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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data



# Endocrine Disruptor Screening Program

Contract No: EP-W-11-063

Task Order No: 2

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 Anal ysis for SMV Lindane 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 for Mysid: SMV Lindane Data

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane 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 ........................................................................................................ 7
3.1.2 Normality ............................................................................................................... .. 7

3.1.3 Transformation of data ............................................................................................ 8
3.1.4 Heterogeneous variances ......................................................................................... 8
3.1.5 Monotonicity of response ........................................................................................ 8
3.1.6 Comparisons of test chemical to controls ................................................................ 8
3.2 Comparisons of the F0 and F1 generations ................................................................ 10

3.2.1 Outlier screens ....................................................................................................... 1 0
3.2.2 Normality ............................................................................................................... 10

3.2.3 Transformation of data .......................................................................................... 11
3.2.4 Heterogeneou s variances ....................................................................................... 11
3.2.5 Monotonicity Test ................................................................................................. 11
3.2.6 F0 generation vers us F1 generation ....................................................................... 11

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

Tables

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

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

## ...................................................................................................... 14
Table 1C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: SMV, Test Chemical: Lindane, Ge neration: F0 ........................................................................... 15
Table 1D. Statistical Analysis Results for Reproduction Endpoints. Laboratory: SMV, Test Chemical: Lindane, Ge neration: F1 ........................................................................... 16

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

iv Table 1E. Comparisons between Two Generations for Reproduction Endpoints. Laboratory: SMV, Test Chemical: Lindane. ................................................................................ 17
Table 2A. Summary of Growth Endpoints. Laboratory: SMV, Test Chemical: Lindane, Generation: F0 1,2,3,4 

## .................................................................................................... 18
Table 2B. Summary of Growth Endpoints. Laboratory: SMV, Test Chemical: Lindane, Generation: F1 1,2,3 

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

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

## ...................................................................................................... 24
Table 3C. Statistical Analysis Results for Survival Endpoints. Laboratory: SMV, Test Chemical: Lindane, Ge neration: F0 ........................................................................... 25
Table 3D. Statistical Analysis Results for Survival Endpoints. Laboratory: SMV, Test Chemical: Lindane, Ge neration: F1 ........................................................................... 26
Table 3E. Comparisons between Two Generation Su rvival Endpoints. Laboratory: SMV, Test Chemical: Lindane. .................................................................................................... 27

Figures

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

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

v Figure 7. Means ± Standard Errors of Untransformed data: Days Between Broods 1 and 2 By Generation and Group For Chemical Lindane From Lab SMV. ............................... 31
Figure 8. Means ± Standard Errors of Untransformed data: Total Reproductive Days for the First Two Broods By Generation and Gr oup For Chemical Lindane From Lab
SMV. .......................................................................................................................... 31
Figure 9. Means ± Standard Errors of Untransf ormed data: Number of Offspring per Female per Reproductive Day for the First Tw o Broods By Generation and Group For Chemical Lindane From Lab SMV. ........................................................................... 32
Figure 10. Means ± Standard Errors of Untran sformed data: Percent of Females Productively Active By Generation and Group For Chem ical Lindane From Lab SMV. .............. 32
Figure 11. Means ± Standard Errors of Untr ansformed data: Sex Ratio By Generation and Group For Chemical Lindane From Lab SMV. ......................................................... 33
Figure 12. Means ± Standard Errors of Untransformed data: Dry Weight (mg) (day 0) By Generation and Group For Chemical Lindane From Lab SMV. ............................... 33
Figure 13. Means ± Standard Errors of Untransformed data: Dry Weight (mg) (day 7) By Generation and Group For Chemical Lindane From Lab SMV. ............................... 34
Figure 14. Means ± Standard Errors of Untransformed data: Dry Weight (mg) (day 14) By Generation and Group For Chemical Lindane From Lab SMV. ............................... 34
Figure 15. Means ± Standard Errors of Untransf ormed data: Dry Weight (mg) (Termination) By Generation and Group For Chemical Lindane From Lab SMV. ............................... 35
Figure 16. Means ± Standard Errors of Untransformed data: Length (mm) (day 0) By Generation and Group For Chemical Lindane From Lab SMV. ............................... 35
Figure 17. Means ± Standard Errors of Untransformed data: Length (mm) (day 7) By Generation and Group For Chemical Lindane From Lab SMV. ............................... 36
Figure 18. Means ± Standard Errors of Untransformed data: Length (mm) (day 14) By Generation and Group For Chemical Lindane From Lab SMV. ............................... 36
Figure 19. Means ± Standard Errors of Untran sformed data: Length (mm) (Termination) By Generation and Group For Chemical Lindane From Lab SMV. ............................... 37
Figure 20. Means ± Standard Errors of Untransformed data: Reproduction Survival Rate (%) (day 7) By Generation and Group For Ch emical Lindane From Lab SMV. ............. 37
Figure 21. Means ± Standard Errors of Untransformed data: Reproduction Survival Rate (%) (day 14) By Generation and Group For Ch emical Lindane From Lab SMV. ........... 38
Figure 22. Means ± Standard Errors of Untransformed data: Reproduction Survival Rate (%) (Termination) By Generation and Group For Chemical Lindane From Lab SMV. .. 38
Figure 23. Means ± Standard Errors of Untransfor med data: Total Survival Rate (%) (day 7) By Generation and Group For Chemical Lindane From Lab SMV. ............................... 39

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

vi Figure 24. Means ± Standard Errors of Untransf ormed data: Total Survival Rate (%) (day 14) By Generation and Group For Chemi cal Lindane From Lab SMV. .......................... 39
Figure 25. Means ± Standard Errors of Untr ansformed data: Total Survival Rate (%) (Termination) By Generation and Group For Chemical Lindane From Lab SMV. .. 40

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane 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. All three laboratories conducted two-gene ration mysid survival and growth tests following a similar protocol with the test chemical lindane. Statistical analyses are being carried out on the SMV and UNT lindane study data. The draft an alysis documented in this report is based on the SMV lindane test.

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 lindane and an untreated control wi th 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 baskets. At termination (around Day 27 for SMV), mysids from the reproduction baskets are also measured for weights and lengths.

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

2 Intra-laboratory statistical analysis of the SM V lindane mysid study was performed according to a common statistical analysis plan. For this intra-la boratory analysis comparisons were made between the test chemical concentration groups and the concu rrent control group to assess the mysid responses to graded concentrations of lindane. The results of the two-generations we re 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:

(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 tank)

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)

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

1 

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

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

3 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 survival mysids on day 14), and for the surviving reproduction animals at termination. For the weight da ta, there are a total of 27 composites (3 sets and 9 composites per set) for the F0 generation and a total of 18 composites (3 replicates per concentration group and 1 composite per replicate among the six con centration groups) for the F1 generation on day 0. On day 7, there are 3 composite weight data per repli cate for 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 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.

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

4 Table 0. Data information for SMV Lindane

Data Files and data sheets Endpoints Comments Original- Biological Data.xls:

- F0 Reproduction

- F1 Reproduction

- F0 Summary

- F1 Summary Reformatted-:

 SMVlindanefecunity.xls:

- F0Reproduction

- F1Reproduction;

 SMVlindaneSexRatio.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.

 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- Biological Data.xls: One sheet for each generation and each day 0, 7, 14 and termination

Reformatted- SMV lindane Growth.xls One sheet for each generation and each day 0, 7, 14 and termination 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 three sets of mysids, each set with 9 composites (5 mysids per composite).

 Length are 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.

 Genders of mysids are provided for day 14 and termination. Original- Biological Data.xls:

- F0 Reproduction Survival

- F0 Growth Survival

- F0 Total Survival

- F1 Reproduction Survival

- F1 Growth Survival

- F1 Total Survival

Reformatted- SMV lindane Survival.xls:

- F0ReproductionSurvival;

- F0GrowthSurvival  Survival (reproduction) (day 7, day 14 and termination)

 Survival (total) (day 7, day 14 and termination)  Survival endpoints are adjusted for the non-treatment 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 lindane data set. Thus the total survival at day 27

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

5 Data Files and data sheets Endpoints Comments

- F0TotalSurvival

- F1ReproductionSurvival

- F1GrowthSurvival

- F1TotalSurvival 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 statis tical procedures for analyses within the F0 and F1 generations as compared to comparisons between the F0 and F1 generations. Monotonicity tests and concentration trend te sts are only made available under the oneway ANOVA in STATCHARRMS. Thus for th e analyses within generations it was elected to use the one-way ANOVA pro cedure. In the one-way ANOVA Model, STATCHARRMS pools the results across replicate tanks (and individual mysids (if the data were presented for each individual mysi d)) within test or c ontrol groups. The oneway ANOVA (parametric or nonparametric forms) is applied to the one generation data and there are no random replicate effects. For the comparison between the F0 and F1 generations STATCHARRMS utilizes a two-way mi xed effects analysis of variance, with replicate tanks within treatment groups treate d as random effects. The objectives of the analyses differ. The analyses within gene rations compare the test group results to the control group results. The analyses between generations compare the results between generations, either pooled across groups or separately within test or control groups.

 The preliminary tests for outliers, normality, heterogeneity of vari ance, and monotonicity were carried out independently for the analys es within the F0 generation, the analyses within the F1 generation, and the analyses be tween the F0 and F1 generations. There is no test for monotonicity within the two-way ANOVA for comparisons between generations. The results of the preliminary te sts may differ among the F0 analysis, the F1 analysis, and the analysis for comparison betw een F0 and F1. Thus the approaches for the principal tests used are independently de termined among these analyses. For example, for a particular endpoint some of the analys es 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 de tection procedures with the one-way ANOVA and with the two-way ANOVA. Theref ore the potential outlie rs screened out may not necessarily be consistent among the F0 and F1 within-gener ation analysis (oneway ANOVA), and the F0 and F1 comparis on between-generation analysis (two-way

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

6 

# ANOVA). The statistical potential outliers are screened values base d on the statistical model applied, and they may not be in fact biologically important. The potential outliers from both one-way and two-way ANOVA’s should be join tly considered. EPA will determine whether the flagged values should be included in the subseque nt analyses or should be treated as outliers.



#  The statistical analysis plan called for the scr eened 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 however 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 lindane data. This is because survival for the growth mysids is recorded only through day 14. The total survival at termination therefore tacitly assu mes that there is no mortality in the growth mysids between day 14 and termination (about study day 27). This is an underestimate of growth mysid mortality beyond day 14.

 For the reproductive response when “NA” is re ported 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 livi ng the 0 brood size should be counted as 0. If the female died before the recorded “NA” brood size is generated, 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 spreadshee t according to this suggestion. In the reformatted data, the changes are also made to the affected endpoi nts: total number of offspring in the first two broods, and the numbe r of offspring per female per reproductive day during the two broods. If the data entries for a given brood size is zero or “NA”, the times for the brood was also recorded as “NA” in the original data file and they are treated as missing in this analysis. If the brood size is missing in eith er 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 reproductive days is recorded as “NA”, and thus the number of offspri ng per female per reproductive day is treated as missing.

 In the SMV spreadsheets the 14 day and te rmination length and weight responses are presented by gender. In the preliminary draft analyses the 14 day and termination weights and lengths are pooled across ma le and female mysids. STATCHARRMS will not accommodate testing for gender effects in the one-way or two-way ANOVA models.

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

7 3. STATISTICAL METHODS

Statistical analysis procedures used conform to the analysis procedure 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 procedure were applied to per mysid data without first averaging across the mysids in the same the replicate baskets.

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

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 re view to determine if the deviati ons, although statistically screened, are in fact biologically unimportant. EPA will de termine whether the flagge d values should be included in the subsequent analyses or should be treated as outliers. The subsets of potential outliers are responsespecific. Due to time constraints, the statistical su mmaries 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.

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

8 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 outlier test were included in the Levene’s tests. If the normality assumption was invalid for both the untransformed and the transformed data heter ogeneity of variances among th e concentration groups was not assessed as the nonparametric procedures were applied.

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 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 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

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

9 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 alternative 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.

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 Jonckheere-

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

10 Terpstra 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. Analys is 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, assuming unequal variances between the two generati ons. Residual variance was treated as homogeneous or heterogeneous between ge nerations 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 outcomes of the prelim inary tests. All test procedures described in this report are based on the default auto option programs in STATCHARRMS.

3.2.1 Outlier screens

In STATCHARMS, the outliers screened for the comparisons of two gene rations were based on the two-way mixed effects 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.

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.

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.

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

11 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 transformed 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.

3.2.5 Monotonicity Test

A test for monotonicity was not carried out as or dered alternatives procedures were not available in the 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 carried out to identify the concen tration groups for which the 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 across all sa mples and all baskets for each concentration group and for each generation. Summary results for the tw o generations are presented in Tables 1A and 1B (reproduction endpoints), Tables 2A and 2B (growth e ndpoints), Tables 3A and 3B (survival endpoints).

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

12 These means and standard deviations are also displaye d, sided-by-side for the two generations in Figures 1 through 25. The summaries and 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 endpoints), Tables 2C and 2D (growth endpoints), and Tables 3C and 3D (survival endpoints). Comparisons between the F0 and F1 generations are summarized in Tables 1E (reproduction endpoints), 2E (growth endpoint s) and 3E (survival endpoints).DRAFTIntra-Laboratory Analysis Reportfor Mysid: SMV Lindane Data13Table 1A. Summary of Reproduction Endpoints. Laboratory: SMV, Test Chemical: Lindane, Generation: F01,2,3Concentration StatisticsTime toMature(females)NumberOffspring(brood 1)NumberOffspring(brood 2)NumberOffspring(twobroods)Days toBrood 1Days toBrood 2DaysBetweenBroods 1and 2TotalReproductiveDaysfor the FirstTwo Broods# of OffspringPer Female PerReproductionDay for the FirstTwo Broods% of FemalesProductivelyActiveSex Ratio(males/females)Control(Group 1)Mean10.3335.4768.80014.30015.81021.2005.4009.2001.567100.0001.268StandardDev.0.5771.6012.7072.7360.8141.7351.4291.7350.2410.0000.635N3212020212020202033CV (%)5.5929.2330.7619.135.158.1826.4618.8615.350.0050.0732 ng/L(Group 2)Mean9.6676.61910.44417.33316.04820.6114.5008.6112.028100.0001.334StandardDev.0.5772.8542.1484.3791.9621.5771.6541.5770.3950.0000.241N3211818211818181833CV(%)5.9743.1220.5725.2612.227.6536.7518.3119.500.0018.0780 ng/L(Group 3)Mean9.6676.3818.76215.14315.15020.2505.1008.2501.98395.2381.357StandardDev.0.5772.4593.4485.3221.8431.7730.4471.7730.6108.2480.566N3212121202020202033CV(%)5.9738.5439.3635.1512.178.768.7721.5030.768.6641.72200 ng/L (Group4)Mean9.3336.3817.94714.15815.33320.3895.0008.3891.801100.0000.696StandardDev.0.5772.0374.4034.1671.1551.2430.0001.2430.4950.0000.279N3211919211818181833CV(%)6.1931.9255.4029.437.536.100.0014.8227.480.0040.07500 ng/L(Group 5)Mean9.0008.04810.55618.61114.19019.4445.2787.4442.554100.0001.052StandardDev.0.0001.6583.2404.0310.9281.0420.9581.0420.6640.0000.557N3211818211818181833CV(%)0.0020.6030.6921.666.545.3618.1613.9925.980.0052.951250 ng/L(Group 6)Mean9.6676.6679.00016.12515.23820.9296.1549.0002.003100.0001.014StandardDev.0.5771.9584.2904.9381.2212.0562.1932.1210.5470.0000.349N3211616211413131333CV(%)5.9729.3747.6630.628.019.8235.6323.5727.280.0034.441.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 aremeasured per mating pair.3.Summaries are based on untransformed datawith no screened outliers removed.DRAFTIntra-Laboratory Analysis Reportfor Mysid: SMV Lindane Data14Table 1B. Summary of Reproduction Endpoints. Laboratory: SMV, Test Chemical: Lindane, Generation: F11,2, 3Concentration StatisticsTime toMatureNumberOffspring(brood 1)NumberOffspring(brood 2)NumberOffspring(twobroods)Days toBrood1Days toBrood2Days BetweenBroods 1 and 2Total ReproductiveDaysfor the First TwoBroods# of OffspringPer Female PerReproductionDayfor the FirstTwo Broods% of FemalesProductivelyActiveSexRatioControl(Group 1)Mean9.6676.5719.60015.95014.85720.0505.2008.0501.997100.0001.364StandardDev.0.5772.0392.9273.8181.1950.8261.1520.8260.5200.0000.688N3212020212020202033CV(%)5.9731.0330.4923.948.044.1222.1510.2626.050.0050.4332 ng/L(Group 2)Mean8.6676.0488.15014.30016.57120.9444.7788.9441.699100.0001.694StandardDev.1.1552.6553.6314.0412.2711.4741.2151.4740.4840.0000.488N3212020211818181833CV(%)13.3243.9044.5628.2613.707.0425.4416.4828.450.0028.8280 ng/L(Group 3)Mean10.6674.8107.10011.90016.66721.8955.2639.8951.269100.0001.430StandardDev.1.1551.9403.1774.0511.4611.6291.1471.6290.4160.0000.439N3212020211919191933CV(%)10.8340.3344.7534.048.767.4421.7916.4732.760.0030.72200 ng/L(Group 4)Mean9.6673.9475.3139.37516.73721.5634.9389.5631.004100.0001.022StandardDev.0.5771.3533.0053.2632.2322.0970.2502.0970.3580.0000.264N3191616191616161633CV(%)5.9734.2756.5634.8113.349.725.0621.9335.660.0025.80500 ng/L(Group 5)Mean10.0004.70010.63215.42115.65021.1055.4749.1051.772100.0000.985StandardDev.1.0001.8384.5125.1022.0332.2831.4292.2830.6130.0000.347N3201919201919191933CV(%)10.0039.1142.4433.0912.9910.8226.1025.0734.590.0035.211250 ng/L(Group 6)Mean10.6675.3007.53313.20016.00021.1675.4179.1671.62095.2380.931StandardDev.0.5773.0804.7794.6481.4531.8011.4431.8010.5838.2480.265N3201515191212121233CV(%)5.4158.1163.4435.219.088.5126.6519.6435.978.6628.471.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 aremeasured per mating pair.3.Summaries are based on untransformed datawith no screened outliers removed.

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

15 Table 1C. Statistical Analysis Results for Re production Endpoints. Laboratory: SMV, Test Chemical: Lindane, 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 Normal Homogeneous NonMonotonic NA No NA

Number Offspring (brood 1) NonNormal NA Monotonic Increasing trends for the two

highest concentration
groups Yes NA

Number Offspring (brood 2) Normal Homogeneous NonMonotonic NA No NA

Number Offspring (two broods) Normal Homogeneous NonMonotonic NA Yes Group 5

Days to Brood 1 NonNormal NA NonMonotonic NA Yes Groups 3, 5 and 6 Days to Brood 2 NonNormal NA NonMonotonic NA Yes Groups 3 and 5 Days Between Broods 1 and 2 NonNormal NA NonMonotonic NA No NA
Total Reproductive Days for the First

Two Broods NonNormal NA NonMonotonic NA Yes Groups 3 and 5
# of Offspring Per Female Per Reproduction Day

for the First Two Broods 8 Normal Homogeneous Monotonic Increasing trends for the two

highest concentration
groups Yes NA

% of Females Productively Active NonNormal 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. Normal when log-transformed.

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

16 Table 1D. Statistical Analysis Results for Re production Endpoints. Laboratory: SMV, Test Chemical: Lindane, 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 NonNormal NA Non-Monotonic NA No NA
Number Offspring (brood 1) NonNormal NA Non-Monotonic NA Yes Groups 4 and 5
Number Offspring (brood 2) Normal Homogeneous NonMonotonic NA Yes Group 4
Number Offspring (two broods) Normal Homogeneous Non-Monotonic NA Yes Groups 3 and 4

Days to Brood 1 NonNormal NA Non-Monotonic NA Yes Groups 2, 3 and 4

Days to Brood 2 NonNormal NA Non-Monotonic NA Yes Group 3
Days Between Broods 1 and 2 NonNormal NA Non-Monotonic NA No NA
Total Reproductive Days for the First Two Broods NonNormal NA Non-Monotonic NA Yes Group 3
# of Offspring Per Female

Per Reproduction Day for the First Two Broods Normal Homogeneous Non-Monotonic NA Yes Groups 3 and 4

% of Females Reproductively Active NonNormal NA NonMonotonic NA No NA

Sex Ratio Normal Homogeneous Non-Monotonic 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.

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

Parameters Normality Test 1 Homogeneity of Variance Test 2 Parametric/ Nonparametric Concentration Main Effect Generation Main Effect Concentration by Generation

Interaction Groups with Significant Difference between Generations 3
Time to Mature Normal Homogeneous Parametric No No No NA

Number Offspring (brood 1 Normal Homogeneous Parametric No Yes Yes Groups 4 and 5

Number Offspring (brood 2) Normal Homogeneous Parametric Yes Yes No Group 4

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

Total Reproductive Days for the

First Two

Broods Normal Homogeneous Parametric No Yes No Groups 3 and 5

# of Offspring Per Female Per Reproduction Day for the First Two Broods Normal Homogeneous Parametric No Yes Yes Groups 3, 4 and 5

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

Sex Ratio 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.

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

18 Table 2A. Summary of Growth Endpoints. Laboratory: SMV, Test Chemical: Lindane, 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) Length (mm) (day 7) Length (mm) (day 14) Length (mm) (Termination)

Control (Group 1) Mean 0.035 0.297 0.642 1.098 1.843 4.458 6.303 7.421

Standard

Dev. 0.010 0.052 0.142 0.267 0.129 0.415 0.317 0.326

N 27 9 32 39 135 18 32 39

CV(%) 28.21 17.44 22.20 24.30 7.01 9.31 5.03 4.40

32 ng/L (Group 2) Mean 0.357 0.616 1.188 4.466 6.568 7.488

Standard

Dev. 0.050 0.156 0.280 0.255 0.412 0.361

N 9 38 35 17 38 35

CV(%) 14.00 25.41 23.55 5.71 6.27 4.82

80 ng/L (Group 3) Mean 0.281 0.675 1.069 4.670 6.636 7.370

Standard

Dev. 0.079 0.207 0.295 0.536 0.561 0.377

N 9 36 48 17 36 48

CV(%) 28.01 30.60 27.62 11.48 8.46 5.12

200 ng/L (Group 4) Mean 0.316 0.891 1.047 4.791 6.815 7.368

Standard

Dev. 0.054 0.191 0.254 0.421 0.335 0.421

N 9 31 41 18 31 41

CV(%) 17.17 21.47 24.24 8.78 4.91 5.71

500 ng/L (Group 5) Mean 0.359 0.997 1.288 4.838 6.971 7.662

Standard

Dev. 0.075 0.190 0.312 0.360 0.302 0.406

N 9 31 32 18 31 32

CV(%) 20.80 19.10 24.19 7.45 4.33 5.30

1250 ng/L (Group 6) Mean 0.304 1.031 1.018 4.583 6.582 7.385

Standard

Dev. 0.058 0.254 0.322 0.229 0.501 0.451

N 9 32 23 18 32 23

CV(%) 19.13 24.64 31.63 5.00 7.62 6.11

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 are measured per mysid. Weight were measured on averag es per (replicate) tank on day 0, per mating pair on day 7, and p er 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 for Mysid: SMV Lindane Data

19 Table 2B. Summary of Growth Endpoints. Laboratory: SMV, Test Chemical: Lindane, 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 0.029 0.380 0.805 1.046 1.633 4.715 6.388 7.403

Standard

Dev. 0.005 0.142 0.178 0.238 0.125 0.260 0.311 0.243

N 3 9 18 37 15 18 18 37

CV(%) 17.56 37.49 22.11 22.76 7.66 5.52 4.87 3.28

32 ng/L (Group 2) Mean 0.030 0.383 0.783 1.022 1.671 4.622 6.333 7.351

Standard

Dev. 0.011 0.149 0.174 0.191 0.064 0.433 0.171 0.298

N 3 9 16 38 15 18 16 38

CV(%) 35.28 38.90 22.25 18.70 3.84 9.36 2.69 4.05

80 ng/L (Group 3) Mean 0.027 0.294 0.588 0.978 1.627 4.644 6.111 7.230

Standard

Dev. 0.007 0.075 0.152 0.220 0.097 0.444 0.268 0.330

N 3 9 26 37 15 18 26 37

CV(%) 25.70 25.44 25.85 22.54 5.95 9.56 4.39 4.57

200 ng/L (Group 4) Mean 0.033 0.307 0.622 0.986 1.705 4.544 6.058 7.124

Standard

Dev. 0.010 0.077 0.186 0.245 0.062 0.570 0.347 0.318

N 3 9 24 33 15 18 24 33

CV(%) 30.79 24.96 29.94 24.86 3.64 12.54 5.73 4.47

500 ng/L (Group 5) Mean 0.025 0.337 0.788 1.137 1.689 4.863 6.474 7.673

Standard

Dev. 0.011 0.065 0.144 0.271 0.070 0.422 0.341 0.475

N 3 9 18 30 15 18 18 30

CV(%) 43.48 19.42 18.29 23.79 4.16 8.67 5.27 6.19

1250 ng/L (Group 6) Mean 0.025 0.318 0.688 0.937 1.699 4.334 6.547 7.220

Standard

Dev. 0.009 0.035 0.171 0.246 0.094 0.400 0.543 0.522

N 3 9 16 21 15 18 16 21

CV(%) 38.32 11.16 24.84 26.29 5.52 9.23 8.29 7.23

1. Summary statistics are calculated across all replicates and all samples in the entire concentration group.
2. Length are measured per mysid. Weight were measured on averag es per (replicate) tank on day 0, per mating pair on day 7, and p er mysid 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 for Mysid: SMV Lindane Data

20 Table 2C. Statistical Analysis Results for Growth Endpoints. Laboratory: SMV, Test Chemical: Lindane, 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) Normal Homogeneous NonMonotonic NA No NA

Dry Weight(mg)

(day 14) Normal Heterogeneous Monotonic Increasing trend for the three

highest concentration
groups Yes NA

Dry Weight (mg) (Termination) NonNormal NA NonMonotonic NA Yes Group 5

Length (mm) (day 7) NonNormal NA Monotonic No Yes NA
Length (mm)

(day 14) NonNormal NA NonMonotonic NA Yes Groups 2, 3, 4, 5 and 6 Length (mm) (Termination) Normal Homogeneous NonMonotonic NA Yes Group 5

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 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 for Mysid: SMV Lindane Data

21 Table 2D. Statistical Analysis Results for Growth Endpoints. Laboratory: SMV, Test Chemical: Lindane, 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) Normal Homogeneous NonMonotonic NA No NA

Dry Weight (mg) (day 7) Normal 8 Homogeneous NonMonotonic NA No NA

Dry Weight (mg) (day 14) Normal Homogeneous NonMonotonic NA Yes Groups 3 and 4
Dry Weight (mg) (Termination) Normal 9 Homogeneous NonMonotonic NA No NA

Length (mm)

(day 0) NonNormal NA NonMonotonic No No NA
Length (mm)

(day 7) Normal Heterogeneous NonMonotonic NA Yes Group 6
Length (mm)

(day 14) Normal Heterogeneous NonMonotonic NA Yes Groups 3 and 4 Length (mm) (Termination) NonNormal NA NonMonotonic NA Yes Groups 4 and 5 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. After log-transformed, normal and homogeneous variance assumptions hold
9. After log-transformed, normal and homogeneous variance assumptions hold.

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# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

Parameters Normality Test 1 Homogeneity of Variance Test 2 Parametric/ 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 No No NA
Dry Weight (mg) (day 14) Normal Homogeneous Parametric Yes Yes Yes Groups 1, 2, 4, 5 and 6
Dry Weight (mg) (Termination) Normal Homogeneous Parametric Yes Yes No Groups 2 and 5
Length (mm)

(day 7) Normal Homogeneous Parametric No No No NA
Length (mm)

(day 14) Normal Homogeneous Parametric No Yes Yes Groups 3, 4 and 5 Length (mm) (Termination) Normal Homogeneous Parametric Yes 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 for Mysid: SMV Lindane Data

23 Table 3A. Summary of Survival Endpoints. Laboratory: SMV, Test Chemical: Lindane, 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 96.667 89.027 75.784 96.667 89.638 75.784

Standard

Dev. 5.774 6.208 9.413 5.774 3.745 9.413

N 3 3 3 3 3 3

CV(%) 5.97 6.97 12.42 5.97 4.18 12.42

32 ng/L (Group 2) Mean 90.000 84.298 72.729 92.500 86.958 72.729

Standard

Dev. 5.000 10.658 9.940 0.000 4.747 9.940

N 3 3 3 3 3 3

CV(%) 5.56 12.64 13.67 0.00 5.46 13.67

80 ng/L (Group 3) Mean 98.333 91.818 85.279 97.500 90.196 85.279

Standard

Dev. 2.887 3.149 4.829 2.500 4.493 4.829

N 3 3 3 3 3 3

CV(%) 2.94 3.43 5.66 2.56 4.98 5.66

200 ng/L (Group 4) Mean 88.333 87.895 80.370 89.167 83.001 80.370

Standard

Dev. 2.887 3.201 8.340 5.204 8.696 8.340

N 3 3 3 3 3 3

CV(%) 3.27 3.64 10.38 5.84 10.48 10.38

500 ng/L (Group 5) Mean 95.000 86.619 76.111 94.167 85.817 76.111

Standard

Dev. 5.000 6.659 3.469 3.819 11.398 3.469

N 3 3 3 3 3 3

CV(%) 5.26 7.69 4.56 4.06 13.28 4.56

1250 ng/L (Group 6) Mean 95.000 86.475 69.697 95.000 86.608 69.697

Standard

Dev. 5.000 6.523 13.887 5.000 1.573 13.887

N 3 3 3 3 3 3

CV(%) 5.26 7.54 19.92 5.26 1.82 19.92

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 for Mysid: SMV Lindane Data

24 Table 3B. Summary of Survival Endpoints. Laboratory: SMV, Test Chemical: Lindane, 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 97.778 95.956 92.308 95.833 91.667 92.308

Standard

Dev. 3.849 3.507 7.692 4.167 7.217 7.692

N 3 3 3 3 3 3

CV(%) 3.94 3.65 8.33 4.35 7.87 8.33

32 ng/L (Group 2) Mean 93.333 88.487 77.835 91.667 87.247 77.835

Standard

Dev. 6.667 4.846 3.836 4.167 3.794 3.836

N 3 3 3 3 3 3

CV(%) 7.14 5.48 4.93 4.55 4.35 4.93

80 ng/L (Group 3) Mean 97.778 97.917 89.744 98.611 97.222 89.744

Standard

Dev. 3.849 3.608 11.750 2.406 2.406 11.750

N 3 3 3 3 3 3

CV(%) 3.94 3.69 13.09 2.44 2.47 13.09

200 ng/L (Group 4) Mean 100.000 100.000 86.931 98.611 97.101 86.931

Standard

Dev. 0.000 0.000 5.250 2.406 5.020 5.250

N 3 3 3 3 3 3

CV(%) 0.00 0.00 6.04 2.44 5.17 6.04

500 ng/L (Group 5) Mean 100.000 94.118 85.769 95.833 88.889 85.769

Standard

Dev. 0.000 5.882 9.398 7.217 6.365 9.398

N 3 3 3 3 3 3

CV(%) 0.00 6.25 10.96 7.53 7.16 10.96

1250 ng/L (Group 6) Mean 95.556 91.667 66.667 89.683 86.905 66.667

Standard

Dev. 3.849 3.608 10.497 5.401 1.031 10.497

N 3 3 3 3 3 3

CV(%) 4.03 3.94 15.75 6.02 1.19 15.75

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 for Mysid: SMV Lindane Data

25 Table 3C. Statistical Analysis Results for Survival Endpoints. Laboratory: SMV, Test Chemical: Lindane, 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 NonMonotonic NA No NA

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

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

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

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

Total Survival Rate (%) (Termination) 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.

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

26 Table 3D. Statistical Analysis Results for Survival Endpoints. Laboratory: SMV, Test Chemical: Lindane, Generation: F1

Parameters Normality Test 1 Homogeneity of Variance Test 2 Monotonicity Test Concentration Trend Tests Overall Concentration Effect Significant Pairwise Comparisons to Control 3 Reproduction Survival Rate (%) (day 7) Normal Homogeneous NonMonotonic NA No NA

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

Reproduction Survival Rate (%) (Termination) Normal Homogeneous Monotonic Decreasing for the highest concentration
group Yes NA

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

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

Total Survival Rate (%) (Termination) Normal Homogeneous NonMonotonic NA Yes No

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.

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

Parameters Normality Test 1 Homogeneity of Variance Test 2 Parametric/ 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 Yes No Group 4

Reproduction Survival Rate (%) (day 14) Normal Homogeneous Parametric No Yes No Group 4

Reproduction Survival Rate (%) (Termination) Normal Homogeneous Parametric Yes Yes No Group 1

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

Total Survival Rate (%) (day 14) Normal Homogeneous Parametric No Yes No Group 4

Total Survival Rate (%) (Termination) Normal Homogeneous Parametric Yes 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 for Mysid: SMV Lindane Data

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

Figure 2. Means ± Standard Errors of Untransf ormed data: Number Offspring (brood 1) By Generation and Group For Chemical Lindane From Lab SMV.Time To Mature (Days)

8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



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

3 4 5 6 7 8 9

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

29 Figure 3. Means ± Standard Errors of Untransf ormed data: Number Offspring (brood 2) By Generation and Group For Chemical Lindane From Lab SMV.

Figure 4. Means ± Standard Errors of Untran sformed data: Number Offspring (two broods) By Generation and Group For Chemical Lindane From Lab SMV.Number Offspring (brood 2)

4 5 6 7 8 9 10 11 12

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



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

8 9 10 11 12 13 14 15 16 17 18 19 20

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

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

13 14 15 16 17 18

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1Days to Brood 2

19 20 21 22 23

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

31 Figure 7. Means ± Standard Errors of Untran sformed data: Days Between Broods 1 and 2 By Generation and Group For Chemical Lindane From Lab SMV.

Figure 8. Means ± Standard Errors of Untransf ormed data: Total Reproductive Days for the First Two Broods By Generation and Gr oup For Chemical Lindane From Lab SMV.Days Between Broods 1 and 2

4 5 6 7

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1Total Reproductive Days

7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

32 Figure 9. Means ± Standard Errors of Untransf ormed data: Number of Offspring per Female per Reproductive Day for the First Tw o Broods By Generation and Group For Chemical Lindane From Lab SMV.

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

0.5 1.0 1.5 2.0 2.5 3.0

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



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

90 91 92 93 94 95 96 97 98 99 100

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

Figure 12. Means ± Standard Errors of Untransformed data: Dry Weight (mg) (day 0) By Generation and Group For Chemical Lindane From Lab SMV.Sex Ratio

0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



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

0.018 0.020 0.022 0.024 0.026 0.028 0.030 0.032 0.034 0.036 0.038 0.040

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

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

0.24 0.26 0.28 0.30 0.32 0.34 0.36 0.38 0.40 0.42 0.44

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



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

0.5 0.6 0.7 0.8 0.9 1.0 1.1

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

Figure 16. Means ± Standard Errors of Untransformed data: Length (mm) (day 0) By Generation and Group For Chemical Lindane From Lab SMV.Dry Weight (mg)(Termination)

0.88 0.92 0.96 1.00 1.04 1.08 1.12 1.16 1.20 1.24 1.28 1.32 1.36

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



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

1.60 1.62 1.64 1.66 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

36 Figure 17. Means ± Standard Errors of Untransformed data: Length (mm) (day 7) By Generation and Group For Chemical Lindane From Lab SMV.

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

4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



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

5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

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

7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



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

86 88 90 92 94 96 98 100

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

Figure 22. Means ± Standard Errors of Untran sformed data: Reproduction Survival Rate (%) (Termination) By Generation and Group For Chemical Lindane From Lab SMV.Reproduction Survival Rate (%)(day 14)

70 75 80 85 90 95 100

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



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

60 65 70 75 80 85 90 95 100

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

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

86 88 90 92 94 96 98 100

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



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

76 80 84 88 92 96 100

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1

## DRAFT



# Intra-Laboratory Analysis Report for Mysid: SMV Lindane Data

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

68 72 76 80 84 88 92 96

Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L Control 32 ng/L 80 ng/L 200 ng/L 500 ng/L 1250 ng/L



## Group Generation F0 Generation F1