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## 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 WLI Octylphenol in Mysids

U.S. Environmental Protection Agency

May 17, 2013

BATTELLE

505 King Avenue

Columbus, Ohio 43201

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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 ................................................................................................... ......... 6

3. Statistical Methods ........................................................................................................ ............ 7 3.1 Comparisons of test chemical with the control ......................................................................... 7

3.1.1 Outlier Screens ......................................................................................................... ............. 7 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

4. Results .................................................................................................................... ................. 11

Tables

Table 0. Data information for SMV Octylphe nol (Only control data were available for F1 generation)................................................................................................................. ........... 4
Table 1A. Summary of Reproduction Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F0 1,2 

### ................................................................................................ 12
Table 1B. Summary of Reproduction Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F1 1,2,3 

### .............................................................................................. 13
Table 1C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F0 .................................................................................. 14
Table 2A. Summary of Growth Endpoints. Labor atory: WLI, Test Chemical: Octylphenol, Generation: F0 1,2,3 

### .................................................................................................................... 15
Table 2B. Summary of Growth Endpoints. Labor atory: WLI, Test Chemical: Octylphenol, Generation: F1 1,2,3 

### .................................................................................................................... 16
Table 2C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F0 .................................................................................. 17

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iv Table 3A. Summary of Survival Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F0 1,2 

### ...................................................................................................................... 18
Table 3B. Summary of Survival Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F1 1,2 

### ...................................................................................................................... 19
Table 3C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F0 .................................................................................. 20

Figures

Figure 1. Time to Mature for Generation F0 by Group for WLI Lab, Chemical Octylphenol. .............. 21
Figure 2. Number Offspring (Brood 1) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. .................................................................................................................. .......... 21
Figure 3. Number Offspring (Brood 2) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. .................................................................................................................. .......... 22
Figure 4. Number Offspring (Two Broods) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. .................................................................................................................. .......... 22
Figure 5. Days to Brood 1 for Generation F0 by Group for WLI Lab, Chemical Octylphenol. ............. 23
Figure 6. Days to Brood 2 for Generation F0 by Group for WLI Lab, Chemical Octylphenol. ............. 23
Figure 7. Days Between Broods 1 and 2 for Ge neration F0 by Group for WLI Lab, Chemical Octylphenol. .................................................................................................................. .......... 24
Figure 8. Percent of Females Productively Active for Generation F0 by Group for WLI Lab, Chemical Octylphenol. .................................................................................................................. .......... 24
Figure 9. Sex Ratio for Generation F0 by Gro up for WLI Lab, Chemical Octylphenol. ....................... 25
Figure 10. Dry Weight (mg) (day 0) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. .................................................................................................................. .......... 25
Figure 11. Dry Weight (mg) (day 7) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. .................................................................................................................. .......... 26
Figure 12. Dry Weight (mg) (day 14) for Generation F0 by Group for WLI Lab, Chemical Octylphenol ................................................................................................................... ......... .26
Figure 13. Dry Weight (mg) (Termination) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. .................................................................................................................. .......... 27
Figure 14. Length (mm) (day 0) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. ..... 27
Figure 15. Length (mm) (day 7) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. ..... 28
Figure 16. Length (mm) (day 14) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. ... 28
Figure 17. Length (mm) (Termination) for Gene ration F0 by Group for WLI Lab, Chemical Octylphenol. .................................................................................................................. .......... 29

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v Figure 18. Reproduction Juvenile Survival Rate - Da y 6 (%) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. ......................................................................................................... ... 29
Figure 19. Reproduction Juvenile Survival Rate - Day 14 (%) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. ......................................................................................................... ... 30
Figure 20. Total Juvenile Survival Rate - Day 6 (%) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. .................................................................................................................. .......... 30
Figure 21. Total Juvenile Survival Rate - Day 14 (%) for Generation F0 by Group for WLI Lab, Chemical Octylphenol. ......................................................................................................... ... 31
Figure 22. Reproduction Adult Survival Rate at Term ination (%) for Generation F0 by Group for WLI Lab, Chemical Octylphenol ..................................................................................................... 31

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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. Two laboratories, SMV and WLI, conducte d two-generation mysid survival and growth tests following a similar protocol with the test chemical octylphenol. Statistical an alyses were carried out on the SMV and WLI octylphenol study data. The draft analysis documented in this report is based on the WLI octylphenol test data. Statistical analysis of th e octylphenol effects in the WLI study is being carried out with the F0 generation data. The reproductive ou tput from the F0 generation mysids treated with octylphenol in this study was not sufficient to pro duce an F1 generation. Only the negative control replicates were carried to the F1 generation and ar e included in the F1 portion of the summarization and analysis.

The two-generation test is initiated with newly -released (<24 hours old) offspring (F0 generation) that are grown to sexual 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 sexual maturity and their offspring are collected (F1 offs pring). The F0 test is conducted with five graded concentrations octylphenol and an untreated control w ith 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. In this st udy only the control mysids were carried to the F1 generation

The F1 generation (control mysids only) is be gun with newly hatched F0 control offspring. Within each replicate tank the growth and reproduc tion baskets each initially include 15 newly hatched mysids, or the maximum available. With the a ppearance of a marsupial pouch on females (sexual maturity), mature females are isolated as pairs w ith males in separate brood cups. Seven breeding pairs are selected in each replicate reproduction basket. The exposure of these F1 mysids is continued until two weeks after the median time to the second brood re lease in the control group. Offspring are collected from the F1 generation each morning from the control group brood cups and transferred immediately to baskets with untreated negative control water.

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

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2 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 baskets. At termination (around Day 46 for WLI octylphenol), mysids from the reproduction basket s are also measured for weights and lengths.

For the intra-laboratory analysis of the WLI octylphenol mysid study comparisons were made between the F0 test chemical concentration groups and the concurrent F0 control group to assess the mysid responses to graded concentrations of octy lphenol. The F1 generation control data were summarized but were not statistically analyzed.

2. STUDY DATA

2.1 Data Endpoints



### The test method specifies 25 endpoints. Some of the endpoints are specified per mysid or composite of mysids (e.g. day 0 weight, day 7 wei ght), some are specified per breeding pair, and some are specified per replicate tank per basket (i .e. per replicate tank). Not all of the specified responses were determined in the WLI octylphe nol study (e.g. number of reproductive days per female.

These endpoints were divide d among three categories:

(i) Reproduction (F0) (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 th e first brood (per breeding pair)
g. Number of young per female in th e second brood (per breeding pair) h. Total number of young in the first two br oods per female (p er breeding pair) i. Percentage of females that are reproductiv ely active in the firs t two broods (per basket) j. Total reproduction days in the firs t two broods (per breeding pair) k. Number of young per female per reproduc tive day in the first two brood (per breeding pair)

(ii) Survival (F0) (6)

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

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### The survival rates were adjusted for the number of F0 mysids that were removed for weight and length determinations (i.e. were omitted from both the numerator and denominator) and for other non-treatm ent related deaths

(iii) Growth (F0) (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)

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 gene ration mysids were kept in untreated water. Growth measurements were determined for mysids on day 0 before they were assigned to growth and reproduction baskets, for mysids from the grow th baskets at day 7 (6 per replicate tank) and day 14 (all surviving growth basket mysids on day 14), and for the surviving reproduction mysids at termination. For the weight data on day 0, there were a total of 9 composites (5 mysids per composite). For day 7 there were 3 com posite weights per replicate (2 mysids per composite) and three replicates per concentration group.

Data were a available for F1 generation fo r the above parameters for the control group only.

The test laboratory ran a conc urrent control and five targeted test concentration levels for the F0 generation. The actual exposure concentrati on levels in general differed from the targeted levels. The statistical analysis procedures do not utilize the actual conc entration levels. The concentrations are labeled by thei r targeted levels but the labels are not actually used in the analyses.

2.2 Data Issues and Data Handling



### Data were received in Excel ta ble format not directly suitabl e for analysis. They were reformatted into STACHARRMS friendly forma tted spreadsheets. SAS and STATA programs were used to read in the raw data, define progr am friendly variable names, and add new character variables to the data (laboratory name, chemical name, generation). The reformatted data sheets were saved into three files according to th e endpoint category: reproduction, growth, and survival.

1 Day 14 and termination weights and lengths were reported by gender.

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4 Table 0. Data information for SMV Octylphenol (Only control data we re available for F1 generation)

Data Files and data sheets Endpoints Comments Original-

 Mys Repro Data.xls

− Repro Spreadsheet

 Mys Repro Data F1.xls

− Repro Spreadsheet

 Mys Repro Time.xls these information are available in the Mys Repro Data.xls, Thus this data does not need to be read in

 Sex ration Sas.xls there are two data sheets SASdata (2) and SASdata. Th ey seems not the same. For now we will use SASdata (2)

 Growth & Survival F1.xls (last datasheet for F1 sex ratio);

Reformatted-:

 WLI Octylphenol Reproduction F0.xlsx

 WLI Octylphenol Reproduction F0.xlsx

 WLI Octylphenol Sex Ratio F0.xlsx

 WLI Octylphenol Sex Ratio F1.xlsx  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

 Sex ratio The following two endpoints are not available:

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

 Total reproductively active days for the first two broods  If there are no offspring for a brood. The data are recorded as zero for the brood sizes. Per EPA suggestion, such data are changed as: zero if female is still living, and “NA” (recorded as missing) for a brood (1 st or 2 nd brood) if female died without releasing the brood.

 The total offspring for the first 2 broods are therefore recalculated and displayed in the reformatted data.

 Time to maturity is same for all animals as “14”.

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

Original-

 Growth & Survival.xls

 Growth & Survival F1.xls The following extra data files provided the same information included in the above two files:

 Day 0 Weight and lengths Data.xls (it look like that this is F0 data);

 Day 0 Lengths Raw Data.xls (it is control data, it looks like F1 data. It actually include both lengths and weights) ;

 Day 7 Weights and lengths Raw Data.xls (F0 data);

 Day 7 Growth Measurements Raw Data F1.xls;

 Day 14 Weights and lengths Raw Data.xls (F0 data);

 Day 14 Growth Measurements Raw Data.xls (F1 data);

 Term Weights and lengths.xls (termination for F0)

 Mysid-Term Growth Raw Data NC Rep. A.xls (termination for F1: Weight (dry weight) and length for days 0, 7, 14 and termination  For F0 generation all mysids on day 0 are kept in untreated water. There were 9 composites (5 mysids per composite).

 Length are reported per mysid.

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

 Genders of mysids are provided for day 14 and termination.

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5 Data Files and data sheets Endpoints Comments control replicate A, the next two files for replicates B and C);

 Mysid-Term Growth Raw Data_NCB.xls;

 Mysid-Term Growth Raw Data_NCC.xls Reformatted-

 WLI Octylphenol Growth F0_Created From SAS.xlsx

 WLI Octylphenol Growth F1_Export From SAS.xlsx

Original-

 Growth & Survival.xls

− Juvenile Survival-Day 6

− Juvenile Survival-Day 7-Pair

− Adult Survival

 Growth & Survival F1.xls

Reformatted- SMV Octylphenol Survival.xls:

- F0ReproductionSurvival;

- F0GrowthSurvival

- F0TotalSurvival

- F1ReproductionSurvival

- F1GrowthSurvival

- F1TotalSurvival  For F0, Survival (reproduction) (day 6 before pair, survival on day 14)

 For F0, Survival (total) (day 6 before pair, survival on day 14),reproductive adult survival at termination.

 For F1, Survival (reproduction) (day 7, day 14, and at termination) – controls only

 For F1, Survival (total) (day 7, day 14) – controls only  For F0 generation:

− Data sheet “Juvenile SurvivalDay 6”. We labeled them as Juvenile Survival (Day 6). Both reproductive survival and total survival are given in this data sheet.

− Sheet “Juvenile Survival-Day 7-Pair” has title l “Juvenile Survival Day 7 - Pairing on Day 14”. We labeled them as “Juvenile Survival (Day 7)”. It is actually survival on day 14. Both reproductive survival and total survival are given in this worksheet.

 Worksheet “Adult Survival” has title “total adult survival after pairing”. It is in fact reproduction survival at termination. There is a column called “Total Adult Survival After Pairing Excluding Impinged Mysids”, which are not in the other two survival sheets. It is in fact reproduction termination survival excluding nontoxic related mortalities..

 For the F1 generation:

− Worksheet “Juvenile SurvivalDay 6 (2)” is for Survival on Day 7.

− Juvenile Survival-Day14 (2) is for day7 to 14.

− Both reproductive survival and total survival are given on these two data sheets.

− Sheet “Adult Survival” has title “total adult survival after

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6 Data Files and data sheets Endpoints Comments pairing”. It is in fact reproductive survival for day 14 to termination. There is a column called “Total Adult Survival After Pairing Excluding Impinged Mysids”. It is in fact reproduction termination survival excluding nontoxic related mortalities.



### We have changed the denominators in the survival rate calcul ations for day 14 and termination to reflect total surv ival from day 0, adjusting for the nontoxic related mortality. The survival rates presented in this report differ fr om those included in the WLI spreadsheet because they represent survival from day 0.

2.3 Analysis Considerations



### Several analysis considerations need to be noted. These are discussed below.

 Since only F0 generation data are availabl e for the intra-laboratory analysis, the STATCHARRMS one-way ANOVA procedur e is used. In the one-way ANOVA STATCHARRMS pools the results acr oss replicate tanks within test or control groups. The one-way ANOVA (parametric or nonparametric form) was applied to the one generation data and there are no random replicat e effects. The within generation analysis compares the test group results to the control group results.

 The preliminary tests for outliers, normality, heterogeneity of vari ance, and monotonicity were carried out independently for the an alyses within the F0 generation only.

 Reproductive response was observed for 46 days , which is much longer than the other laboratory studies (around 30 da ys). It might have increas ed the opportunity to observe the second broods for some mysids. Terminati on survival is also at 46 days. This may impact the survival rates.

 Survival is calculated by WLI differently than the other laboratories. Survival rates are presented in the WLI spreadsheet at day 6, between days 7 and 14, and between days 14 and termination. The denominators of the survival rates are th e numbers of mysids surviving to the beginnings of the survival periods. This ha s been modified in this report to represent survival from day 0.

 For the reproductive response when “0” is reporte d 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 brood size should be counted as 0. If the female died before the recorded “0” brood size is generated, the brood size should be counted as missing. Battelle has changed the data entries in the columns for brood1 size and brood2 size in the Excel spreadsheet accordi ng to this suggestion. In the reformatted

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### data, the changes are also made to the affected endpoints: to tal number of offspring in the first two broods, and the numb er of offspring per female per reproductive day during the two broods. If the data entries for a given brood size is zero or missing, the times for the brood was also recorded as “-“ in the original data file and they are treated as missing in this analysis. If the brood size is missing in either brood 1 or brood 2, then the sum of the two broods is also treated as missing.

 In the WLI octylphenol spreadsheets the 14 day and termination length and weight responses are presented by gender. In the preliminary draft analyses the 14 day and termination weights and lengths are pool ed across male and female mysids.

 There is no information about number of re productive days per female or number of offspring per reproductive day per female. These endpoints are not included in the analysis in this report.

3. STATISTICAL METHODS


### Statistical analysis procedures used conf orm to the analysis procedures in the STATCHARRMS package. Statistical analyses co mpare the test chemical responses with the control responses within the F0 generation. There is no analys is of the F1 generation. The statistical analyses were carried out in a univariate fashion, in dependently for each endpoint. There is no comparison of the F0 and F1 generations.

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 procedure were applied to pe r mysid or per mysid-pair data without first averaging across the mysids in the same the replicate baskets.

3.1 Comparisons of test chemical with the control



### Analyses were carried out on a per mysid, per breeding pair, or per basket basis depending on how the data were re ported. Preliminary tests were first carried out to detect outliers, departure from normality, heterogeneity of variance, and departure from monotonicity of response. Subsequent comparisons among grou ps were carried out including all the values (including the screened outlier values) usi ng procedures depending on the outcomes of the preliminary analyses. All the test procedures de scribed here are based on the default auto option programs in the STATCHARRMS

3.1.1 Outlier Screens



### Outlier screens were carried out using Tukey’s outlier rule on the untransformed data. Outliers were assessed from residuals (results of subtracting treatment group means from individual values) to avoid confounding outliers a nd treatment effects. Any response more than

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### 1.5 times the interquartile rang e 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 results of the statistical outlier screens were to be documented and reported to EPA for review to determine if the deviations, although statistically significant, are in fact biologica lly important. EPA w ill determine whether the flagged values should be included in the subsequent analyses or s hould be treated as outliers. The subsets of potential outliers are response speci fic. Due to time constraints, the statistical 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 procedure 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). Scr eened 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 tr ansformation. Under the auto option in STATCHARRMS, the analysis procedure is firs t applied to the untransformed data. If the normality assumption is valid, then no transforma tion is carried out. If the normality assumption is invalid, then normality tests are again performe d on the log and square root transformed data. If the normality assumption is va lid 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 pro cedures are applied. Transformation is not necessary for nonparametr ic procedures as any transformation does not change the rank scores for the data wh en nonparametric procedures are used.

3.1.4 Heterogeneous variances



### If the normality assumption was accepted for ei ther the untransformed or the transformed data heterogeneity of variances among the c oncentration groups was assessed in the ANOVA procedure on the untransformed or the transfor med data with Levene’s test with absolute residuals (Levene, 1960). Screened outliers were included in the Levene’s tests. If the normality assumption was invalid for both the untransformed and the transformed da ta, heterogeneity of variances among the concentration groups was not assessed as nonparametric procedures were applied.

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9 3.1.5 Monotonicity of response



### In STATCHARRMS, a monotonicity test usi ng linear and quadratic contrasts under oneway ANOVA was applied to normalized rank statisti cs (to avoid the complications that can arise from non-normal or heterogeneous data). If the linear contrast was signi ficant but the quadratic contrasts 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 monotonici ty either ordered alternatives or general alternatives analysis procedures were used. Ordered alternatives procedures assume that the response is monotonically increasin g or monotonically decreasing with increasing test chemical concentration. Ordered alternativ es procedures were always carri ed out on the ranks of the data. If there was significant departur e from monotonicity, general a lternatives procedures were carried out.

If the test for departure from normality was not significant for either the untransformed or the transformed data normal theory parametric methods with homogeneous or heterogeneous variances were used. If the test for depart ure from normality was significant for both the untransformed and the transformed data nonpara metric 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 was accepted, ordered alternatives procedures were used. Step down multiple comp arison procedures are particularly useful for ordered alternatives situations. The test is carried out as long as there are significant effects, starting from all concentration groups (includi ng the control), successively omitting the highest concentration group. If at any stage the compar ison is nonsignificant, the process stops and all means in that concentration group and lower ar e 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-Terp stra (Jonckheere (1954)) test of trend was used on the rank data.

Additionally, global overall con centration effects were tested. These were based on the parametric analysis of variance F-test if the normality assumption held or the Kruskal-Wallis test if the normal assumption did not hold for both th e untransformed and the transformed data. See the general alternatives section for more details.

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### General alternatives – parametric procedure

If the hypothesis of monotonicity of res ponse was rejected and the normality assumption was not rejected, parametric general alternat ive procedures were used. If the overall concentration effect with the ANOVA was significant, comparisons of test concentrations to the control were conducted using Dunnett’s test with homogeneous within-tre atment variances or using the Tamhane-Dunnett test with hetero geneous within-treatme nt variances.

General alternatives – nonparametric procedure

If the hypothesis of monotonicity of res ponse was rejected and the normality assumption was rejected for both the untransformed and the transformed data, nonparametric general alternative procedures were used. The Kruskal-Wallis test was applied to test for the overall concentration effect. If the ove rall concentration effect was signi ficant, the comparisons of test concentration responses to the control responses were conducted using a modified Dunn’s test.

Exact permutation tests

For nonparametric procedures, if a large pe rcentage of the responses have the same measured value (ties), specifically in the case of very small samples, say less than three per concentration for most concentrations, it is may no t be possible to assess in a meaningful way the true distribution of responses acr oss test concentration groups. One can either use an assumed distribution or treat this situation in the same wa y as data with massive ties. Exact permutation test methodology is an appropria te tool to use in these ca ses. The ANOVA component of STATCHARRMS automatically uses exact methods when deemed appropriate. In the ANOVA procedure, there are two conditi ons under which exact permutati on methods are used: (1) There are no more than three experimental units in any treatment group and non-parametric procedures are found appropriate. (2) There are “massive ties” in the responses, i.e., 25% or more of the experimental units have the same response or 40 % or more have one of only two response values or 65% or more have one of only three respons e values. An exact version of the JonckheereTerpstra test is used for the step-down trend te st 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



### Comparisons of the F0 and F1 generation were not carried out as only the F0 generation was available for the intra-laboratory analysis.

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

11 4. RESULTS


### For each endpoint the summary tables presen t 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 across all samples and all baskets for each concentration group. Summary results for the F0 generation are presented in Table 1A (reproduction endpoints), Table 2A (growth endp oints), and Table 3A (survival endpoints). These means and standard deviations are also displayed in Figures 1 through 22. Summary results for the control group in the F1 generation are presen ted in Table 1B (reproduction endpoints), Table 2B (growth e ndpoints), and Table 3B (surviva l endpoints). The summaries and figures are based on untransformed da ta with no screened outliers removed.

Statistical analysis was performed according to the statistical met hods described in the methods section using STATCHARRMS. The re sults of statistical analyses for the F0 generation are summarized in Table 1C (repr oduction), Table 2C (growth), and Table 3C (survival).DRAFTIntra-Laboratory Analysis Report for Mysid: WLI Octylphenol Data12Table 1A. Summary of Reproduction Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F01,2Dose Concentration StatisticsTime to Mature(Females)NumberOffspring(Brood 1)NumberOffspring(Brood 2)NumberOffspring(Two Broods)Days toBrood 1Days toBrood 2Days BetweenBroods 1 and 2% of FemalesProductivelyActiveSex Ratio(males/females)Control(Group 1)Mean14.000 3.471 5.800 9.067 26.250 34.077 9.000 76.167 1.064Standard Dev.0.0002.375 4.554 5.0075.871 6.251 4.243 8.256 0.403N21 17 15 15 16 13 13 3 3CV (%)068.43 78.52 55.2222.37 18.34 47.14 10.84 37.850.26 μg/L (Group 2)Mean14.000 3.150 2.167 5.278 26.778 33.222 9.000 85.700 0.878Standard Dev.0.0002.498 2.834 4.0996.656 5.167 4.062 14.300 0.257N21 20 18 18 18 9 9 3 3CV(%)079.3 130.78 77.6624.86 15.55 45.13 16.69 29.280.64 μg/L (Group 3)Mean14.000 3.211 1.722 4.944 28.938 35.100 7.900 76.167 1.245Standard Dev.0.0002.507 2.347 3.7967.362 7.094 4.841 8.256 0.043N21 19 18 18 16 10 10 3 3CV(%)078.1 136.25 76.7725.44 20.21 61.28 10.84 3.461.6 μg/L (Group 4)Mean14.000 2.300 1.222 3.667 26.438 38.167 10.000 71.400 0.896Standard Dev.0.0002.452 2.045 3.49813.038 6.735 5.550 14.300 0.193N21 20 18 18 16 6 6 3 3CV(%)0106.59 167.34 95.449.32 17.65 55.5 20.03 21.564.0 μg/L (Group 5)Mean14.000 2.056 0.500 2.556 30.357 35.000 6.667 66.633 1.094Standard Dev.0.0002.508 1.465 2.9357.880 7.937 2.887 8.256 0.455N2118 18 1814 3 3 3 3CV(%)0122.02 293.06 114.8525.96 22.68 43.3 12.39 41.5810 μg/L (Group 6)Mean14.0003.867 3.100 7.20025.800 36.000 9.000 71.400 0.920Standard Dev.0.0003.701 3.604 5.6535.226 4.690 4.050 14.300 0.033N2115 10 1015 6 6 3 3CV(%)095.71 116.26 78.5120.26 13.03 45 20.03 3.581.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.DRAFTIntra-Laboratory Analysis Report for Mysid: WLI Octylphenol Data13Table 1B. Summary of Reproduction Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F11,2,3Dose Concentration StatisticsTime to Mature(Females)NumberOffspring(Brood 1)NumberOffspring(Brood 2)NumberOffspring(Two Broods)Days toBrood 1Days toBrood 2Days BetweenBroods 1 and 2% of FemalesProductivelyActiveSex Ratio(males/females)Control(Group 1)Mean14.000 1.364 1.818 4.364 19.286 25.250 6.500 61.667 2.029Standard Dev.0.000 1.433 2.994 5.732 4.716 4.573 3.000 12.583 1.593N11 11 11 11 7 4 4 3 3CV (%)0 105.11 164.67 131.36 24.45 18.11 46.15 20.4 78.521.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.Only control data were available for F1 generation.

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

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

Parameters Normality Test 1 Homogeneous of Variance Test 2 Monotonicity Test 3 Concentration Trend Tests 4 Overall Concentration Effect 5 Pairwise comparisons to Control 6,7
Time to Mature NA NA No NA No NA Number Offspring (Brood 1) Normal 8 Homogeneous NonMonotonic NA No NA

Number Offspring (Brood 2) NonNormal NA NonMonotonic NA Yes Group 2, 3, 4 & 5

Number Offspring (Two Broods) Normal 8 Homogeneous NonMonotonic NA Yes Group 4 & 5

Days to Brood 1 NonNormal NA NonMonotonic NA No NA
Days to Brood 2 Normal Homogeneous NonMonotonic NA No NA
Days Between Broods 1 and 2 Normal 9 Homogeneous NonMonotonic NA No NA

% of Females Productively Active Normal Homogeneous 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.
8. Normal when square root-transformed.
9. Normal when log-transformed.DRAFTIntra-Laboratory Analysis Report for Mysid: WLI Octylphenol Data15Table 2A. Summary of Growth Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F01,2,3Dose ConcentrationStatisticsDryWeight (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)Mean0.045 0.597 0.662 1.407 1.916 5.550 5.837 8.829Standard Dev.0.010 0.062 0.102 0.241 0.111 0.368 0.235 0.397N9 9 41 32 45 18 41 32CV(%)22.6 10.33 15.4 17.15 5.78 6.63 4.03 4.50.26 μg/L(Group 2)Mean0.508 0.598 1.462 5.083 6.090 8.915Standard Dev.0.042 0.086 0.240 0.257 0.366 0.394N9 40 39 18 40 39CV(%)8.28 14.4 16.43 5.06 6.01 4.420.64 μg/L(Group 3)Mean0.488 0.643 1.400 5.094 6.234 8.830Standard Dev.0.118 0.107 0.240 0.472 0.284 0.409N9 38 28 17 38 28CV(%)24.23 16.64 17.12 9.27 4.56 4.631.6 μg/L(Group 4)Mean0.600 0.624 1.427 5.233 6.220 8.959Standard Dev.0.073 0.107 0.266 0.270 0.374 0.447N9 40 36 18 40 36CV(%)12.13 17.08 18.62 5.16 6.02 4.994.0 μg/L(Group 5)Mean0.533 0.590 1.394 4.911 6.217 8.948Standard Dev.0.095 0.114 0.234 0.478 0.396 0.357N9 41 33 18 41 33CV(%)17.84 19.39 16.81 9.72 6.37 3.9910 μg/L(Group 6)Mean0.518 0.560 1.326 4.989 6.185 8.936Standard Dev.0.087 0.098 0.333 0.459 0.332 0.567N9 39 26 18 39 26CV(%)16.72 17.49 25.14 9.2 5.37 6.351.Summary statistics are calculated across all replicatesand all samples in theentire concentration group.2.On day 0, all mysids are in control environments.3.Length is measured per mysid. Weight were measured on averages per (replicate) tank on day 0,per mating pair on day 7, and per mysid on day 14 and termination.DRAFTIntra-Laboratory Analysis Report for Mysid: WLI Octylphenol Data16Table 2B. Summary of Growth Endpoints. Laboratory: WLI, Test Chemical: Octylphenol, Generation: F11,2,3Dose ConcentrationStatisticsDry 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)Mean0.044 0.340 0.847 1.394 1.889 5.200 8.040 8.207Standard Dev.0.011 0.045 0.111 0.217 0.129 0.486 0.394 0.935N4 6 15 20 19 12 15 20CV(%)24.72 13.15 13.12 15.56 6.81 9.35 4.9 11.391.Summary statistics are calculated across all replicatesand all samples in theentire concentration group.2.Length is measured per mysid. Weight were measured on averages per (replicate) tank on day 0,per mating pair on day 7, and per mysid on day 14 and termination.3.Only control data were available for F1 generation.

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

Parameters Normality Test 1 Homogeneous 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) Normal Homogeneous Non-Monotonic NA Yes Group 3

Dry Weight(mg) (day 14) Normal Homogeneous Monotonic Decreasing trend for the two highest dose
groups Yes NA

Dry Weight (mg)

(Termination) Non-Normal NA Non-Monotonic NA No NA

Length (mm) (day 7) Non-Normal NA Monotonic Decreasing trend for the two highest dose
groups Yes NA

Length (mm)

(day 14) Non-Normal NA Non-Monotonic NA Yes Groups 2, 3, 4, 5 & 6 Length (mm) (Termination) Non-Normal NA 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 varian ces and using Tamhane-Dunnett testwith heterogeneous withintreatment 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: WLI Octylphenol Data

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

Dose Concentration Statistics Reproduction Juvenile Survival rate (%) (day 6) Reproduction Juvenile Survival rate (%) (day 14) Total Juvenile Survival rate (%) (day 6) Total Juvenile Survival rate (%) (day 14) Reproductive Adult Survival rate (%)

(Termination)

Control (Group 1) Mean 100.000 91.667 100.000 95.098 69.583

Standard

Dev. 0.000 7.638 0.000 4.493 3.411

N 3 3 3 3 3

CV(%) 0 8.33 0 4.72 4.9

0.26 μg/L (Group 2) Mean 98.333 96.667 99.167 96.078 82.185

Standard

Dev. 2.887 5.774 1.443 3.396 8.791

N 3 3 3 3 3

CV(%) 2.94 5.97 1.46 3.53 10.7

0.64 μg/L (Group 3) Mean 100.000 86.667 100.000 90.196 57.778

Standard

Dev. 0.000 14.434 0.000 9.455 10.184

N 3 3 3 3 3

CV(%) 0 16.65 0 10.48 17.63

1.6 μg/L (Group 4) Mean 100.000 95.000 99.167 96.078 75.000

Standard

Dev. 0.000 5.000 1.443 1.698 10.825

N 3 3 3 3 3

CV(%) 0 5.26 1.46 1.77 14.43

4.0 μg/L (Group 5) Mean 100.000 98.333 100.000 98.039 74.644

Standard

Dev. 0.000 2.887 0.000 3.396 4.707

N 3 3 3 3 3

CV(%) 0 2.94 0 3.46 6.31

10 μg/L (Group 6) Mean 96.667 95.000 97.500 94.118 60.513

Standard

Dev. 2.887 0.000 0.000 2.941 13.363

N 3 3 3 3 3

CV(%) 2.99 0 0 3.12 22.08

1. Summary statistics are calculated across all replicates and all samples in the entire concentration group.
2. Survival rates were measured per tank.

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

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

Dose Concentration Statistics Reproduction Juvenile Survival rate (%) (day 6) Reproduction Juvenile Survival rate (%) (day 14) Total Juvenile Survival rate (%) (day 6) Total Juvenile Survival rate (%) (day 14) Reproductive Adult Survival rate (%) (Termination)

Control (Group 1) Mean 88.533 86.325 89.103 90.103 61.640

Standard

Dev. 7.407 6.161 7.997 2.156 14.425

N 3 3 3 3 3
CV(%) 8.37 7.14 8.98 2.39 23.4

1. Summary statistics are calculated across all replicates and all samples in the entire concentration group.
2. Survival rates were measured per tank.
3. Only control data were available for F1 generation.

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

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

Parameters Normality Test 1 Homogeneous of Variance Test 2 Monotonicity Test 3 Concentration Trend Tests 4 Overall Concentration Effect 5 Pairwise comparisons to

Control 6,7 Reproduction

Juvenile Survival Rate (%) (day 6) Non-Normal NA Non-Monotonic NA No NA

Reproduction

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

Total Juvenile Survival Rate (%) (day 6) Non-Normal NA Monotonic No No NA

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

Reproductive Adult Survival rate (%)

(Termination) Normal Homogeneous Non-Monotonic NA No NA

1. Shapiro-Wilk test for normality.
2. Levene Test for homogeneous 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: WLI Octylphenol Data

21 Figure 1. Time to Mature for Generation F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 2. Number Offspring (Brood 1) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.Time To Mature (Days)

13 14 15

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



### Group Generation F0Number Offspring (brood 1)

1 2 3 4 5 6

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



### Group Generation F0

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

22 Figure 3. Number Offspring (Brood 2) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 4. Number Offspring (Two Broods) fo r Generation F0 by Group for WLI Lab, Chemical Octylphenol.Number Offspring (brood 2)

0 1 2 3 4 5 6 7 8

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



### Group Generation F0Number Offspring (two broods)

0 2 4 6 8 10 12 14 16

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



### Group Generation F0

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

23 Figure 5. Days to Brood 1 for Generation F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 6. Days to Brood 2 for Generation F0 by Group for WLI Lab, Chemical Octylphenol.Days to Brood 1

20 22 24 26 28 30 32 34

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



### Group Generation F0Days to Brood 2

28 30 32 34 36 38 40 42 44

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



### Group Generation F0

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

24 Figure 7. Days Between Broods 1 and 2 for Generation F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 8. Percent of Females Productively Active for Generation F0 by Group for WLI Lab, Chemical Octylphenol.Days Between Broods 1 and 2

4 6 8 10 12 14

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



### Group Generation F0% of Females Productively Active

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



### Group Generation F0

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

25 Figure 9. Sex Ratio for Generation F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 10. Dry Weight (mg) (day 0) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.Sex Ratio

0.6 0.8 1.0 1.2 1.4 1.6

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



### Group Generation F0Dry Weight (mg)(day 0)

0.040 0.041 0.042 0.043 0.044 0.045 0.046 0.047 0.048 0.049 0.050

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



### Group Generation F0

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

26 Figure 11. Dry Weight (mg) (day 7) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 12. Dry Weight (mg) (day 14) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.Dry Weight (mg)(day 7)

0.40 0.42 0.44 0.46 0.48 0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66

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



### Group Generation F0Dry Weight (mg)(day 14)

0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70

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



### Group Generation F0

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

27 Figure 13. Dry Weight (mg) (Termination) fo r Generation F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 14. Length (mm) (day 0) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.Dry Weight (mg)(Termination)

1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60

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



### Group Generation F0Length (mm)(day 0)

1.89 1.90 1.91 1.92 1.93 1.94

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



### Group Generation F0

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

28 Figure 15. Length (mm) (day 7) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 16. Length (mm) (day 14) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.Length (mm)(day 7)

4.6 4.8 5.0 5.2 5.4 5.6 5.8

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



### Group Generation F0Length (mm)(day 14)

5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4

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



### Group Generation F0

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

29 Figure 17. Length (mm) (Termination) for Gene ration F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 18. Reproduction Juvenile Survival Rate - Day 6 (%) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.Length (mm)(Termination)

8.7 8.8 8.9 9.0 9.1

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



### Group Generation F0Reproduction Juvenile Survival Rate - Day 6 (%)

94 95 96 97 98 99 100

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



### Group Generation F0

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

30 Figure 19. Reproduction Juvenile Survival Rate - Day 14 (%) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 20. Total Juvenile Survival Rate - Day 6 (%) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.Reproduction Juvenile Survival Rate - Day 14 (%)

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

Group Generation F0Total Juvenile Survival Rate - Day 6 (%)

97.0 97.5 98.0 98.5 99.0 99.5 100.0

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

Group Generation F0

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

31 Figure 21. Total Juvenile Survival Rate - Day 14 (%) for Generation F0 by Group for WLI Lab, Chemical Octylphenol.

Figure 22. Reproduction Adult Survival Rate at Termination (%) for Generation F0 by Group for WLI Lab, Chemical OctylphenolTotal Juvenile Survival Rate - Day 14 (%)

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



### Group Generation F0Reproductive Adult Survival Rate (%) (Termination)

50 55 60 65 70 75 80 85 90

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



### Group Generation F0