. When the K-W (a comparison among all treatments) was significant, a 

K-W multiple comparison test was used to evaluate the median responses between the control and 

treatment doses and to estimate the LOEC/NOEC. No identified outliers were removed from the 

analyses. Time to metamorphosis at NF stage 62 was analyzed by following the statistical procedures 

used for the other study parameters, and the median time to metamorphosis was determined by a 

Kaplan-Meier product-limit estimator and was analyzed statistically. A binomial probability was 

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

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

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

replicates within treatments. The mean and standard deviation of the treatment-related responses for 

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

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

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

both NF stage 66 cull parameters, and termination whole body weight, snout-to-vent length, and plasma 

thyroxine levels were not separated by frog sex because there is no expectation that these parameters 

would vary by sex at the particular times of development at which they were measured. Statistics were 

done and graphs prepared in Minitab v16, except for the analytical chemistry data and nuptial pad 

binomial probability, which were analyzed in Microsoft Excel. Table mean or median values for Time 

to NF stage 62 were rounded to the nearest whole number; however boxplot values were not because 

Minitab creates the plots by using the values it calculates.

Results

4-tert-octylphenolAnalytical Chemistry

The measured 4-tert-octylphenol concentrations for treatment Level 1 and Level 3 were relatively 

variable during the study, as indicated by CV value of 21% and 25%, respectively (Table 1; Figure 1). 

These CV values were greater than the guideline acceptability criterion of CV ≤20%. The percent 

recoveries based on the mean 4-tert-octylphenolconcentrations ranged from 106% to 143% of the 

nominal values. 

Table 1. Summary of analytical results for the Xenopus laevis 4-tert-octylphenol in-life exposure.

Treatment

Nominal

Concentration 

(μg/L)

Mean Measured

Concentration 

(μg/L) StDev CV Recovery

Control 0 <MQL 1 – – NA

Level-1 6.25 8.93 1.86 21% 143%

Level-2 12.5 15.8 1.67 11% 126%

Level-3 25 29.3 7.23 25% 117%
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Level-4 50 53.0 5.80 11% 106%

1 MQL = Minimum Quantifiable Limit = 3.08 μg/L

Figure 1. Analytical results for the Xenopus laevis 4-tert-octylphenolin-life exposure. Thick horizontal 

lines indicate the nominal doses.

4-tert-octylphenol Biological Studies—NF Stage 62

Survival.—Survival through NF Stage 62 was 100 percent for the control and all 4-tert-octylphenol

treatments.

Time To Metamorphosis.—The average time to metamorphosis at NF Stage 62 for Xenopus laevis

exposed to 4-tert-octylphenol based on the replicate analysis ranged from 38 to 43 days (Table 2; Figure 

2a). The median time to metamorphosis based on the replicate analysis was 40 days in the control 

treatment and ranged from 38 to 43 days in the 4-tert-octylphenol treatments. No significant differences 

among treatments were detected at p = 0.05. The median time to metamorphosis based on the KaplanMeier product-limit estimator analysis was 35 days in the control treatment and ranged from 35 to 38 

days in the 4-tert-octylphenol treatments (Table 2; Figure 2b). No significant differences among 

treatments were detected at p = 0.05. A comparative evaluation of the two methods of estimating time 

to NF stage 62 is provided in Appendix 1 of the statistical report.

0

10

20

30

40

50

60

70

0 7 14 21 28 35 42 49 56 63 70 77 81 91 98 105 109

4-Tert-Octylphenol Concentration (ug/L)

Study Day

Level 1

Level 2

Level 3

Level 4
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Table 2. Mean (and standard deviation) time to metamorphosis (NF Stage 62) during the Xenopus laevis 4-

tert-octylphenol exposure.

Mean Measured

Concentration (μg/L)

Time to Metamorphosis (d)

Mean (StDev) Median 1 Median 2

<MQL 40 (2.9) 40 35

8.93 43 (3.8) 43 38

15.8 38 (1.3) 39 35

29.3 39 (0.9) 39 35

53.0 38 (2.3) 38 35

p-value – 0.187 0.770

Test: – K-W K-W 1 Median of treatment replicates 2 Median of replicate medians based on the Kaplan-Meier product-limit estimator analysis

<MQL 8.93 15.8 29.3 53.0

47.5

45.0

42.5

40.0

37.5

35.0

Mean Measured Dose (μg/L)

Time to Metamorphosis (d)

Figure 2a. Boxplot of time to metamorphosis (NF Stage 62) during the Xenopus laevis 4-tert-octylphenol

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

median.
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<MQL 8.93 15.8 29.3 53.0

44

42

40

38

36

34

32

Mean Measured Dose (μg/L)

Median Kaplan-Meier Estimate (d)

Figure 2b. Boxplot of time to metamorphosis (NF Stage 62) during the Xenopus laevis 4-tert-octylphenol 

exposure based on the Kaplan-Meier product-limit estimator analysis. The box represents the 

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

Growth.—Mean whole body (wet) weight of NF Stage 62 frogs was 0.461 g in the control treatment 

and ranged from 0.466 to 0.540 g in the 4-tert-octylphenol treatments (Table 3; Figure 3). No 

significant differences in whole body weight among treatments were detected at p = 0.05. Mean snoutto-vent length of NF Stage 62 frogs was 1.52 cm in the control treatment and ranged from 1.52 to 1.60

cm in the 4-tert-octylphenol treatments (Table 3; Figure 4). No significant differences in snout-to-vent 

length among treatments were detected at p = 0.05.

Table 3. Mean (and standard deviation) whole body weight and snout-to-vent length for NF Stage 62 frogs

during the Xenopus laevis 4-tert-octylphenol exposure.

Mean Measured

Concentration (μg/L)

Whole Body Weight 

(g)

Snout-to-Vent Length 

(cm)

<MQL 0.461 (0.027) 1.52 (0.035)

8.93 0.475 (0.063) 1.52 (0.073)

15.8 0.540 (0.081) 1.60 (0.054)

29.3 0.494 (0.083) 1.55 (0.075)

53.0 0.466 (0.041) 1.55 (0.036)

p-value 0.537 0.188

Test: K-W K-W
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<MQL 8.93 15.8 29.3 53.0

0.60

0.55

0.50

0.45

0.40

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 3. Boxplot of whole body weight for NF Stage 62 frogs during the Xenopus laevis 4-tert-octylphenol 

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

median.

<MQL 8.93 15.8 29.3 53.0

1.65

1.6

1.55

1.5

1.45

1.4

Mean Measured Dose (μg/L)

Snout-to-Vent Length (cm)

Figure 4. Boxplot of snout-to-vent length for NF Stage 62 frogs during the Xenopus laevis 4-tertoctylphenol exposure. The box represents the interquartile range, the circle is the mean, and the horizontal 

line is the median.

Biochemistry.—The mean plasma thyroxine (T4) concentration for NF Stage 62 frogs was 104 ng/mL 

in the control treatment and ranged from 106 to 130 ng/mL in the 4-tert-octylphenol treatments (Table 4; 

Figure 5). No significant differences in T4 concentration among treatments were detected at p = 0.05. 

The plasma vitellogenin (VTG) concentration for NF Stage 62 frogs was 4701 ng/mL in the control 
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treatment and ranged from 5399 to 15,005 ng/mL in the 4-tert-octylphenol treatments (Table 4; Figure 

6). Significant differences in plasma VTG concentrations among treatments were detected. VTG 

concentrations in the 29.3- and 53.0-μg/L concentrations were significantly greater than that in the 

control treatment (K-W multiple comparisons; p < 0.001 for both treatments).

Table 4. Mean (and standard deviation) plasma thyroxine and vitellogenin concentration for NF Stage 62 

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

Mean Measured

Concentration (μg/L)

Plasma Thyroxine

(ng/mL)

Plasma Vitellogenin

(ng/mL)

<MQL 104 (16.7) 4701 (645)

8.93 109 (18.8) 5399 (354)

15.8 106 (18.1) 7085 (1000)

29.3 106 (11.1) 13,867 (744)

53.0 130 (52.3) 15,005 (2300)

p-value 0.932 <0.001

Test: K-W K-W

<MQL 8.93 15.8 29.3 53.0

220

200

180

160

140

120

100

80

Mean Measured Dose (μg/L)

Thyroxine Concentration (ng/mL)

Figure 5. Boxplot of plasma thyroxine concentration for NF Stage 62 frogs during the Xenopus laevis 4-

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

horizontal line is the median.

 
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<MQL 8.93 15.8 29.3 53.0

17500

15000

12500

10000

7500

5000

Mean Measured Dose (μg/L)

Vitellogenin Concentration (ng/mL)

Figure 6. Boxplot of plasma vitellogenin concentration for NF Stage 62 frogs during the Xenopus laevis 4-

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

horizontal line is the median.

4-tert-octylphenol Biological Studies—Juveniles

Survival.—Survival from NF Stage 62 to NF Stage 66 and from NF Stage 66 to test termination was

100 percent for the control and all 4-tert-octylphenol treatments.

Growth.—Mean whole body (wet) weight of frogs measured at the NF Stage 66 cull was 2.42 g in the 

control treatment and ranged from 1.84 to 2.62 g in the 4-tert-octylphenol treatments (Table 5; Figure 7). 

No significant differences in whole body weight among treatments were detected for these frogs at p = 

0.05. Mean snout-to-vent length of frogs measured at the NF Stage 66 cull was 2.69 cm in the control 

treatment and ranged from 2.46 to 2.78 cm in the 4-tert-octylphenol treatments (Table 5; Figure 8). No 

significant differences among treatments were detected in snout-to-vent length for frogs at the NF Stage 

66 cull. 

Mean whole body (wet) weight of frogs measured at the termination of the 4-tert-octylphenol exposure 

was 8.48 g in the control treatment and ranged from 7.82 to 8.99 g in the 4-tert-octylphenol treatments 

(Table 5; Figure 9). Significant differences among treatments were detected in whole body weight for 

frogs at the termination of the exposure. However, the Dunnett Method 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 frogs measured at the termination of the exposure was 4.09 cm in the control 

treatment and ranged from 4.11 to 4.30 cm in the 4-tert-octylphenol treatments (Table 5; Figure 10). 

Significant differences among treatments were detected in snout-to-vent length for frogs at the 

termination of the exposure. The median snout-to-vent length for the 15.8-μg/L 4-tert-octylphenol 

concentration was significantly greater than that for the control treatment (K-W multiple comparisons; p

= 0.003).
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Table 5. Mean (and standard deviation) whole body weight and snout-to-vent length for frogs at the NF

Stage 66 cull and at the termination of the Xenopus laevis 4-tert-octylphenol exposure.

NF Stage 66 Cull Termination

Mean Measured

Concentration (μg/L)

Whole Body 

Weight (g)

Snout-to-Vent 

Length (cm)

Whole Body 

Weight (g)

Snout-to-Vent 

Length (cm)

<MQL 2.42 (0.377) 2.69 (0.159) 8.48 (0.577) 4.09 (0.117)

8.93 2.62 (0.058) 2.78 (0.051) 8.99 (0.410) 4.17 (0.017)

15.8 2.12 (0.435) 2.53 (0.172) 8.80 (0.669) 4.30 (0.102)

29.3 1.98 (0.834) 2.54 (0.324) 7.82 (0.299) 4.14 (0.046)

53.0 1.84 (0.519) 2.46 (0.225) 7.84 (0.540) 4.11 (0.042)

p-value 0.239 0.190 0.013 0.028

Test: K-W K-W 1-Way ANOVA K-W

<MQL 8.93 15.8 29.3 53.0

2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4

1.2

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 7. Boxplot of whole body weight for frogs at the NF Stage 66 cull of the Xenopus laevis 4-tertoctylphenol exposure. The box represents the interquartile range, the circle is the mean, the asterisk is an 

outlier, and the horizontal line is the median.
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<MQL 8.93 15.8 29.3 53.0

2.9

2.8

2.7

2.6

2.5

2.4

2.3

2.2

Mean Measured Dose (μg/L)

Snout-to-Vent Length (cm)

Figure 8. Boxplot of snout-to-vent length for frogs at the NF Stage 66 cull 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.

<MQL 8.93 15.8 29.3 53.0

10.0

9.5

9.0

8.5

8.0

7.5

7.0

Mean Measured Dose (μg/L)

Whole Body Weight (g)

Figure 9. Boxplot of whole body weight for 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.
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<MQL 8.93 15.8 29.3 53.0

4.5

4.4

4.3

4.2

4.1

4.0

3.9

Mean Measured Dose (μg/L)

Snout-to-Vent Length (cm)

Figure 10. Boxplot of snout-to-vent length for 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.

Nuptial pad development.—The mean proportion of male frogs in the control treatment that had 

nuptial pads at the termination of the 4-tert-octylphenol exposure was 0.72 (Table 6; Figure 11). No 

females from the control treatment had nuptial pads. The mean proportion of male frogs from the 4-tertoctylphenol treatments that had nuptial pads at the termination of the exposure ranged from 0.50 to 1.0

(Table 6; Figure 11). Significant differences among treatment means were detected for the proportion of 

males having nuptial pads. However, the K-W multiple comparisons test did not identify the differences 

between any individual treatment value and the control treatment. The proportion of male frogs for 

which presence or absence of nuptial pads could not be determined was 0.11 for frogs from the control 

treatment and ranged from 0.10 to 0.81 for frogs from the 4-tert-octylphenol treatments (Table 6; Figure 

12). Significant differences among treatment means were detected for the proportion of males for which 

the occurrence of nuptial pads could not be determined; the proportion for frogs from the 53.0-μg/L 

treatment was significantly greater than that for the control treatment (Dunnett’s Method). Only two 

females in the 4-tert-octylphenol treatments had nuptial pads; one each in the 8.93- and 29.3-μg/L 

treatments). No statistical analysis was conducted on the proportion of females with nuptial pads. 

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 males 

(Table 7). The expected proportion of nuptial pad occurrence was calculated for males from the mean 

proportion from the control replicates (probability of nuptial pads occurring by chance = 0.71 for males; 

n = 8). The probability of obtaining the total number of occurrences out of 40 frogs (the number in each 

treatment) from chance alone was calculated independently for each exposure concentration. The 

proportions of males in the 8.93- and 29.3-μg/L 4-tert-octylphenol concentrations having nuptial pads 

were 1.0, which were considered significantly greater than chance (p = 0.009 and 0.048, respectively). 

A binomial probability was not calculated for females.
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Table 6. 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)

Nuptial Pads 

(proportion of 

females)

Nuptial Pads 

(proportion of 

males)

Total Number of 

Frogs with Nuptial 

Pads 1

Nuptial Pads 

Not 

Determined 2

<MQL 0 0.72 (0.16) 25 0.11 (0.16)

8.93 0.04 3 1.00 (–) 15 0.10 (0.20)

15.8 0 0.76 (0.25) 12 0.10 (0.13)

29.3 0.04 3 1.00 (–) 10 0.40 (0.27)

53.0 0 0.50 (0.58) 3 0.81 (0.13)

p-value – 0.036 4 – 0.001

Test: – K-W – 1-Way ANOVA

1 Total number of frogs = 80 for control and 40 for each 4-tert-octylphenol dose.

2 The proportion of male frogs for which presence or absence of nuptial pads could not be determined.

This is not an endpoint included in the LAGDA.

3 Only one female in each treatment had nuptial pads.

4 Adjusted for ties.

0.00 8.93 15.8 29.3 53.0

1.0

0.8

0.6

0.4

0.2

0.0

Mean Measured Dose (μg/L)

Proportion of Males with Nuptial Pads

Figure 11. Boxplot of the proportion of male frogs having nuptial pads at the termination of the Xenopus 

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

the horizontal line is the median.
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0.00 8.93 15.8 29.3 53.0

1.0

0.8

0.6

0.4

0.2

0.0

Mean Measured Dose (μg/L)

Proportion with Nuptial Pads Not Determined

Figure 12. Boxplot of the proportion of male frogs for which nuptial pads could not be determined 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.

Table 7. Proportion of frogs within each treatment having nuptial pads at the termination of the Xenopus 

laevis 4-tert-octylphenol exposure.

Mean Measured

Concentration (μg/L)

Proportion of 

Females with 

Nuptial Pads

Binomial 

Probability

Proportion of 

Males with 

Nuptial Pads

Binomial 

Probability

<MQL 0 – 0.71 –

8.93 0.04 1 – 1.00 0.009

15.8 0 – 0.71 0.647

29.3 0.04 1 – 1.00 0.048

53.0 0 – 0.75 0.677

1 Only one female in each treatment had nuptial pads.

Liver weight.—Mean liver weight:whole body weight ratios of frogs measured for the control treatment 

at the termination of the 4-tert-octylphenol exposure were 0.072 for males and 0.075 for females. The 

ratio for males ranged from 0.064 to 0.071 in the 4-tert-octylphenol treatments (Table 8; Figure 13). 

The ratio for females ranged from 0.068 to 0.075 in the 4-tert-octylphenol treatments (Table 8; Figure 

14). Significant differences among treatments were detected in liver weight:whole body weight for male 

and female frogs at the termination of the exposure. However, in neither case could the individual 

differences between any treatment and the control be determined (p > 0.013).
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Table 8. 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 0.072 (0.004) 0.075 (0.004)

8.93 0.065 (0.003) 0.073 (0.004)

15.8 0.071 (0.003) 0.075 (0.005)

29.3 0.066 (0.007) 0.069 (0.002)

53.0 0.064 (0.007) 0.068 (0.004)

p-value 0.048 0.037

Test: K-W K-W

0.00 8.93 15.8 29.3 53.0

0.080

0.075

0.070

0.065

0.060

0.055

Mean Measured Dose (μg/L)

Liver Weight:Whole Body Weight

Figure 13. Boxplot of liver weight:whole body weight for male frogs at the termination of the Xenopus 

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

the horizontal line is the median.
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0.00 8.93 15.8 29.3 53.0

0.085

0.080

0.075

0.070

0.065

Mean Measured Dose (μg/L)

Liver Weight:Whole Body Weight

Figure 14. 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.

Biochemistry.—The mean plasma thyroxine concentration in the control treatment at the termination of 

the 4-tert-octylphenol exposure was 5.27 ng/mL. Mean T4 concentrations for frogs in the 4-tertoctylphenol treatments at termination of the exposure ranged from 5.48 to 6.82 ng/mL (Table 9; Figure 

15). No significant differences in T4 concentration among treatments were detected at p = 0.05. The 

plasma vitellogenin concentration for male and female frogs in the control treatment at the termination 

of the 4-tert-octylphenol exposure were 4585 and 36,811 ng/mL, respectively. VTG concentrations for 

frogs in the 4-tert-octylphenol treatments at termination of the exposure ranged from 7348 to 32,974

ng/mL for males and from 42,266 to 141,624 ng/mL for females (Table 9; Figures 16, 17). Significant 

differences in plasma VTG concentrations among treatments for males and females were detected. VTG 

concentrations in male frogs from all concentrations were significantly greater than that in the control 

treatment (1-Way ANOVA; p < 0.001). VTG concentrations in female frogs from the 29.3- and 53.0-

μg/L concentrations were significantly greater than that in the control treatment (K-W multiple 

comparisons; p =0.003 and p < 0.001, respectively). 
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Table 9. Mean (and standard deviation) plasma thyroxine and vitellogenin concentration for frogs at the 

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

Mean Measured

Concentration (μg/L)

Plasma

Thyroxine

(ng/mL)

Male Plasma 

Vitellogenin 

(ng/mL)

Female Plasma 

Vitellogenin 

(ng/mL)

<MQL 5.27 (1.198) 4585 (909) 36,811 (7340)

8.93 5.99 (0.500) 7348 (1646) 42,266 (10,567)

15.8 5.48 (0.998) 8724 (1909) 55,862 (4954)

29.3 6.19 (0.377) 20,371 (7529) 98,434 (9894)

53.0 6.82 (0.936) 32,974 (8539) 141,624 (9789)

p-value 0.165 <0.001 <0.001

Test: K-W 1-Way ANOVA K-W

<MQL 8.93 15.8 29.3 53.0

8

7

6

5

4

Mean Measured Dose (μg/L)

Thyroxine Concentration (ng/mL)

Figure 15. Boxplot of plasma thyroxine concentration for 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.
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0.00 8.93 15.8 29.3 53.0

40000

30000

20000

10000

0

Mean Measured Dose (μg/L)

Vitellogenin Concentration (ng/mL)

Figure 16. Boxplot of plasma vitellogenin 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, and 

the horizontal line is the median.

0.00 8.93 15.8 29.3 53.0

160000

140000

120000

100000

80000

60000

40000

20000

Mean Measured Dose (μg/L)

Vitellogenin Concentration (ng/mL)

Figure 17. Boxplot of plasma vitellogenin 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.

Sex ratios.—The phenotypic and genotypic sex ratios at the termination differed within the control 

treatment and all treatment levels (Table 10). 
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Table 10. Frog phenotypic and genotypic sex ratios (average number per replicate) at the termination of 

the Xenopus laevis 4-tert-octylphenol exposure.

Mean Measured

Concentration (μg/L) N

Phenotype Genotype

Males Females Males Females

<MQL 80 5.13 4.88 5.00 5.00

8.93 40 4.25 5.75 4.00 6.00

15.8 40 3.75 6.25 5.00 5.00

29.3 40 4.00 6.00 3.75 6.25

53.0 40 4.50 5.50 4.75 5.25

No-Observable-Effect Concentration/ Lowest-Observable-Effect Concentration.—The NoObservable-Effect Concentration and Lowest-Observable-Effect Concentration were calculated as mean 

measured concentrations of 53 μg/L 4-tert-octylphenol and >53 μg/L 4-tert-octylphenol for several

Xenopus laevis parameters (Table 11). 
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Table 11. Calculated No-Observable-Effect Concentration and Lowest-Observable-Effect Concentration 

based on mean measured 4-tert-octylphenol concentrations for parameters measured for Xenopus laevis

exposed to 4-tert-octylphenol.

Parameter No-Observable-Effect 

Concentration (μg/L)

Lowest-Observable- Effect Concentration 

(μg/L)

NF Stage 62

Time to Metamorphosis (Replicate Analysis) 53 >53

Time to Metamorphosis (Kaplan-Meier) 53 >53

Whole Body Weight 53 >53

Snout-to-Vent Length 53 >53

Plasma Thyroxine Level 53 >53

Plasma Vitellogenin Level 15.8 29.3

NF Stage 66 Cull

Whole Body Weight 53 >53

Snout-to-Vent Length 53 >53

Frogs at Termination

Whole Body Weight 53 >53

Liver Weight:Whole Body Weight (Male) 1 53 >53

Liver Weight:Whole Body Weight (Female) 1 53 >53

Snout-to-Vent Length 53 >53

Nuptial Pad Development (Male) 2 53 >53

Plasma Thyroxine Level 53 >53

Plasma Vitellogenin Level (Male) <8.93 8.93

Plasma Vitellogenin Level (Female) 15.8 29.3

1 K-W tests were significant for males (p = 0.048) and females (p = 0.037), but in neither case could the 

individual differences between any treatment and the control be determined (p > 0.013). 2 Only two females had nuptial pads.
FEL

Appendix E. RAW DATA – Summary of Revised Results from FEL Study BPNL01-00242