## DOCUMENT AMENDMENT FORM Fort Environmental Laboratories

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

Amendment Number : 0 7 Document ID Number : BPNL01

- 3 ( 0024 2 )

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

Amendment Relating To :

[ ] Protocol [ ] Study Plan [ ] QAPP [ ] Q A MP [ ] SOP

[ X ] Other (describe): Final Report

Original Specifications : The following Compliance Statement, t ables

, 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 repo rted 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 ...”

A dditional pages/sections, tables, figures and appendices affected:

Page 10 Section 1.2. Results and Conclusions, Page 27 Section 5.5.3. Nuptial Pad Development, P age 28 Section 5.5.5. Biochemistry, Page 28 5.5.6. Gender Ratios, Page 29 Section 5.6. Concent ration 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 characteriz ed, 3) a reserve sample of the test article was not retained, and 4) re

- analysis of selected hind foot samples for genotyp ing by US EPA (Duluth, MN). ”

Page 32, Section 9. Study Protocol Adherence

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

Protocol deviation 02 form attached.

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

See attach ments 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 addre ss 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 analy ses 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 mal es. 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. S ignificant 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 a ny treatment and the control be determ ined (p > 0.013). Plasma T4 concentrations in 10 weeks post

metamorphosis (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 tre atments 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). Th e 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 p lasma 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 complet ion.

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 o f 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 signif icantly 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 statis tical analys e s w ere 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 concentrat ions 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 1 6; 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 me an 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; Figur e 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 t reatments 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 ran ged 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 RECOMMENDATION S

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

– go nadal (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 wee k 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 males 3 

### Total Number of

Frogs with Nuptial Pads 4 

### Nuptial Pads

Not Determined 5 

### 0. 0 0 [< 3

. 1 ] 0 0.7 2 (0.1 6 ) 25 0.1 1 (0.1 6 ) 

### 6.25 [ 8.93 ] 0 .04 6 

### 1.00 (

– ) 15 0.10 (0.20) 

### 12.5 [ 15.8 ] 0 0. 76 (0.25) 12 0.1 0 (0.1 3 ) 

### 25.0 [ 29.3 ] 0.04 6 

### 1.00 (

– ) 10 0.40 (0.27) 

### 50.0 [ 53.0 ] 0 0. 50 (0. 58 ) 3 0. 81 (0.1 3 ) 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 .0 36 (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

- o ctylphenol 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. 0 0 [< 3

. 1 ] 0

– 0. 71

– 6.25 [ 8.93 ] 0.04 9



### – 1.00 0.00 9 12.5 [ 15.8 ] 0

– 0. 71 0. 647 25.0 [ 29.3 ] 0.04 9



### – 1.00 0. 048 50.0 [ 53.0 ] 0

– 0. 7 5 0. 677

8 Nominal concentration with mean measured concen tration 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

Mean 2 SEM 3 Replicate Mean Treatment

Mean 4 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 individ ual individ ual 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 a nd treatment and the control could not be determined (p = 0.048).Table 1 6 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) SEM 2 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

7348 6 823 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

8724 6 954 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

98434 5 4947 10.1 29172

20371 6 3765 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

141624 5 4894 6.9 43491

32974 6 4270 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 n ot 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: 5 7.5 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: 6 2.5 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 Observ ed Effect Concentration and Lowest Observ ed Effect Concentration s for Xenopus laevis exposed to 4

- tert

- octylphenol in LAGDA

.

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) 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 the treatment and the control be determined (p > 0.013).
2 Only two females had nu ptial 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.

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.

### 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 th e horizontal line is the median.

Figure 1 4

.

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

### Figure 1 7

.

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 1 8

.

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. BPNL 0 1

- 0024 2

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, W ashington 98382



# March 2 5 , 201 3

### PNWD

- 4374

LEGAL NOTICE

This report was prepared by Battelle Memorial Institute (Battelle) as an account of sponsored research activities. Neither Cl ient 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 infrin ge privately owned rights: or

Assumes any liabilities with respect to the use of or for damages resulting from the use of any information, apparatus, proce ss, or composition disclosed in this report.

Reference herein to any specific commercial product, pr ocess, 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 reflec t 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 Disrup tor 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

s ubcontracted 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 disrupt ing in vertebrates.

Th is Task Order 31 study evaluate d 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. T his r eport 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 . T he reanalyses changed the lowest observed effects concentration (LOEC) and the no observed effects concentration (NOEC) values for female l iver 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 transformat ion. 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 arcsi ne 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 withi n class variances were met

, otherwise t he 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 wh en the assumptions of normality and homogeneity of within class variances we re met. When these assumptions were not met, t he K ruskal

- Wallis (K

- W) test followed by a step

- down Dunn’s test w as 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

. H owever, because this test does not account for the difference in the number of replicates between the control (n=8) and the test

### PNWD

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

- W multiple comparisons test of the control median compared to the test m edians. 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 significan t, 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 treatmen ts) 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 prob ability was calculate d 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 th e 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. Whe n 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 p repared 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 va lues were not because Minitab creates the plots by using the values it calculates.

Results

4

- tert

- octylphenol Analytical Chemistry

The measured 4

- tert

- octylphenol concentrations for treatment L evel 1 and Level 3 were relatively variable during the study, as indicated by CV value of 21 % and 25%, respectively (Table 1; Figure 1). Th ese CV value s were greater than the guideline acceptability criterion of CV ≤20%. The percent recoveries based on the mean 4

- tert

- octylphenol concentrations ranged fro m 106 % to 14 3 % 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) St D ev 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%

### PNWD

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3 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

- octylphenol in

- 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.

T ime T o M etamorphosis

. — 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 2 a ). The median time to metamorphosis based on the replicate analysis was 40 day s 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 Kaplan

Meier 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
trea tments 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 1094-Tert-Octylphenol Concentration (ug/L)

Study Day Level 1

Level 2

Level 3

Level 4

### PNWD

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4 Table 2

. Mean (and standard deviation) time to metamorphosis (NF Stage 62) during the Xe nopus 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 replicate s 2 Median of replicate medians based on the Kaplan

- Meier product

- limit estimator analysis

5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 4 7

. 5

4 5

. 0

4 2

. 5

4 0

. 0

3 7

. 5

3 5

. 0

M e a n M e a s u r e d D o s e ( μ g / L )TimetoMetamorphosis(d)



### Figure 2 a . 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.

### PNWD

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5 5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 4 4

4 2

4 0

3 8

3 6

3 4

3 2

M e a n M e a s u r e d D o s e ( μ g / L )MedianKaplan-MeierEstimate(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 t o 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 snout

to

- vent length of NF Stage 62 frogs was 1

. 52 c m in the control treatment and ranged from 1

. 52 to 1

. 60

c m in the 4

- tert

- octylphenol treatments (Table 3; Figure 4). No significant differences in snout

- to

- vent length am ong 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 ( c m) <MQL 0.46 1 ( 0.027 ) 1

. 52 ( 0. 0 35 )
8.93 0.475 (0.063) 1.5 2 ( 0. 0 7 3 )
15.8 0.5 40 (0.081) 1

. 60 ( 0. 0 5 4 )
29.3 0.494 ( 0.08 3) 1.5 5 ( 0. 0 7 5 )
53.0 0.46 6 ( 0.041 ) 1

. 5 5 ( 0. 0 3 6 )

p

- value 0.537 0.188
Test: K

- W K

- W

### PNWD

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6 5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 0

. 6

0

0

. 5 5

0

. 5

0

0

. 4 5

0

. 4

0

M e a n M e a s u r e d D o s e ( μ g / L )WholeBodyWeight(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.

5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 1

. 6 5

1

. 6

1

. 5 5

1

. 5

1

. 4 5

1

. 4

M e a n M e a s u r e d D o s e ( μ g / L )Snout-to-VentLength(cm)



### Figure 4 . Boxplot of snout

- to

- vent length 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.



### 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; F igure 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

### PNWD

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7 treatment and ranged from 5399 to 15,005 ng/mL in the 4

- tert

- octylphenol treatments (Table 4; Figure 6 ). S ignificant differences in plasma VTG concentration s among treatments were detected. VTG concentrations in the 29.3

- and 53.0

- μg/L concentrations were significantly greater than that in the control treat ment ( K

- W multiple comparisons; p < 0.001 for both treatments).

Table 4

. Mean (and standard deviation) plasma thyroxin e and vitellogenin concentration for NF Stage 62 frogs during the Xenopus laevis 4

- tert

- octylphenol exposure

.

Mean Measured Concentration ( μg/L ) Plasma Thyroxin e ( 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

5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 2 2

0

2 0 0

1 8

0

1 6

0

1 4

0

1 2

0

1 0

0

8 0

M e a n M e a s u r e d D o s e ( μ g / L )ThyroxineConcentration(ng/mL)



### Figure 5 . Boxplot of plasma thyroxin e 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.

### PNWD

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8 5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 1 7 5 0 0

1 5

0 0 0

1 2 5 0 0

1 0

0 0 0

7 5

0 0

5 0 0 0

M e a n M e a s u r e d D o s e ( μ g / L )VitellogeninConcentration(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.

— S urvival from NF Stage 62 to NF Stage 66 and from NF Stage 66 to test termination w as 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 (Tab le 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

s ignificant 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 termin ation 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 c m in the control treatment and ranged from 4

. 11 to 4

. 30 c m 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).

### PNWD

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9 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 2.4 2 ( 0.377 ) 2.69 ( 0.159 ) 8.48 ( 0.577 ) 4

. 09 (0. 11 7)
8.93 2.62 ( 0.05 8) 2.78 (0.051) 8.9 9 ( 0.4 10) 4

. 1 7 ( 0. 0 1 7)
15.8 2.1 2 ( 0.43 5) 2.5 3 ( 0.172 ) 8. 80 ( 0.66 9) **4.30 (0. 102**  **)**
29.3 1.9 8 ( 0.83 4) 2.5 4 ( 0.324 ) 7.82 ( 0.299 ) 4

. 1 4 ( 0. 0 4 6)
53.0 1.84 ( 0.51 9) 2.46 ( 0.22 5) 7.84 ( 0.5 40) 4

. 1 1 ( 0. 0 42 )

p

- value 0.239 0.190 0.013 **0.028**
Test: K

- W K

- W 1

- Way ANOVA K

- W

5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 2

. 8

2

. 6

2

. 4

2

. 2

2

. 0

1

. 8

1

. 6

1

. 4

1

. 2

M e a n M e a s u r e d D o s e ( μ g / L )WholeBodyWeight(g)



### Figure 7 . Boxplot of whole body weight for frogs at the NF Stage 66 cull of the Xenopus laevis 4

- tert

octylphenol exposure. The box represents the interquartile range, the circle is the mean , the asterisk is an outlier, and the horizontal line is the median.

### PNWD

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10 5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 2

. 9

2

. 8

2

. 7

2

. 6

2

. 5

2

. 4

2

. 3

2

. 2

M e a n M e a s u r e d D o s e ( μ g / L )Snout-to-VentLength(cm)



### Figure 8 . Boxplot of snout

- to

- vent length for frogs at the NF Stage 66 cull of the Xenopus laevis 4

- tert

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

5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 1 0

. 0

9

. 5

9

. 0

8

. 5

8

. 0

7

. 5

7

. 0

M e a n M e a s u r e d D o s e ( μ g / L )WholeBodyWeight(g)



### Figure 9 . Boxplot of whole body weight 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.

### PNWD

- 4374

11 5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 4

. 5

4

. 4

4

. 3

4

. 2

4

. 1

4

. 0

3

. 9

M e a n M e a s u r e d D o s e ( μ g / L )Snout-to-VentLength(cm)



### Figure 10 . Boxplot of snout

- to

- vent length 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.



### Nuptial pad developme nt

. — 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. 7 2 (Table 6 ; Figure 1 1 ) . No females from the control treatment had nuptial pads. The mean proportion of male frogs from the 4

- tert

octylphenol treatments that had nuptial pads at the termination of the exposure ranged from 0. 50 to 1.0 (Table 6 ; Figure 1 1 )

. 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. 1 1 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 1 2 ). 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 e ach 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. T he 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 = 0.0 0 9 and 0.0 48 , respectively ). A binomial probability was not calculated for females.

### PNWD

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12 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) Nuptia l Pads (proportion of males) Total Number of Frogs with Nuptial Pads 1 Nuptial Pads Not Determined 2
<MQL 0 0. 72 ( 0.1 6) 25 0. 11 ( 0.1 6)
8.93 0.04 3 1.00 (

– ) 1 5 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. 2 7 )
53.0 0 0.50 (0.58) 3 **0.81 (0.13)**

p

- value

– **0.036 4** 

– **0.0 0**  **1**
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.

5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 0

. 0

0 1

. 0

0

. 8

0

. 6

0

. 4

0

. 2

0

. 0

M e a n M e a s u r e d D o s e ( μ g / L )ProportionofMaleswithNuptialPads



### Figure 1 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.

### PNWD

- 4374

13 5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 0

. 0

0 1

. 0

0

. 8

0

. 6

0

. 4

0

. 2

0

. 0

M e a n M e a s u r e d D o s e ( μ g / L )ProportionwithNuptialPadsNotDetermined



### Figure 1 2

. 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 Proporti on 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 f emales. The ratio for males ranged from 0. 06 4 to 0. 07 1 in the 4

- tert

- octylphenol treatments (Table 8 ; Figure 1 3 ). The ratio for females ranged from 0. 06 8 to 0. 07 5 in the 4

- tert

- octylphenol treatments (Table 8 ; Figure 1 4 ). 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 > 0.0 13 )

.

### PNWD

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14 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 0.07 2 ( 0.00 4 ) 0.07 5 ( 0.00 4)
8.93 0.065 ( 0.003 ) 0.07 3 ( 0.00 4 )
15.8 0.07 1 ( 0.00 3 ) 0.07 5 ( 0.00 5 )
29.3 0.06 6 ( 0.00 7 ) 0.06 9 ( 0.002 )
53.0 0.06 4 ( 0.00 7 ) 0.06 8 ( 0.00 4 )

p

- value **0.0 48**  **0.0 37** 
Test: K

- W K

- W

5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 0

. 0

0 0

. 0

8 0

0

. 0

7 5

0

. 0

7 0

0

. 0

6 5

0

. 0

6 0

0

. 0

5 5

M e a n M e a s u r e d D o s e ( μ g / L )LiverWeight:WholeBodyWeight



### Figure 1 3 . 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.

### PNWD

- 4374

15 5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 0

. 0

0 0

. 0

8 5

0

. 0

8 0

0

. 0

7 5

0

. 0

7 0

0

. 0

65

M e a n M e a s u r e d D o s e ( μ g / L )LiverWeight:WholeBodyWeight



### Figure 1 4 . 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 c oncentration 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

- tert

octylphenol treatments at termination of the exposure ranged from 5.48 to 6.82 ng/mL ( Table 9; Figur e 1 5 ). 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 3 6

, 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 ; Figure s 1 6

, 1 7 ). Significa nt 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 concent rations 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 =0.00 3 and p < 0.001, respectively).

### PNWD

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16 Table 9

. Mean (and standard deviation) plasma thyroxin e 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 ( n g/mL) Female Plasma Vitellogenin ( n g/mL) <MQL 5.27 ( 1.198 ) 4585 ( 909 ) 36

, 811 ( 7340 )
8.93 5.9 9 ( 0. 500) **7348 (1646)** 42 ,266 ( 10

, 567 )
15.8 5.48 ( 0.998 ) **8724 (1909)** 55 ,862 ( 4954 )
29.3 6.1 9 (0.37 7 ) **20,371 (7529) 98**  **,434 ( 9894**  **)**
53.0 6.8 2 (0.93 6 ) **32,974 (8539) 14**  **1 ,**  **624 ( 9789**  **)**

p

- value 0.165 **<0.001 <0.001** 
Test: K

- W 1

- Way ANOVA K

- W

5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 < M Q L 8

7

6

5

4

M e a n M e a s u r e d D o s e ( μ g / L )ThyroxineConcentration(ng/mL)



### Figure 1 5 . Boxplot of plasma thyroxin e 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.

### PNWD

- 4374

17 5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 0

. 0

0 4 0 0 0 0

3 0 0 0 0

2 0 0 0 0

1 0

0 0 0

0

M e a n M e a s u r e d D o s e ( μ g / L )VitellogeninConcentration(ng/mL)



### Figure 1 6 . 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.

5 3

. 0 2 9

. 3 1 5

. 8 8

. 9 3 0

. 0

0 1 6

0 0 0 0

1 4

0 0 0 0

1 2

0 0 0 0

1 0

0 0 0 0

8 0 0 0 0

6 0 0 0 0

4 0 0 0 0

2 0 0 0 0

M e a n M e a s u r e d D o s e ( μ g / L )VitellogeninConcentration(ng/mL)



### Figure 1 7 . Boxplot of plasma vitellogenin concentration for female frogs at the termination of the Xenopus laevis 4

- tert

- octylphenol exp osure. 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 1 0 ).

### PNWD

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18 Table 1 0

. 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 No

Observable

- Effect Concentration and Lowest

- Observable

- Effect Concentration were calculated as mean measured concentrations of 53 μg/L 4

- tert

- octylphenol and > 5 3 μg/L 4

- tert

- octylphenol for several Xenopus laevis parameter s (Table 11 ).

### PNWD

- 4374

19 Table 11

. Calculated No

- Observable

- Effect Concentration and Lowest

- Observable

- Effect Concentration based on mean measured 4

- tert

- octylphenol concentrations for parameter s measured for Xenopus laevis exposed to 4

- tert

- octylphenol

.

Parameter No

- Observable

- Effect Con centration ( 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