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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data



# Endocrine Disruptor Screening Program

Contract No: EP-W-11-063

Task Order No: 2

Maintain the Data Coordination Center

Statistical Support for Technical Directive #3
Data Analysis Support fo r Invertebrate Tier 2 Te st and Generation of Integrated Summary Report

Intra-Laboratory Statistical Analysis for SMV Octylphenol in Mysids

U.S. Environmental Protection Agency

May 17, 2013

BATTELLE
505 King Avenue
Columbus, Ohio 43201

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

iii Table of Contents Page 1. Introduction ............................................................................................................... ............................ 1 2. Study Data ................................................................................................................. ........................... 2

2.1 Data Endpoints ............................................................................................................ .............. 2 2.2 Data Issues and Data Handling.............................................................................................. .... 3 2.3 Analysis Considerations ................................................................................................... ......... 5
3. Statistical Methods ........................................................................................................ ........................ 7 3.1 Comparisons of test chemical with the control ......................................................................... 7
3.1.1 Outlier Screens ......................................................................................................... .... 8 3.1.2 Normality ............................................................................................................... ...... 8
3.1.3 Transformation of data ................................................................................................. 8 3.1.4 Heterogeneous variances .............................................................................................. 8 3.1.5 Monotonicity of response ............................................................................................. 9 3.1.6 Comparisons of test chemical to controls ..................................................................... 9 3.2 Comparisons of the F0 and F1 generations ............................................................................. 10
3.2.1 Outlier screens ......................................................................................................... ... 11 3.2.2 Normality ............................................................................................................... .... 11
3.2.3 Transformation of data ............................................................................................... 11 3.2.4 Heterogeneous variances ............................................................................................ 11 3.2.5 Monotonicity test ....................................................................................................... . 12 3.2.6 F0 generation vers us F1 generation ............................................................................ 12
4. Results .................................................................................................................... ............................. 12

Tables
Table 0. Data information for SMV Octylphenol ................................................................................ .... 4 Table 1A. Summary of Reproduction Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F0 1,2,3 

## .................................................................................................................... 13 Table 1B. Summary of Reproduction Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F1 1,2,3 

## .................................................................................................................... 14 Table 1C. Statistical Analysis Results for Reproducti on Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F0 ................................................................................................... 15 Table 1D. Statistical Analysis Results for Reproducti on Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F1 ................................................................................................... 16

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

iv Table 1E. Comparisons between Two Generations fo r Reproduction Endpoints. Laboratory: SMV, Test Chemical: Octylphenol. ................................................................................................... 17 Table 2A. Summary of Growth Endpoints. Laborat ory: SMV, Test Chemical: Octylphenol, Generation: F0 1,2,3,4



## .............................................................................................................................. ........ 18 Table 2B. Summary of Growth Endpoints. La boratory: SMV, Test Chemical: Octylphenol, Generation: F1 1,2,3 

## .................................................................................................................... 19 Table 2C. Statistical Analysis Results for Reproducti on Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F0 ................................................................................................... 20 Table 2D. Statistical Analysis Results for Reproducti on Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F1 ................................................................................................... 21 Table 2E. Comparisons between Two Generations fo r Growth Endpoints. Laboratory: SMV, Test Chemical: Octylphenol. ........................................................................................................ ... 22 Table 3A. Summary of Survival Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F0 1,2,3 

## .................................................................................................................... 23 Table 3B. Summary of Survival Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F1 1,2,3 

## .................................................................................................................... 24 Table 3C. Statistical Analysis Results for Reproducti on Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F0 ................................................................................................... 25 Table 3D. Statistical Analysis Results for Reproducti on Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F1 ................................................................................................... 26 Table 3E. Comparisons between Two Generation Surv ival Endpoints. Laboratory: SMV, Test Chemical: Octylphenol. ........................................................................................................ ... 27

Figures
Figure 1. Means ± Standard Errors of Untransformed data: Time to Mature By Generation and Group For Chemical Octylphenol From Lab SMV. ........................................................................... 28 Figure 2. Means ± Standard Errors of Untransformed data: Number Offspring (brood 1) By Generation and Group For Chemical Octylp henol From Lab SMV. ......................................................... 28 Figure 3. Means ± Standard Errors of Untransformed data: Number Offspring (brood 2) By Generation and Group For Chemical Octylp henol From Lab SMV. ......................................................... 29 Figure 4. Means ± Standard Errors of Untransformed data: Number Offspring (two broods) by Generation and Group For Chemical Octylphenol From Lab SMV. ...................................... 29 Figure 5. Means ± Standard Errors of Untransformed data: Days to Brood 1 By Generation and Group For Chemical Octylphenol From Lab SMV. ........................................................................... 30 Figure 6. Means ± Standard Errors of Untransformed data: Days to Brood 2 By Generation and Group For Chemical Octylphenol From Lab SMV. ........................................................................... 30

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

v Figure 7. Means ± Standard Errors of Untransformed data: Days Between Broods 1 and 2 By Generation and Group For Chemical Octylphenol From Lab SMV. ...................................... 31 Figure 8. Means ± Standard Errors of Untransformed data : Total Productive Days By Generation and Group For Chemical Octylphenol From Lab SMV. ................................................................ 31 Figure 9. Means ± Standard Errors of Untransformed data : Offspring per Female per Productive Day By Generation and Group For Chemical Octylphenol From Lab SMV. ................................. 32 Figure 10. Means ± Standard Errors of Untransformed data : Percent of Females Productively Active By Generation and Group For Chemical Octylphenol From Lab SMV. ...................................... 32 Figure 11. Means ± Standard Errors of Untransformed da ta: Sex Ratio By Generation and Group For Chemical Octylphenol From Lab SMV. ................................................................................. 33 Figure 12. Dry Weight (mg) (day 0) By Generati on and Group For Chemical Octylphenol From Lab SMV. .......................................................................................................................... ............. 33 Figure 13. Means ± Standard Errors of Untransformed data: Dry Weight (mg) (day 7) By Generation and Group For Chemical Octylp henol From Lab SMV. ......................................................... 34 Figure 14. Means ± Standard Errors of Untransformed data: Dry Weight (mg) (day 14) By Generation and Group For Chemical Octylp henol From Lab SMV. ......................................................... 34 Figure 15. Means ± Standard Errors of Untransformed da ta: Dry Weight (mg) (Termination) By Generation and Group For Chemical Octylphenol From Lab SMV. ...................................... 35 Figure 16. Means ± Standard Errors of Untransformed data : Length (mm) (day 0) By Generation and Group For Chemical Octylphenol From Lab SMV. ................................................................ 35 Figure 17. Means ± Standard Errors of Untransformed data : Length (mm) (day 7) By Generation and Group For Chemical Octylphenol From Lab SMV. ................................................................ 36 Figure 18. Means ± Standard Errors of Untransformed data : Length (mm) (day 14) By Generation and Group For Chemical Octylphenol From Lab SMV. ................................................................ 36 Figure 19. Means ± Standard Errors of Untransformed data : Length (mm) (Termination) By Generation and Group For Chemical Octylp henol From Lab SMV. ......................................................... 37 Figure 20. Means ± Standard Errors of Untransformed data: Reproduction Survival Rate (%) (day 7) By Generation and Group For Chemical Octylphenol From Lab SMV. ...................................... 37 Figure 21. Means ± Standard Errors of Untransformed data: Reproduction Survival Rate (%) (day 14) By Generation and Group For Chemical Octylphenol From Lab SMV. ................................. 38 Figure 22. Means ± Standard Errors of Untransformed data: Reproduction Survival Rate (%) (Termination) By Generation and Group For Ch emical Octylphenol From Lab SMV. ......... 38 Figure 23. Means ± Standard Errors of Untransformed data : Total Survival Rate (%) (day 7) By Generation and Group For Chemical Octylphenol From Lab SMV. ...................................... 39

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

vi Figure 24. Means ± Standard Errors of Untransformed data : Total Survival Rate (%) (day 14) By Generation and Group For Chemical Octylphenol From Lab SMV. ...................................... 39 Figure 25. Means ± Standard Errors of Untransformed data: Total Survival Rate (%) (Termination) By Generation and Group For Chemical Octylphenol From Lab SMV. ...................................... 40

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

1 1. INTRODUCTION
To evaluate the effects of endocrine disruptin g chemicals, EPA is currently developing and validating a Tier 2, two-generation mysid toxicity test to characterize the adverse consequences of exposure to putative endocrine active substances. The t est is being carried out in three labs: SMV, UNT and WLI. SMV and WLI conducted two-generation mysi d survival and growth tests following a similar protocol with the test chemical octylphenol. Statis tical analyses are being carried out on the SMV and WLI octylphenol study data. The draft analysis documented in this report is based on the SMV octylphenol test data.
The two-generation test is initiated with newly -released (<24 hours old) offspring (F0 generation) that are grown to sexually maturity (~13 to 16 days) and then their offspring are collected (F0 offspring). The F1 generation test is started with newly hatched F0 offspring. The F1 generation is also grown to maturity and their offspring are collected (F1 offs pring). The test is conducted with five graded concentrations of octylphenol and an untreated control with three replicate tanks per test concentration or control group. Each replicate tank contains two retention baskets, one basket housing mysids for reproduction observations and a second basket for grow th and optional biochemical measurements.
For the F0 test the growth and reproducti on baskets each initially in clude 20 newly hatched mysids. With the appearance of a marsupial pouch on females (sexual maturity), mature females are isolated as pairs with males in separate brood cups. Seven breeding pairs are selected in each replicate reproduction basket. The exposure of these F0 mysids is continued until two weeks after the median time to the second brood release in the control group. Offspring (F1 generation) are collected from the F0 generation each morning from the brood cups and tran sferred immediately to baskets with the same F0 exposure conditions as their F0 parents.
The F1 generation is begun with newly hatched F0 of fspring. Within each replicate tank the growth and reproduction baskets each initially include 15 newly hatched mysids. With the appearance of a marsupial pouch on females (sexual maturity), matu re females are isolated as pairs with males in separate brood cups. Seven breeding pairs are selected in each replicate reproduction basket. The exposure of these F1 mysids is continued until tw o weeks after the median time to the second brood release in the control group. Offspring are collected from the F1 generation each morning from the brood cups and transferred immediately to baskets with th e same exposure conditions as their F1 parents.
Development, sexual maturation, reproduction, growth, and survival endpoints are observed in the F0 and F1 generations. Measurements are ma de of a number of endpoints in both F0 and F1 generations reflective of functional endocrine regul ation of development, gr owth, reproduction, and survival. They include sex ratio, time to maturity , time to brood release for the first two broods, and interbrood duration between the fi rst two broods. Observations are asses sed and recorded daily for each individual or breeding pair on mortality, sex determin ation, reproductive condition of female, and number of young released in each of the first two broods. My sids are sampled to determine weight and length at the beginning (Day 0) before being placed into the growth and reproduction baskets, and then at weekly intervals (Day 7 and Day 14) from the growth b askets. At termination (around Day 30 for SMV octylphenol), mysids from the reproduction basket s are also measured for weights and lengths.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

2 Intra-laboratory statistical analysis of th e SMV octylphenol mysid study was performed according to a common statistical analysis plan. For this intra-laboratory analysis comparisons were made between the test chemical concentration gr oups and the concurrent control group to assess the mysid responses to graded concentrations of octyl phenol. The results of th e two-generations were compared to assess the benefits of a two-generation study as compared to a one-generation life cycle study.

2. STUDY DATA

2.1 Data Endpoints
The test method specifies 25 endpoints. Some of the endpoints are recorded per mysid or composite of mysids (e.g. day 0 weight), some are re corded per breeding pair, and some are recorded per basket (i.e. per replicate tank). These endpoints are divided among three categories:
The test method specifies 25 endpoints. Some of the endpoints are recorded per mysid or composite of mysids (e.g. day 0 weight), some are re corded per breeding pair, and some are recorded per basket (i.e. per replicate tank). These endpoints are divided among three categories:
(i) Reproduction (F0, F1) (11)

a. Sex ratio (per basket) b. Time to mature (per basket)

c. Time to first brood release (per breeding pair) d. Time to second brood release (per breeding pair)

e. Interbrood duration, time between release of first and second brood (per breeding pair) f. Number of young per female in the first brood (per breeding pair) g. Number of young per female in the second brood (per breeding pair) h. Total number of young in the first two broods per female (per breeding pair) i. Percentage of females that are reproductively active in the first two broods (per basket) j. Total reproduction days in the first two broods (per breeding pair) k. Number of young per female per reproductive day in the first two brood (per breeding pair)

(ii) Survival (F0, F1) (6)

a. Survival rate for reproduction animals at days 7, 14 and termination (reproduction survival, per basket) b. Survival rate for all animals including both the growth and reproduction animals at days 7, 14 and termination (total survival, per basket)

The survival rates were adjusted for the numbe r removed for weight and length determinations (i.e. were omitted from both the numerator and denomi nator) and the other non-treatment related deaths

(iii) Growth (F0, F1) (8) 1



## a. Length at day 0 (per mysid) b. Length at day 7 (per mysid)

c. Length at day 14 (per mysid) d. Length at termination. (per mysid)

1 

## Day 14 and terminal weights and lengths were reported by gender.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

3 e. Weight at day 0 (on average per 5 mysids) f. Weight at day 7 (on average per 2 mysids) g. Weight at day 14 (per mysid) h. Weight at termination (per mysid).

Note that on day 0, the newly hatched F0 genera tion mysids were kept in untreated water. By contrast on day 0 the newly hatched F1 generation mysids were kept in water at the same test chemical concentration as their parents’ treatment groups. Gr owth measurements were determined for mysids on day 0 before they were assigned to growth and reprod uction baskets, for mysids from the growth baskets at day 7 (6 per replicate tank) and day 14 (all surviving mysids on day 14), and for the surviving reproduction animals at termination. For the weight da ta, there are a total of 9 composites (5 mysids per composite) for the F0 generation and a total of 54 com posites (9 composites (5 mysids per composite) per concentration group among the six concentration groups) for the F1 generation on day 0. On day 7, there are 3 weight composites (2 mysids per composite) per replicate and three replicates per concentration group.
The test laboratory ran a concurrent control and fi ve targeted test concentration levels. The actual exposure concentration levels in ge neral differed from the targeted levels. The statistical analysis procedures do not utilize the actual concentration levels. The concentrations are labeled by their targeted levels but the labels are not actually used in the analyses.

2.2 Data Issues and Data Handling

Data were received in Excel table format not directly suitable for analysis. They were reformatted into STACHARRMS friendly formatted sp readsheets. SAS and STATA programs were used to read in the raw data, define program friendly vari able names, and add new character variables to the data (laboratory name, chemical na me, generation). The reformatted data sheets were saved into three files according to the endpoint category: reproduction, growth, and survival.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

4 Table 0. Data information for SMV Octylphenol

Data Files and data sh eets Endpoints Comments Original- SMV octylpehnol data.xls:

- F0 Reproduction

- F1 Reproduction

- F0 Summary

- F1 Summary Reformatted-:

 SMV Octylphenol fecunity.xls:

- F0Reproduction

- F1Reproduction;

 SMV Octylphenol SexRatio.xls:

- F0SexRatio

- F1SexRatio  Time to Maturity

 Time to 1 st 

## and 2 nd 

## broods

 # of offspring at each brood

 time between two broods

 # of offspring at two broods

 %of female reproductively active

 Offspring per female per reproductive day for the first two broods

 Total reproductively active days for the first two broods

 Sex ratio  If there are no offspring for a brood. The data are recorded as “NA” for the brood sizes and time to brood. Per EPA suggestion, such data are changed as: zero if female is still living, and “NA” for a brood (1 st 

## or 2 nd 

## brood) if female died without releasing the brood.

 The total offspring for the first 2 broods and the # of offspring per female per reproductive day are therefore recalculated and displayed in the reformatted data.

 For each brood pair, the total reproductive days for the first two broods is the days from the first brood from any female to the days of second broods of the brood pairs. If the broods have a “NA” or zero sizes, the day for the brood is a “NA”, and total reproductive days for the SMV lindane data was recorded as “NA”. The SMV octylphenol used the date until the female died, which is inconsistent from the SMV lindane. Thus, we treated them as “NA” or missing just like the SMV lindane data.

 Time to maturity is given once for each replicate tank.

 %of female reproductively active, sex ratio are reported per tank. The other reproductive endpoints are reported per mating pair. Original- 

## SMV octylpehnol data.xls: One sheet for each generation and Weight (dry weight) and length for days 0, 7, 14 and  For F0 generation all mysids on day 0 are kept in

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

5 each day 0, 7, 14 and termination

Reformatted- SMV Octylphenol Growth.xls One sheet for each generation and each day 0, 7, 14 and termination termination untreated water. There were 9 weight composites (5 mysids per composite).

 For F1 day 0 9 composites (5 mysids per composite) for each control and concentration group.

 Length is reported per mysid.

 Weight is reported per 5 mysid composites for day 0, per 2 mysid composites for day 7, and per mysid for day 14 and for termination.

 Gender of mysid is provided for day 14 and termination data. Original- SMV octylpehnol data.xls:

- F0 Reproduction Survival

- F0 Growth Survival

- F0 Total Survival

- F1 Reproduction Survival

- F1 Growth Survival

- F1 Total Survival

Reformatted- SMV Octylphenol Survival.xls:

- F0ReproductionSurvival;

- F0GrowthSurvival

- F0TotalSurvival

- F1ReproductionSurvival

- F1GrowthSurvival

- F1TotalSurvival  Survival (reproduction) (day 7, day 14 and termination)

 Survival (total) (day 7, day 14 and termination)  Survival endpoints areadjusted for the nontreatment related death,

 The Total survival at day 27 endpoint is incorrect because the total survival is the sum of the reproductive survival at day 27 and the growth survival at day 14

. Growth survival is not carried beyond day 14, at least in the SMV Octylphenol data set. Thus the total survival at day 27 assumes that there is no lethality in the growth bin between day 15 and day 27 and so overestimates the survival.

 Survival endpoints are reported per-tank.

2.3 Analysis Considerations
Several analysis considerations need to be noted. These are discussed below.

 STATCHARRMS uses totally independent statistical procedures for analyses within the F0 and F1 generations as compared to comparisons betw een the F0 and F1 generations. Monotonicity tests and Concentration trend tests are available only with the one-way ANOVA in STATCHARRMS. Thus for the analyses within gene rations it was elected to base analyses on the one-way ANOVA procedure. In the one wa y ANOVA STATCHARRMS pools the results across

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

6 replicate tanks (and individual mysids (if the data were presented for each individual)) within test or control groups. The one way ANOVA (parametri c or nonparametric forms) is applied to the one generation data and there ar e no random replicate effects. For the comparison between the F0 and F1 generations STATCHARRMS utilizes a two-way mixed effects ANOVA model, with replicate tanks within treatment groups treated as random effects. The objectives of the analyses differ from those of the within generation analysis . The analyses within generations compare the test group results to the control group results. The analyses between generations compare the results between generations, with in test or control groups.

 The preliminary tests for outliers, normality, heterogeneity of variance, and monotonicity were carried out independently for the analyses within the F0 generation, the analyses within the F1 generation, and the analyses between the F0 a nd F1 generations. In STATCHARRMS, there is no test for monotonicity within the mixed effects models (thus, the two-way ANOVA for comparisons between generations). The results of the preliminary tests may differ among the F0 analysis, the F1 analysis, and the analysis for comparison between F0 and F1. Thus the approaches for the principal tests used are inde pendently determined among these analyses. For example, for a particular endpoint some of the analyses may be parametric for one-generation and nonparametric for the other generation, or they may be based on different transformations of the endpoint (or none).

 STATCHARRMS uses independent outlier detec tion procedures with the one-way ANOVA and with the two-way ANOVA. Therefore the potentia l outliers screened out may not necessarily be consistent among the F0 and F1 within-genera tion analysis (one-way ANOVA), and the F0 and F1 comparison between-generation analysis (two -way ANOVA). The statistical potential outliers are screened values based on the statistical mode l applied, and they may not be in fact biologically important. The potential outliers from both one-way and two-way ANOVA’s should be jointly considered. EPA will determine whethe r the flagged values should be included in the subsequent analyses or should be treated as outliers.

 The statistical analysis plan called for the screen ed values to be sent to EPA for review. EPA would determine which screened values to include in the analyses, which to exclude from the analyses, and which to both include in and exclude from the analyses. In the interests of time for the draft analyses all the screened values were included in the analyses. The screened values were enumerated in the STATCHARRMS outputs, and will be sent to EPA for review on a later date.

 Total survival at termination is an upwardly biased value in the SMV octyl phenol data. This is because survival for the growth mysids is record ed only through day 14. The total survival at termination therefore tacitly assumes that there is no mortality in the growth mysids between day 14 and termination (about study day 30). This is clearly an underestimate of growth mysid mortality beyond day 14.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

7  For the reproductive response when “NA” is reported as a brood size the question arises as to whether or not to treat this as a missing value or as a zero value in the analysis. EPA has suggested that if the female is living the 0 broo d size should be counted as 0. If the female died before the recorded “NA” brood size is generate d, the brood size should be counted as missing. Battelle has changed the data entries in the columns for brood size 1 and brood size 2 in the Excel spreadsheet according to this suggestion. In th e reformatted data, the changes are also made to the affected endpoints: total number of offspr ing in the first two broods and the number of offspring per female per reproductive day during the two broods. If the data entry for a given brood size is zero or “NA”, the time for the brood w as also recorded as “NA” in the original data file and treated as missing in this analysis. If th e brood size is missing in either brood 1 or brood 2, then the sum of the two broods is also treated as missing. If the brood size is missing or zero in either brood 1 or brood 2, the number of reproductiv e days is recorded as “NA” and the number of offspring per female per reproductive day is treated as missing.

 In the SMV spreadsheets the 14 day and termina tion length and weight responses are presented by gender. In the preliminary draft analyses th e 14 day and termination weights and lengths are pooled across male and female mysids.

3. STATISTICAL METHODS
Statistical analysis procedures used conform to the analysis procedures in the STATCHARRMS package. Statistical analyses include two components: comparisons of test chemical with the control within each generation and comparison of the F0 and F1 generations. The statistical analysis was carried out in a univariate fashion, independently for each endpoint.
Statistical summaries and statistical analyses were applied to the data as reported without first summarizing within baskets. For example if the data were reported on a per mysid basis then the analysis procedures were applied to per mysid or per mysid-pair data without first averaging across the mysids in the same replicate basket.

3.1 Comparisons of test chemical with the control
Mysid data for the multi-concentration test chemical were analyzed according to a common procedure for each generation. Analyses were carried out on a per mysid, per breeding pair, or per basket basis depending on how the data were reported. Prelim inary tests were first carried out to detect outliers, departure from normality, heterogeneity of variance, and departure from monotonicity of response. Subsequent comparisons among groups were carried out including all the values (including the screened outlier values) using procedures depe nding on the outcomes of the preliminary analyses. All the test procedures described here are based on the de fault auto option programs in STATCHARRMS.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

8 3.1.1 Outlier Screens
For each generation, outlier screens were carried out using Tukey’s outlier rule on the untransformed data. Outliers were assessed from resi duals (results of subtracting treatment group means from individual values) to avoid confounding outliers and treatment effects. Any response more than 1.5 times the interquartile range above the third quartile (75 th 

## percentile) or below the first quartile (25 th 

## percentile) is considered an outlier by Tukey’s rule.
The statistical analysis plan states that the r esults of the statistical outlier screens were to be documented and reported to EPA for review to dete rmine if the deviations , although statistically significant, are in fact biologically important. EPA will determine whether th e flagged values should be included in the subsequent analyses or should be tr eated as outliers. The subsets of potential outliers are response specific. Due to time constraints, the sta tistical summaries and analyses in this draft report were carried out including the flagged values. The list of screened values is including in the STATACHARRMS outputs and will be sent to EPA.

3.1.2 Normality
Normality was formally assessed in the ANOVA pr ocedure on the untransformed data using the Shapiro-Wilk test (Shapiro and Wilk ( 1965)). It was assessed from residuals (results of subtracting treatment group means from individual values). Scre ened potential outliers were included in the tests.

3.1.3 Transformation of data

STATCHARRMS includes three data transfor mations: log-transformation, square root transformation, and arc-sin square-root transforma tion. Under the auto option in STATCHARRMS, the analysis procedure is first applied to the untransfo rmed data. If the normality assumption is valid, then no transformation is carried out. If the normality assump tion is invalid, then normality tests are again performed on the log and square root transformed data. If the normality assumption is valid after either transformation, then further procedures are applied to the transformed data. If the normality assumption is invalid for both the untransformed data and the transformed data, then nonparametric procedures are applied. Transformation is not necessary for nonpa rametric procedures as any transformation does not change the rank scores for the data when nonparametric procedures are used.

3.1.4 Heterogeneous variances
If the normality assumption was accepted for either the untransformed or the transformed data heterogeneity of variances among the concentrati on groups was assessed in the ANOVA procedure on the untransformed or the transformed da ta with Levene’s test with abso lute residuals (Levene, 1960). Screened outliers were included in the Levene’s t ests. If the normality assumption was invalid for both the untransformed and the transformed data, heter ogeneity of variances among the concentration groups was not assessed as nonparametric procedures were applied.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

9 3.1.5 Monotonicity of response
In STATCHARRMS, a monotonicity test using linear and quadratic contrasts under one-way ANOVA was applied to normalized rank statistics (to avoid the complications that can arise from nonnormal or heterogeneous data). If the linear contrast was significant but the quadratic contrast was not significant, then that was taken as evidence of monotonicity.

3.1.6 Comparisons of test chemical to controls
Depending on the results of the test for monotoni city either ordered alternatives or general alternatives analysis procedures were used. Ordere d alternatives procedures assume that the response is monotonically increasing or monotonically decreasing with increasing test chemical concentration. Ordered alternatives procedures were always carried out on the ranks of the data. If there was significant departure from monotonicity, general alte rnatives procedures were carried out.
If the test for departure from normality was not si gnificant for either the untransformed or the transformed data normal theory parametric methods with homogeneous or hete rogeneous variances were used. If the test for departure from normality w as significant for both the untransformed and the transformed data nonparametric methods were used. The analysis procedures fall into three categories:

 Ordered alternatives

− nonparametric

 General alternatives

− parametric

 General alternatives

− nonparametric
Ordered alternatives – step down procedure
If the hypothesis of monotonicity of response w as accepted, ordered alternatives procedures were used. Step down multiple comparison procedures ar e particularly useful for ordered alternatives situations. The test is carried out as long as there are significant effects, starting from all concentration groups (including the control), successively omitting the highest concentration group. If at any stage the comparison is nonsignificant, the process stops and a ll means in that concentration group and lower are declared to be compatible with the null hypothesis of equality to the control group mean. No adjustment for simultaneity is required. In STATCHARRMS, the nonparametric Jonckheere-Terpstra (Jonckheere (1954)) test of trend was used on the rank data.
Additionally, global overall concentration effects were tested. These were based on the parametric analysis of variance F-test if the norma lity assumption held or the Kruskal-Wallis test if the normal assumption did not hold for both the untransfo rmed and the transformed data. See the general alternatives section for more details.

General alternatives – parametric procedure
If the hypothesis of monotonicity of response w as rejected and the normality assumption was not rejected, parametric general alternative procedures were used. If the overall concentration effect with the ANOVA was significant, comparisons of test concen trations to the contro l were conducted using Dunnett’s test with homogeneous within-treatment variances and using the Tamhane-Dunnett test with heterogeneous within-treatment variances.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

10 General alternatives – nonparametric procedure
If the hypothesis of monotonicity of response was rejected and the normality assumption was rejected for both the untransformed and the tran sformed data, nonparametr ic general alternative procedures were used. The Kruskal-Wallis test was app lied to test for the overall concentration effect. If the overall concentration effect was significant, the comp arisons of test concentration to the control were conducted using a modified Dunn’s test.

Exact permutation tests
For nonparametric procedures, if a large per centage of the responses have the same measured value (ties), specifically in the case of very small samp les, say less than three per concentration for most concentrations, it is may not be possible to assess in a meaningful way the true distribution of responses across test concentration groups. One can either use an assumed distribution or treat this situation in the same way as data with massive ties. Exact permutati on test methodology is an appropriate tool to use in these cases. The ANOVA component of STATCHARRM S automatically uses exact methods when deemed appropriate. In the ANOVA procedure, ther e are two conditions under which exact permutation methods are used: (1) There are no more than thr ee experimental units in any treatment group and nonparametric procedures are found appropriate. (2) Ther e are “massive ties” in the responses, i.e., 25% or more of the experimental units have the same res ponse or 40% or more have one of only two response values or 65% or more have one of only three r esponse values. An exact version of the JonckheereTerpstra test is used for the step-down trend test and an exact version of the Mann-Whitney test with Bonferroni-Holm adjusted p-values is used in place of Dunn’s test.

3.2 Comparisons of the F0 and F1 generations
Mysid data for the two generation multi-concentr ation test were analyzed according to a common procedure for each generation as described above. Analy ses were also carried out compare the results in the F0 and F1 generations. Analyses were carried out using data reported on a per mysid, per breeding pair, or per basket basis. The comparisons of the F0 and F1 generations were performed with a two way mixed effects ANOVA model, which included generation, concentration group, and the interaction of generation and concentration as fixed effects and replicate (concentration, generation) as a random effect, allowing unequal variances between the two generati ons. Residual variance was treated as homogeneous or heterogeneous between generations based on the results of Levene’s test.
Preliminary tests were carried out to detect outliers, departures from nor mality, and heterogeneity of variance. These analyses were independent of tho se carried out for the analy ses within generations and did not necessarily arrive at the same conclusions. Subsequent comparisons among groups were carried out using procedures based on the outcome of the prelim inary tests. All test procedures described here are based on the default auto options in STATCHARRMS.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

11 3.2.1 Outlier screens

In STATCHARMS, the outliers screened for the comparisons of two gene rations were based on the two-way mixed effect model in section 3.2.6. Outliers were detected based on the studentized residuals from the mixed effects model fit using Tuke y’s outlier rule on the untransformed data. Any response more than 1.5 times the interqua rtile range above the third quartile (75 th 

## percentile) or below the first quartile (25 th 

## percentile) is considered an outlier by Tukey’s rule. These outliers were documented together with those identified in the one generation analyses and will be reported to EPA for review. EPA will determine if the deviations, although statistically significant, are in fact biologically important. EPA will determine whether the flagged values should be included in the subsequent analyses or should be treated as outliers. The subsets of potential outliers ar e response specific. Due to time constraints, the subsequent summaries and statistical analyses in this re port were carried out including the flagged values. The list of screened values is presented in the STATACHARRMS output.

3.2.2 Normality
Normality was formally assessed in the mixed e ffects ANOVA procedure using the Shapiro-Wilk test (Shapiro and Wilk, 1965). It was assessed based on residuals from the 2-way mixed effects ANOVA model fit, where group and generation as well as group by generation interaction were fixed effects. The random replicate tank-to-tank variance was assume d by STATCHARRMS to be heterogeneous between generations. Residual variance within replicate tanks was assumed to be homogeneous between generations. Screened potential ou tliers were included in the tests.

3.2.3 Transformation of data
There are three options for data transformati on in STATCHARRMS: log-transformation, square root transformation, and arc-sin square-root transf ormation. Under the auto option in STATCHARRMS, the analysis procedures are first applied to the unt ransformed data. If the normality assumption is valid, then no transformation is necessary. If the normality assumption is invalid, then normality tests are again performed on the log and the square root transforme d data. If the normality assumption is valid after a transformation, then the procedures are applied to the transformed data, including the homogeneity of variance analysis. If the normality assumption is i nvalid for both transformations, then nonparametric procedures are applied. Transformation is then not necessary as any transformation does not change the rank scores for the data when the nonparametric procedures are used.

3.2.4 Heterogeneous variances
If the normality assumption was accepted for either the untransformed or the transformed data heterogeneity of variances among the generations and the concentrati on groups was assessed using the ANOVA procedure and Levene’s test with absolute r esiduals (Levene, 1960). Outliers were included in the test.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

12 3.2.5 Monotonicity test
A test for monotonicity was not carried out as ordered alternatives procedures are not available in the STATCHARRMS two-way mixed effects ANOVA.

3.2.6 F0 generation versus F1 generation
If the normality assumptions held, a parametric two-way mixed effects analysis was applied to the untransformed or transformed data (whichever satisfied the normality assumption). Otherwise, a nonparametric mixed effects analysis was applied to the rank scores. If the generation main effect or the interaction of concentration by generation effect was significant, F-tests for significant differences between generations, within concentr ation groups (including the control group), the so-called slices in the ANOVA table, were tested to identify the concentr ation groups for which th e two generations were significantly different.

4. RESULTS
For each endpoint the summary tables present the following statistical analysis for the control group and each concentration group for each generation:

 Number of samples

 Mean

 Standard deviation

 CV (%)

These summary statistics were calculated acro ss all samples and all baskets for each concentration group and for each generation. Summary results for the two generations are presented in Tables 1A and 1B (reproduction endpoints), Tables 2A and 2B (growth endpoints), Tables 3A and 3B (survival endpoints). These means and standard devia tions are also displayed, sided-by-side for the two generations in Figures 1 through 25. The summaries a nd figures are based on untransformed data with no screened outliers removed.
Statistical analysis was performed according to the statistical methods described in the methods section using STATCHARRMS. The results of statis tical analyses by generation are summarized in Tables 1C and 1D (reproduction), Tables 2C and 2D (growth), and Tables 3C and 3D (survival). Comparisons between the F0 and F1 generations are summarized in Tables 1E (reproduction), 2E (growth), and 3E (survival).DRAFTIntra-Laboratory Analysis Report for Mysid: SMV Octylphenol Data13Table 1A. Summary of Reproduction Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F01,2,3Concentration Statistics Time to MatureNumberOffspring(Brood 1)NumberOffspring(Brood 2)NumberOffspring(twoBroods)DaystoBrood1DaystoBrood2DaysBetweenBroods1 and 2TotalReproductiveDaysfor the FirstTwo Broods# of OffspringPer FemalePerReproductionDayfor the FirstTwo Broods% ofFemalesProductivelyActive Sex RatioControl(Group 1)Mean13.000 6.048 8.238 14.286 19.000 23.500 4.500 9.500 1.636 95.238 1.773StandardDev.0.000 2.519 3.700 5.021 1.487 1.821 0.688 1.821 0.499 8.248 0.452N3 21 21 21 20 20 20 20 20 3 3CV (%)0 41.66 44.91 35.15 7.83 7.75 15.29 19.17 30.46 8.66 25.50.26 μg/L(Group 2)Mean13.000 5.050 5.889 10.944 17.500 23.778 6.278 9.778 1.126 95.238 2.300StandardDev.0.000 1.669 4.213 4.569 1.147 2.777 2.675 2.777 0.354 8.248 1.006N3 20 18 18 20 18 18 18 18 3 3CV(%)0 33.06 71.55 41.75 6.55 11.68 42.6 28.4 31.46 8.66 43.730.64 μg/L(Group 3)Mean13.000 4.450 6.389 10.944 18.450 23.053 4.579 9.053 1.242 95.238 1.783StandardDev.0.000 1.820 3.806 4.783 2.350 2.345 0.961 2.345 0.347 8.248 0.811N3 20 18 18 20 19 19 19 18 3 3CV(%)0 40.9 59.57 43.7 12.74 10.17 20.99 25.9 27.91 8.66 45.491.6 μg/L(Group 4)Mean13.000 4.524 6.842 11.579 19.238 24.278 5.222 10.278 1.194 100.000 1.457StandardDev.0.000 1.601 3.219 3.322 2.755 2.421 1.060 2.421 0.269 0.000 1.010N3 21 19 19 21 18 18 18 18 3 3CV(%)0 35.38 47.05 28.69 14.32 9.97 20.3 23.55 22.54 0 69.314.0 μg/L(Group 5)Mean13.000 6.000 5.238 11.238 18.143 24.000 6.050 10.000 1.229 100.000 1.613StandardDev.0.000 2.757 2.548 4.098 2.308 3.061 2.328 3.061 0.423 0.000 0.280N3 21 21 21 21 20 20 20 20 3 3CV(%)0 45.95 48.64 36.46 12.72 12.75 38.48 30.61 34.37 0 17.3610 μg/L(Group 6)Mean13.000 4.842 6.111 11.056 19.895 25.500 5.500 11.500 0.975 90.476 1.607StandardDev.0.000 1.463 3.306 4.165 2.747 2.407 1.886 2.407 0.345 8.248 0.521N3 19 18 18 19 18 18 18 18 3 3CV(%)0 30.21 54.09 37.68 13.81 9.44 34.3 20.93 35.36 9.12 32.411.Summary statistics are calculated across all replicatesand all samples in theentire concentration group.2.Time to mature, % of females productively active, sex ratio are measured per tank (replicate), Number of offspring, day to broods, days between broods are measured per mating pair.3.Summaries are based on untransformed datawith no screened outliers removed.DRAFTIntra-Laboratory Analysis Report for Mysid: SMV Octylphenol Data14Table 1B. Summary of Reproduction Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F11,2,3Concentration StatisticsTime toMatureNumberOffspring(Brood 1)NumberOffspring(Brood 2)NumberOffspring(twoBroods)DaystoBrood1DaystoBrood2Days BetweenBroods 1 and 2TotalReproductiveDaysfor the First TwoBroods# of OffspringPer Female PerReproductionDayfor the FirstTwo Broods% of FemalesProductivelyActiveSexRatioControl(Group 1)Mean10.667 6.313 7.154 13.077 16.333 21.182 5.364 9.182 1.754 88.571 1.183StandardDev.0.577 2.869 4.451 6.563 2.717 2.523 1.859 2.523 0.664 10.302 0.652N3 16 13 13 15 11 11 11 11 3 3CV(%)5.41 45.44 62.21 50.19 16.63 11.91 34.65 27.47 37.86 11.63 55.140.26 μg/L(Group 2)Mean12.000 7.842 11.000 19.111 17.789 22.889 4.944 10.889 1.787 100.000 0.927StandardDev.0.000 3.819 4.058 6.902 2.658 2.742 1.110 2.742 0.545 0.000 0.210N3 19 18 18 19 18 18 18 18 3 3CV(%)0 48.7 36.89 36.11 14.94 11.98 22.45 25.18 30.49 0 22.640.64 μg/L(Group 3)Mean12.333 6.056 7.800 13.533 15.778 20.929 5.286 8.929 1.618 100.000 0.640StandardDev.1.528 2.209 3.406 3.962 0.943 1.385 1.139 1.385 0.459 0.000 0.414N3 18 15 15 18 14 14 14 14 3 3CV(%)12.39 36.48 43.67 29.27 5.98 6.62 21.54 15.51 28.35 0 64.671.6 μg/L(Group 4)Mean11.667 5.706 7.500 13.125 16.765 21.333 5.000 9.333 1.669 94.444 0.807StandardDev.0.577 1.795 4.775 4.965 2.562 1.073 1.348 1.073 0.285 9.623 0.453N3 17 16 16 17 12 12 12 12 3 3CV(%)4.95 31.45 63.67 37.83 15.28 5.03 26.97 11.5 17.09 10.19 56.194.0 μg/L(Group 5)Mean13.000 6.450 9.500 15.722 16.750 21.353 4.882 9.353 1.741 100.000 1.003StandardDev.1.732 2.781 3.312 4.860 1.916 1.412 0.600 1.412 0.357 0.000 0.095N3 20 18 18 20 17 17 17 17 3 3CV(%)13.32 43.12 34.87 30.91 11.44 6.61 12.29 15.09 20.48 0 9.4710 μg/L(Group 6)Mean11.000 4.688 6.200 10.933 16.250 20.231 4.385 8.231 1.471 94.444 1.130StandardDev.1.000 1.922 4.109 4.317 2.082 2.279 0.506 2.279 0.568 9.623 0.526N3 16 15 15 16 13 13 13 13 3 3CV(%)9.09 41.01 66.28 39.49 12.81 11.26 11.55 27.68 38.62 10.19 46.521.Summary statistics are calculated across all replicatesand all samples in the entire concentration group.2.Time to mature, % of females productively active, sex ratio aremeasured per tank (replicate), Number of offspring, day to broods, days between broods are measured per mating pair.3.Summaries are based on untransformed datawith no screened outliers removed.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

15 Table 1C. Statistical Analysis Results for Re production Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F0

Parameters Normality Test 1 Homogeneity of Variance Test 2 Monotonicity Test 3 Concentration Trend Tests 4 Overall Concentration Effect 5 Pairwise Comparisons to Control 6,7

Time to Mature 8 Non-Normal NA NonMonotonic NA No NA

Number Offspring (Brood 1) Non-Normal NA NonMonotonic NA Yes Groups 3 & 4

Number Offspring (Brood 2) Normal 9 Homogeneous NonMonotonic NA Yes Group 5

Number Offspring (two Broods) Normal Homogeneous NonMonotonic NA Yes Groups 2 and 3, 5 and 6 Days to Brood 1 Non-Normal NA NonMonotonic NA Yes Group 2 Days to Brood 2 Normal 10 Homogeneous NonMonotonic NA No NA Days Between Broods 1 and 2 Non-Normal NA NonMonotonic NA Yes Groups 2 & 5 Total Reproductive Days for the First Two Broods Non-Normal NA NonMonotonic NA No NA

# of Offspring Per Female Per Reproduction Day

for the First Two Broods Normal Homogeneous Monotonic Decreasing trend for all concentration groups Yes NA

% of Females Productively Active Non-Normal NA NonMonotonic NA No NA

Sex Ratio Normal Homogeneous NonMonotonic NA No NA

1. Shapiro-Wilk test for normality. 2. Levene Test for Homogeneity of Variances. 3. Monotonicity is tested using linear and quadratic contrasts under one-way ANOVA as applied to normalized rank statistics. 4. Concentration Trends are tested using a step -down Jonckheere-Terpstra test procedure. 5. If normality assumption is valid one way ANOVA is applied to the data and F-test is us ed to determine the concentration effect. If normality assumption is invalid, Kruskal-Wallis test is applied to test for the overall concentration effect. 6. Control is group 1 and test concentration groups are group 2 to 6. 7. If overall concentration of ANOVA is significant and monotonicity is rejected, comparisons of test concentration to the control are conducted:

a. using Dunnett’s test with homogeneous within-treatment va riances and using Tamhane-Dunne tt test with heterogeneous within-treatment variances for normal distributed data b. using a modified (nonparametric) Dunn’s test if data are not normally distributed. 8. All responses are constant. 9. Normal and homogeneous when square root-transformed 10. Normal and homogeneous when log-transformed.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

16 Table 1D. Statistical Analysis Results for Re production Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F1

Parameters Normality Test 1 Homogeneity of Variance Test 2 Monotonicity Test 3 Concentration Trend Tests 4 Overall Concentra tion Effect 5 Pairwise Comparisons to Control 6,7

Time to Mature Normal Homogeneous NonMonotonic NA No NA Number Offspring (Brood 1) Normal Homogeneous Monotonic Decreasing trends for the highest dose group Yes NA Number Offspring (Brood 2) NonNormal NA NonMonotonic NA Yes No Number Offspring (two Broods) Normal Homogeneous NonMonotonic NA Yes Group 2

Days to Brood 1 Normal Homogeneous NonMonotonic NA No NA

Days to Brood 2 NonNormal NA NonMonotonic NA No NA Days Between Broods 1 and 2 NonNormal NA NonMonotonic NA No NA Total Reproductive Days for the First Two Broods NonNormal NA NonMonotonic NA No NA # of Offspring Per Female

Per Reproduction Day for the First Two Broods Normal Homogeneous NonMonotonic NA No NA

% of Females Reproductively Active NonNormal NA NonMonotonic NA No NA

Sex Ratio Normal Homogeneous NonMonotonic NA No NA

1. Shapiro-Wilk test for normality. 2. LeveneTest for Homogeneity of Variances. 3. Monotonicity is tested using linear and quadratic contrasts under one-way ANOVA as applied to normalized rank statistics. 4. Concentration Trends are tested using a step -down Jonckheere-Terpstra test procedure. 5. If normality assumption is valid one way ANOVA is applied to the data and F-test is us ed to determine the concentration effect. If normality assumption is invalid, Kruskal-Wallis test is applied to test for the overall concentration effect. 6. Control is group 1 and test concentration groups are group 2 to 6. 7. If overall concentration of ANOVA is significant and monotonicity is rejected, comparisons of test concentration to the control are conducted:

a. using Dunnett’s test with homogeneous within-treatment va riances and using Tamhane-Dunne tt test with heterogeneous within-treatment variances for normal distributed data b. using a modified (nonparametric) Dunn’s test if data are not normally distributed.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

17 Table 1E. Comparisons between Two Generation s for Reproduction Endpoints. Laboratory: SMV, Test Chemical: Octylphenol.

Parameters Normality Test 1 Homogeneity of variance Test 2 Parameteric/ Nonparametric Concentration Main Effect Generation Main Effect Concentration by Generation

Interaction Groups with Significant Difference between Generations 3 Time to Mature 4 NA NA NA NA NA NA NA

Number Offspring (Brood 1) Normal Homogeneous Parametric Yes Yes No Group 2

Number Offspring (Brood 2) Normal Homogeneous Parametric No Yes Yes Groups 2 and 5

Number Offspring (two Broods) Normal Homogeneous Parametric Yes Yes Yes Groups 2 and 5 Days to Brood 1 Normal Homogeneous Parametric No Yes No Groups 1, 3, 4, and 6 Days to Brood 2 Normal Homogeneous Parametric No Yes No Groups 1, 4, 5 and 6 Days Between Broods 1 and 2 Normal Homogeneous Parametric No No No NA

Total Reproductive Days for the

First Two

Broods Normal Homogeneous Parametric No No No NA

# of Offspring Per Female Per Reproduction Day for the First Two Broods Normal Homogeneous Parametric Yes Yes No Groups 2, 4, 5, 6

% of Females Productively Active NonNormal NA Nonparametric No No No NA

Sex Ratio Normal Homogeneous Parametric No Yes No Groups 2 and 3 1. Shapiro-Wilk test for normality. 2. Levene test for homogeneity of variances. 3. Control is group 1 and test concentration groups are group 2 to 6. 4. Model is not fit due to constant value for time to mature in F0 generation.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

18 Table 2A. Summary of Growth Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F0 1,2,3,4

Concentration Statistics Dry Weight

(mg) (day 0) Dry Weight

(mg) (day 7) Dry Weight

(mg) (day 14) Dry Weight (mg) (Termination ) Length (mm) (day 0) Lengt h (mm) (day

7) Length (mm) (day 14) Length (mm) (Termination )

Control (Group 1) Mean 0.034 0.083 0.440 0.994 1.703 3.469 5.321 7.467 Standard

Dev. 0.005 0.036 0.139 0.205 0.165 0.361 0.379 0.306

N 9 9 36 41 45 18 36 41 CV(%) 13.86 42.64 31.54 20.59 9.66 10.4 7.12 4.1

0.26 μg/L (Group 2) Mean 0.144 0.544 1.032 4.062 5.763 7.412 Standard

Dev. 0.042 0.093 0.250 0.276 0.322 0.355

N 9 32 31 18 32 31 CV(%) 28.88 17.16 24.25 6.8 5.59 4.79

0.64 μg/L (Group 3) Mean 0.124 0.456 1.027 3.926 5.699 7.375 Standard

Dev. 0.057 0.128 0.214 0.307 0.241 0.351

N 9 32 41 18 32 41 CV(%) 45.61 27.95 20.8 7.81 4.22 4.75

1.6 μg/L (Group 4) Mean 0.137 0.440 1.011 3.893 5.649 7.240 Standard

Dev. 0.082 0.096 0.211 0.320 0.300 0.318

N 9 38 38 18 38 38 CV(%) 59.72 21.84 20.83 8.23 5.32 4.39

4.0 μg/L (Group 5) Mean 0.159 0.440 1.012 3.809 5.558 7.313 Standard

Dev. 0.077 0.078 0.207 0.429 0.519 0.476

N 9 37 42 18 37 42 CV(%) 48.49 17.81 20.42 11.27 9.33 6.52

10 μg/L (Group 6) Mean 0.146 0.553 0.995 3.927 5.848 7.128 Standard

Dev. 0.047 0.086 0.203 0.323 0.270 0.300

N 9 33 32 18 33 32 CV(%) 31.97 15.49 20.44 8.24 4.62 4.21

1. Summary statistics are calculated across all replicates and all samples in the entire concentration group. 2. On day 0, all mysids are in control environments. 3. Length is measured per mysid. Weight were measured on averag es per (replicate) tank on day 0, per mating pair on day 7, and pe r mysid on day 14 and termination. 4. Summaries are based on untransformed data with no screened outliers removed.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

19 Table 2B. Summary of Growth Endpoints. La boratory: SMV, Test Chemical: Octylphenol, Generation: F1 1,2,3

Concentration Statistics Dry Weight

(mg) (day 0) Dry Weight

(mg) (day 7) Dry Weight

(mg) (day 14) Dry Weight (mg) (Termination) Length (mm) (day 0) Length (mm) (day 7) Length (mm) (day 14) Length (mm) (Termination)

Control (Group 1) Mean 0.023 0.252 0.560 1.066 1.607 4.446 6.057 7.311 Standard

Dev. 0.004 0.036 0.131 0.274 0.173 0.394 0.367 0.384

N 9 9 19 29 45 18 19 29 CV(%) 15.91 14.26 23.34 25.74 10.76 8.86 6.06 5.25

0.26 μg/L (Group 2) Mean 0.020 0.292 0.553 1.039 1.581 4.943 6.554 7.542 Standard

Dev. 0.004 0.045 0.144 0.216 0.121 0.423 0.686 0.452

N 9 9 22 29 45 18 22 29 CV(%) 22.16 15.38 26 20.79 7.66 8.55 10.47 5.99

0.64 μg/L (Group 3) Mean 0.020 0.263 0.597 1.060 1.586 4.466 6.212 7.479 Standard

Dev. 0.004 0.072 0.139 0.220 0.111 0.421 0.353 0.397

N 9 9 19 27 45 18 19 27 CV(%) 22.74 27.32 23.24 20.77 6.99 9.43 5.68 5.31

1.6 μg/L (Group 4) Mean 0.020 0.252 0.579 0.996 1.610 4.739 6.334 7.296 Standard

Dev. 0.003 0.041 0.146 0.251 0.145 0.527 0.396 0.381

N 9 9 16 29 45 18 16 29 CV(%) 16.58 16.08 25.15 25.18 8.99 11.11 6.25 5.23

4.0 μg/L (Group 5) Mean 0.026 0.230 0.562 1.014 1.608 4.863 6.823 7.399 Standard

Dev. 0.008 0.042 0.113 0.260 0.146 0.350 0.400 0.575

N 9 9 21 35 45 18 21 35 CV(%) 30.25 18.19 20.15 25.66 9.08 7.21 5.86 7.77

10 μg/L (Group 6) Mean 0.021 0.276 0.627 0.989 1.582 4.651 6.412 7.099 Standard

Dev. 0.011 0.040 0.144 0.281 0.130 0.328 0.520 0.368

N 9 9 21 31 45 18 21 31 CV(%) 55.59 14.53 23 28.43 8.23 7.05 8.12 5.18 1. Summary statistics are calculated across all replicates and all samples in the entire concentration group. 2. Length is measured per mysid. Weights were measured on averag es per composite on day 0, per ma ting pair on day 7, and per mysi d on day 14 and termination. 3. Summaries are based on untransformed data with no screened outliers removed.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

20 Table 2C. Statistical Analysis Results for Re production Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F0

Parameters Normality Test 1 Homogeneity of variance Test 2 Monotonicity Test 3 Concentration Trend Tests 4 Overall Concentration Effect 5 Pairwise Comparisons to Control 6,7
Dry Weight(mg) (day 7) Non-Normal NA Monotonic Increasing trend for the two highest concentration groups No NA

Dry Weight(mg) (day 14) Normal Heterogeneous Non-M onotonic NA Yes Groups 2 & 6 Dry Weight (mg)

(Termination) Non-Normal NA Non-Monotonic NA No NA Length (mm) (day 7) Normal Homogeneous Non-Monotonic NA Yes Groups 2, 3, 4, 5 & 6 Length (mm)

(day 14) Non-Normal NA Monotonic Increasing trends for all concentration groups Yes NA

Length (mm) (Termination) Non-Normal NA Monotonic Decreasing trends for the three highest concentration groups Yes NA

1. Shapiro-Wilk test for normality. 2. LeveneTest for Homogeneity of Variances. 3. Monotonicity is tested using linear and quadratic contrasts under one-way ANOVA as applied to normalized rank statistics. 4. Concentration Trends are tested using a step -down Jonckheere-Terpstra test procedure. 5. If normality assumption is valid one way ANOVA is applied to the data and F-test is us ed to determine the concentration effect. If normality assumption is invalid, Kruskal-Wallis test is applied to test for the overall concentration effect. 6. Control is group 1 and test concentration groups are group 2 to 6. 7. If overall concentration of ANOVA is significant and monotonicity is rejected, comparisons of test concentration to the control are conducted:

a. using Dunnett’s test with homogeneous within-treatment va riances and using Tamhane-Dunne tt test with heterogeneous within-treatment variances for normal distributed data b. using a modified (nonparametric) Dunn’s test if data are not normally distributed.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

21 Table 2D. Statistical Analysis Results for Re production Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F1

Parameters Normality Test 1 Homogeneity of variance Test 2 Monotonicity Test 3 Concentration Trend Tests 4 Overall Concentration Effect 5 Pairwise Comparisons to Control 6,7 Dry Weight (mg) (day 0) Non-Normal NA Non-Monotonic NA No NA Dry Weight (mg) (day 7) Normal Homogeneous Non-Monotonic NA No NA Dry Weight (mg) (day 14) Non-Normal NA Non-Monotonic NA No NA Dry Weight (mg) (Termination) Non-Normal NA Non-Monotonic NA No NA

Length (mm)

(day 0) Normal Homogeneous Non-Monotonic No No NA Length (mm)

(day 7) Non-Normal NA Non-Monotonic NA Yes Groups 2 & 5

Length (mm)

(day 14) Normal Heterogeneous Monotonic Increasing dose trends for the two highest concentration

groups. Yes NA

Length (mm) (Termination) Non-Normal NA Non-Monotonic NA Yes No

1. Shapiro-Wilk test for normality. 2. LeveneTest for Homogeneity of Variances. 3. Monotonicity is tested using linear and quadratic contrasts under one-way ANOVA as applied to normalized rank statistics. 4. Concentration Trends are tested using a step -down Jonckheere-Terpstra test procedure. 5. If normality assumption is valid one way ANOVA is applied to the data and F-test is us ed to determine the concentration effect. If normality assumption is invalid, Kruskal-Wallis test is applied to test for the overall concentration effect. 6. Control is group 1 and test concentration groups are group 2 to 6. 7. If overall concentration of ANOVA is significant and monotonicity is rejected, comparisons of test concentration to the control are conducted:

a. using Dunnett’s test with homogeneous within-treatment va riances and using Tamhane-Dunne tt test with heterogeneous within-treatment variances for normal distributed data b. using a modified (nonparametric) Dunn’s test if data are not normally distributed.

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# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

22 Table 2E. Comparisons between Two Generation s for Growth Endpoints. Laboratory: SMV, Test Chemical: Octylphenol.

Parameters Normality Test 1 Homogeneity of variance Test 2 Parameteric/ Nonparametric Concentration Main Effect Generation Main Effect Concentration by Generation

Interaction Groups with Significant Difference between Generations 3 Dry Weight(mg) (day 7) Normal Homogeneous Parametric No Yes No Groups 1, 2, 3, 4 and 6 Dry Weight (mg) (day 14) Normal Homogeneous Parametric No Yes No Groups 3, 4 and 5 Dry Weight (mg) (Termination) Normal Homogeneous Parametric No No No NA

Length (mm)

(day 7) Normal Homogeneous Parametric Yes Yes No Groups 1, 2, 3, 4, 5 and 6 Length (mm)

(day 14) Normal Homogeneous Parametric Yes No No Groups 1, 2, 3, 4, 5 and 6 Length (mm) (Termination) Normal Homogeneous Parametric No No No NA

1. Shapiro-Wilk test for normality. 2. Levene test for homogeneity of variances. 3. Control is group 1 and test concentration groups are group 2 to 6.

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

23 Table 3A. Summary of Survival Endpoints. La boratory: SMV, Test Chemical: Octylphenol, Generation: F0 1,2,3

Concentration Statistics Reproduction Survival Rate (%) (day 7) Reproduction Survival Rate (%) (day 14) Reproduction Survival Rate (%) (Termination) Total Survival Rate (%) (day 7) Total Survival Rate (%) (day 14) Total Survival Rate (%)

(Termination)

Control (Group 1) Mean 91.667 86.667 73.000 94.333 86.000 79.000 Standard

Dev. 10.408 2.887 8.660 4.041 6.245 9.000

N 3 3 3 3 3 3 CV(%) 11.35 3.33 11.86 4.28 7.26 11.39

0.26 μg/L (Group 2) Mean 93.333 91.667 64.333 95.000 86.000 70.333 Standard

Dev. 5.774 5.774 12.662 5.000 3.464 5.508

N 3 3 3 3 3 3 CV(%) 6.19 6.3 19.68 5.26 4.03 7.83

0.64 μg/L (Group 3) Mean 93.333 91.667 78.000 95.333 85.000 77.000 Standard

Dev. 7.638 5.774 10.000 4.619 7.937 8.718

N 3 3 3 3 3 3 CV(%) 8.18 6.3 12.82 4.84 9.34 11.32

1.6 μg/L (Group 4) Mean 91.667 91.667 70.333 94.333 91.000 79.333 Standard

Dev. 7.638 7.638 6.429 5.132 6.000 3.512

N 3 3 3 3 3 3 CV(%) 8.33 8.33 9.14 5.44 6.59 4.43

4.0 μg/L (Group 5) Mean 91.667 83.333 75.333 92.667 85.333 81.000 Standard

Dev. 10.408 15.275 13.051 7.506 13.317 12.124

N 3 3 3 3 3 3 CV(%) 11.35 18.33 17.32 8.1 15.61 14.97

10 μg/L (Group 6) Mean 91.667 86.667 65.000 92.000 83.000 71.667 Standard

Dev. 5.774 2.887 9.539 1.732 1.732 8.145

N 3 3 3 3 3 3 CV(%) 6.3 3.33 14.68 1.88 2.09 11.36 1. Summary statistics are calculated across all replicates and all samples in the entire concentration group. 2. Survival rates were measured per tank. 3. Summaries are based on untransformed data with no screened outliers removed.

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

24 Table 3B. Summary of Survival Endpoints. La boratory: SMV, Test Chemical: Octylphenol, Generation: F1 1,2,3

Concentration Statistics Reproduction Survival Rate (%) (day 7) Reproduction Survival Rate (%) (day 14) Reproduction Survival Rate (%) (Termination) Total Survival Rate (%) (day 7) Total Survival Rate (%) (day 14) Total Survival Rate (%)

(Termination)

Control (Group 1) Mean 95.333 89.333 77.000 92.000 86.000 78.333

Standard

Dev. 4.041 10.066 16.093 1.732 5.196 6.658

N 3 3 3 3 3 3
CV(%) 4.24 11.27 20.9 1.88 6.04 8.5

0.26 μg/L (Group 2) Mean 95.333 95.333 90.000 94.667 90.333 84.000

Standard

Dev. 4.041 4.041 8.660 6.807 6.658 8.185

N 3 3 3 3 3 3
CV(%) 4.24 4.24 9.62 7.19 7.37 9.74

0.64 μg/L (Group 3) Mean 84.333 83.000 75.000 87.667 81.333 76.333

Standard

Dev. 7.506 3.606 7.211 5.033 2.887 7.234

N 3 3 3 3 3 3
CV(%) 8.9 4.34 9.61 5.74 3.55 9.48

1.6 μg/L (Group 4) Mean 88.667 81.667 68.333 87.667 77.667 69.333

Standard

Dev. 7.506 5.508 12.220 6.807 9.238 7.767

N 3 3 3 3 3 3
CV(%) 8.46 6.74 17.88 7.76 11.89 11.2

4.0 μg/L (Group 5) Mean 95.333 93.000 88.000 95.333 90.000 86.333

Standard

Dev. 4.041 0.000 3.606 4.041 1.732 0.577

N 3 3 3 3 3 3
CV(%) 4.24 0 4.1 4.24 1.92 0.67

10 μg/L (Group 6) Mean 100.000 97.667 93.000 94.333 90.333 86.667

Standard

Dev. 0.000 4.041 6.083 5.132 8.737 8.505

N 3 3 3 3 3 3
CV(%) 0 4.14 6.54 5.44 9.67 9.81
1. Summary statistics are calculated across all replicates and all samples in the entire concentration group. 2. Survival rates were measured per tank. 3. Summaries are based on untransformed data with no screened outliers removed.

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

25 Table 3C. Statistical Analysis Results for Re production Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F0

Parameters Normality Test 1 Homogeneity of variance Test 2 Monotonicity Test 3 Concentration Trend Tests 4 Overall Concentration Effect 5 Pairwise Comparisons to Control 6,7 Reproduction Survival Rate (%) (day 7) Normal Homogeneous Non-Monotonic NA No NA

Reproduction Survival Rate (%) (day 14) Normal Homogeneous Non-Monotonic NA No NA

Reproduction Survival Rate (%) (Termination) Normal Homogeneous Non-Monotonic NA No NA

Total Survival Rate (%) (day 7) Normal Homogeneous Non-Monotonic NA No NA

Total Survival Rate (%) (day 14) Normal Homogeneous Non-Monotonic NA No NA

Total Survival Rate (%) (Termination) Normal Homogeneous Non-Monotonic NA No NA

1. Shapiro-Wilk test for normality. 2. LeveneTest for Homogeneity of Variances. 3. Monotonicity is tested using linear and quadratic contrasts under one-way ANOVA as applied to normalized rank statistics. 4. Concentration Trends are tested using a step -down Jonckheere-Terpstra test procedure. 5. If normality assumption is valid one way ANOVA is applied to the data and F-test is us ed to determine the concentration effect. If normality assumption is invalid, Kruskal-Wallis test is applied to test for the overall concentration effect. 6. Control is group 1 and test concentration groups are group 2 to 6. 7. If overall concentration of ANOVA is significant and monotonicity is rejected, comparisons of test concentration to the control are conducted:

a. using Dunnett’s test with homogeneous within-treatment va riances and using Tamhane-Dunne tt test with heterogeneous within-treatment variances for normal distributed data b. using a modified (nonparametric) Dunn’s test if data are not normally distributed.

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

26 Table 3D. Statistical Analysis Results for Re production Endpoints. Laboratory: SMV, Test Chemical: Octylphenol, Generation: F1

Parameters Normality Test 1 Homogeneity of variance Test 2 Monotonicity Test 3 Concentration Trend Tests 4 Overall Concentration Effect 5 Significant Pairwise Comparisons to Control 6,7 Reproduction Survival Rate (%) (day 7) Normal Homogeneous Non-Monotonic NA Yes No

Reproduction Survival Rate (%) (day 14) Normal Homogeneous Non-Monotonic NA Yes No

Reproduction Survival Rate (%) (Termination) Normal Homogeneous Monotonic NA No NA

Total Survival Rate (%) (day 7) Normal Homogeneous Non-Monotonic NA No NA

Total Survival Rate (%) (day 14) Normal Homogeneous Non-Monotonic NA No NA

Total Survival Rate (%) (Termination) Normal Homogeneous Non-Monotonic NA NO NA

1. Shapiro-Wilk test for normality. 2. LeveneTest for Homogeneity of Variances. 3. Monotonicity is tested using linear and quadratic contrasts under one-way ANOVA as applied to normalized rank statistics. 4. Concentration Trends are tested using a step -down Jonckheere-Terpstra test procedure. 5. If normality assumption is valid one way ANOVA is applied to the data and F-test is us ed to determine the concentration effect. If normality assumption is invalid, Kruskal-Wallis test is applied to test for the overall concentration effect. 6. Control is group 1 and test concentration groups are group 2 to 6. 7. If overall concentration of ANOVA is significant and monotonicity is rejected, comparisons of test concentration to the control are conducted:

a. using Dunnett’s test with homogeneous within-treatment va riances and using Tamhane-Dunne tt test with heterogeneous within-treatment variances for normal distributed data b. using a modified (nonparametric) Dunn’s test if data are not normally distributed.

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

27 Table 3E. Comparisons between Two Generation Survival Endpoints. Laboratory: SMV, Test Chemical: Octylphenol.

Parameters Normality Test 1 Homogeneity of variance Test 2 Parameteric/ Nonparametric Concentration Main Effect Generation Main Effect Concentration by Generation

Interaction Groups with Significant Difference between Generations 3 Reproduction Survival Rate (%) (day 7) Normal Homogeneous Parametric No No No NA

Reproduction Survival Rate (%) (day 14) Normal Homogeneous Parametric No No No NA

Reproduction Survival Rate (%) (Termination) Normal Homogeneous Parametric No Yes Yes Groups 2 and 6

Total Survival Rate (%) (day 7) Normal Homogeneous Parametric No No No NA

Total Survival Rate (%) (day 14) Normal Homogeneous Parametric No No No NA

Total Survival Rate (%) (Termination) Normal Homogeneous Parametric No No No NA

1. Shapiro-Wilk test for normality. 2. Levene test for homogeneity of variances. 3. Control is group 1 and test concentration groups are group 2 to 6.

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

28 Figure 1. Means ± Standard Errors of Untransformed data: Time to Mature By Generation and Group For Chemical Octylphenol From Lab SMV.

Figure 2. Means ± Standard Errors of Untransformed da ta: Number Offspring (brood 1) By Generation and Group For Chemic al Octylphenol From Lab SMV.Time To Mature (Days)

10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Number Offspring (brood 1)

3 4 5 6 7 8 9

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

29 Figure 3. Means ± Standard Errors of Untransformed da ta: Number Offspring (brood 2) By Generation and Group For Chemic al Octylphenol From Lab SMV.

Figure 4. Means ± Standard Errors of Untran sformed data: Number Offspring (two broods) by Generation and Group For Chem ical Octylphenol From Lab SMV.Number Offspring (brood 2)

4 5 6 7 8 9 10 11 12

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Number Offspring (two broods)

8 10 12 14 16 18 20 22

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

30 Figure 5. Means ± Standard Errors of Untransformed data: Days to Brood 1 By Generation and Group For Chemical Octylphenol From Lab SMV.

Figure 6. Means ± Standard Errors of Untransformed data: Days to Brood 2 By Generation and Group For Chemical Octylphenol From Lab SMV.Days to Brood 1

15 16 17 18 19 20 21 22

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Days to Brood 2

19 20 21 22 23 24 25 26 27

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

31 Figure 7. Means ± Standard Errors of Untransformed data: Days Between Broods 1 and 2 By Generation and Group For Chemic al Octylphenol From Lab SMV.

Figure 8. Means ± Standard Errors of Untransformed data: Total Productive Days By Generation and Group For Chemic al Octylphenol From Lab SMV.Days Between Broods 1 and 2

4 5 6 7

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Total Reproductive Days

7 8 9 10 11 12 13

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

32 Figure 9. Means ± Standard Errors of Untransforme d data: Offspring per Female per Productive Day By Generation and Gro up For Chemical Octylphenol From Lab SMV.

Figure 10. Means ± Standard Errors of Untransformed data: Percent of Females Productively Active By Generation and Group For Chemical Octylphenol From Lab SMV.Offspring per Female per Reproductive Day

0.5 1.0 1.5 2.0

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1% of Females Productively Active

80 85 90 95 100

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

33 Figure 11. Means ± Standard Errors of Untransformed data: Sex Ratio By Generation and Group For Chemical Octylphenol From Lab SMV.

Figure 12. Dry Weight (mg) (day 0) By Gen eration and Group For Chemical Octylphenol From Lab SMV.Sex Ratio

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Dry Weight (mg)(day 0)

0.014 0.016 0.018 0.020 0.022 0.024 0.026 0.028 0.030 0.032 0.034 0.036 0.038

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

34 Figure 13. Means ± Standard Errors of Untransformed data: Dry Weight (mg) (day 7) By Generation and Group For Chemic al Octylphenol From Lab SMV.

Figure 14. Means ± Standard Errors of Untransformed data: Dry Weight (mg) (day 14) By Generation and Group For Chemic al Octylphenol From Lab SMV.Dry Weight (mg)(day 7)

0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28 0.30 0.32

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Dry Weight (mg)(day 14)

0.40 0.45 0.50 0.55 0.60 0.65 0.70

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

35 Figure 15. Means ± Standard Errors of Untransformed data: Dry Weight (mg) (Termination) By Generation and Group For Chemical Octylphenol From Lab SMV.

Figure 16. Means ± Standard Errors of Untransforme d data: Length (mm) (day 0) By Generation and Group For Chemic al Octylphenol From Lab SMV.Dry Weight (mg)(Termination)

0.90 0.95 1.00 1.05 1.10 1.15

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Length (mm)(day 0)

1.54 1.56 1.58 1.60 1.62 1.64 1.66 1.68 1.70 1.72 1.74

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

36 Figure 17. Means ± Standard Errors of Untransforme d data: Length (mm) (day 7) By Generation and Group For Chemic al Octylphenol From Lab SMV.

Figure 18. Means ± Standard Errors of Untransformed data: Length (mm) (day 14) By Generation and Group For Chemic al Octylphenol From Lab SMV.Length (mm)(day 7)

3.0 3.5 4.0 4.5 5.0 5.5

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Length (mm)(day 14)

5.0 5.5 6.0 6.5 7.0

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

37 Figure 19. Means ± Standard Errors of Untransformed data: Length (mm) (Termination) By Generation and Group For Chemic al Octylphenol From Lab SMV.

Figure 20. Means ± Standard Errors of Untransformed data: Reproduction Survival Rate (%) (day 7) By Generation and Group For Chemical Octylphenol From Lab SMV.Length (mm)(Termination)

7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Reproduction Survival Rate (%)(day 7)

80 82 84 86 88 90 92 94 96 98 100

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

38 Figure 21. Means ± Standard Errors of Untransformed data: Reproduction Survival Rate (%) (day 14) By Generation and Group For Chemical Octylphenol From Lab SMV.

Figure 22. Means ± Standard Errors of Untransformed data: Reproduction Survival Rate (%) (Termination) By Generation and Gro up For Chemical Octylphenol From Lab SMV.Reproduction Survival Rate (%)(day 14)

70 75 80 85 90 95 100

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Reproduction Survival Rate (%)(Termination)

55 60 65 70 75 80 85 90 95 100

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

39 Figure 23. Means ± Standard Errors of Untransformed data: Total Survival Rate (%) (day 7) By Generation and Group For Chemical Octylphenol From Lab SMV.

Figure 24. Means ± Standard Errors of Untransformed data: Total Survival Rate (%) (day 14) By Generation and Group For Chemical Octylphenol From Lab SMV.Total Survival Rate (%)(day 7)

80 82 84 86 88 90 92 94 96 98 100

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1Total Survival Rate (%)(day 14)

70 75 80 85 90 95 100

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1

## DRAFT


# Intra-Laboratory Analysis Report fo r Mysid: SMV Octylphenol Data

40 Figure 25. Means ± Standard Errors of Untransformed data: Total Survival Rate (%) (Termination) By Generation and Gro up For Chemical Octylphenol From Lab SMV.Total Survival Rate (%)(Termination)

60 65 70 75 80 85 90 95 100

Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L Control 0.26 ug/L 0.64 ug/L 1.6 ug/L 4 ug/L 10 ug/L



## Group Generation F0 Generation F1