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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin 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 Analys is for UNT Vinclozolin in Mysids

U. S. Environmental Protection Agency

May 17, 2013

BATTELLE
505 King Avenue
Columbus, Ohio 43201

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin 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 ............................................................................................. 6

3. Statistical Methods ..................................................................................................... 8 3.1 Comparisons of test chemical with the control .......................................................... 8

3.1.1 Outlier Screens ......................................................................................................... .. 8 3.1.2 Normality ............................................................................................................... .... 9

3.1.3 Transformation of data ............................................................................................... 9 3.1.4 Heterogeneous variances ............................................................................................ 9 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 .............................................................. 11

3.2.1 Outlier screens ......................................................................................................... . 11 3.2.2 Normality ............................................................................................................... .. 12

3.2.3 Transformation of data ............................................................................................. 12 3.2.4 Heterogeneou s variances .......................................................................................... 12 3.2.5 Monotonicity Test .................................................................................................... 12 3.2.6 F0 generation vers us F1 generation .......................................................................... 12

4. results .................................................................................................................... ... 13

Tables

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

## ................................................................................ 14 Table 1B. Summary of Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F1 1,2,3 

## ................................................................................ 15 Table 1C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F0 .................................................................... 16

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

iv Table 1D. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F1 .................................................................... 17 Table 1E. Comparisons between Two Generations for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin. .......................................................................... 18 Table 2A. Summary of Growth Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F0 1,2,3,4,5 

## ................................................................................................. 19 Table 2B. Summary of Growth Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F1 1,2,3,4 

## ................................................................................................... 20 Table 2C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F0 .................................................................... 21 Table 2D. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F1 .................................................................... 22 Table 2E. Comparisons between Two Generations for Growth Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin. ..................................................................................... 23 Table 3A. Summary of Survival Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F0 1,2,3 

## ..................................................................................................... 24 Table 3B. Summary of Survival Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F1 1,2,3 

## ..................................................................................................... 25 Table 3C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F0 .................................................................... 26 Table 3D. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F1 .................................................................... 27 Table 3E. Comparisons between Two Generation Su rvival Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin. ............................................................................................. 28

Figures

Figure 1. Time to Mature By Generati on and Group For Chemical Vinclozolin From Lab UNT. ........................................................................................................ 29 Figure 2. Number Offspring (brood 1) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 29 Figure 3. Number Offspring (brood 2) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 30 Figure 4. Number Offspring (two broods) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 30 Figure 5. Days to Brood 1 By Generation and Group For Chemical Vinclozolin From Lab UNT. ........................................................................................................ 31

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

v Figure 6. Days to Brood 2 By Generation and Group For Chemical Vinclozolin From Lab UNT. ........................................................................................................ 31 Figure 7. Days Between Broods 1 and 2 By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 32 Figure 8. Percent of Females Productivel y Active By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................... 32 Figure 9. Sex Ratio By Generation and Gr oup For Chemical Vinclozolin From Lab UNT. ................................................................................................................. 33 Figure 10. Dry Weight (mg) (day 0) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 33 Figure 11. Dry Weight (mg) (day 7) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 34 Figure 12. Dry Weight (mg) (day 14) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 34 Figure 13. Dry Weight (mg) (Termination) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 35 Figure 14. Length (mm) (day 0) By Generation and Group For Chemical Vinclozolin From Lab UNT. ........................................................................................................ 35 Figure 15. Length (mm) (day 7) By Generation and Group For Chemical Vinclozolin From Lab UNT. ........................................................................................................ 36 Figure 16. Length (mm) (day 14) By Genera tion and Group For Chemical Vinclozolin From Lab UNT. ........................................................................................................ 36 Figure 17. Length (mm) (Termination) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 37 Figure 18. Reproduction Survival Rate (%) (day 7) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................... 37 Figure 19. Reproduction Survival Rate (%) (day 14) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................... 38 Figure 20. Reproduction Survival Rate (%) (Termination) By Generation and Group For Chemical Vinclozolin From Lab UNT. ............................................................. 38 Figure 21. Total Survival Rate (%) (day 7) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 39 Figure 22. Total Survival Rate (%) (day 14) By Generation and Group For Chemical Vinclozolin From Lab UNT. .................................................................................... 39

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin 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 with various chemicals. UNT conducted a tw o-generation mysid survival and growth test with vinclozolin. The draft analysis docu mented in this report is based on this UNT vinclozolin 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 vinclozolin and an untreated control, with three replicate tanks per test concentration or control group. Each replicate tank contains two retention baskets, one basket housing mysids for reproduction observations and a second basket for grow th and optional biochemical measurements.

For the F0 test the growth and reproducti on baskets each initially in clude 20 newly hatched mysids. With the appearance of a marsupial pouch on females (sexual maturity), mature females are isolated as pairs with males in separate brood cups. Seven breeding pairs are selected in each replicate reproduction basket. The exposure of these F0 mysids is continued until two weeks after the median time to the second brood release in the control group. Offspring (F1 generation) are collected from the F0 generation each morning from the brood cups and tran sferred immediately to baskets with the same F0 exposure conditions as their F0 parents.

The F1 generation is begun with newly hatched F0 of fspring. Within each replicate tank the growth and reproduction baskets each initially include 15 newly hatched mysids. With the appearance of a marsupial pouch on females (sexual maturity), matu re females are isolated as pairs with males in separate brood cups. Seven breeding pairs are selected in each replicate reproduction basket. The exposure of these F1 mysids is continued until tw o weeks after the median time to the second brood release in the control group. Offspring are collected from the F1 generation each morning from the brood cups and transferred immediately to baskets with th e same exposure conditions as their F1 parents.

Development, sexual maturation, reproduction, growth, and survival endpoints are observed in the F0 and F1 generations. Measurements are ma de of a number of endpoints in both F0 and F1 generations reflective of functional endocrine regul ation of development, gr owth, reproduction, and survival. They include sex ratio, time to maturity , time to brood release for the first two broods, and interbrood duration between the fi rst two broods. Observations are asses sed and recorded daily for each individual or breeding pair on mortality, sex determin ation, reproductive condition of female, and number of young released in each of the first two broods. My sids are sampled to determine weight and length at the beginning (Day 0) before being placed into the growth and reproduction baskets, and then at weekly intervals (Day 7 and Day 14) from the growth baskets. At termination (around Day 32 for UNT vinclozolin), mysids from the reproduction b askets are measured for weights and lengths.

Intra-laboratory statistical analysis of the UNT vinclozolin mysid study was performed according to a common statistical analysis plan. For this intr a-laboratory analysis comparisons were made between

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

2 the test chemical concentration groups and the concurrent control group to assess the mysid responses to graded concentrations of vinclozolin. The results of the two-generations were compared to assess the benefits of a two-generation study as compared to a one-generation life cycle study.

2. STUDY DATA

2.1 Data Endpoints

The test method specified 25 endpoints. Some of the endpoints were recorded per mysid or composite of mysids (e.g. day 0 weight), some were recorded per breeding pair, and some were recorded per basket (i.e. per replicate tank). These endpoints were 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)

Total reproduction days in the first two broods (per breeding pair) and number of young per female per reproductive day in the first two brood (per breeding pair) were not provided for the UNT vinclozolin study Excel spreadsheets. They could be calculated based on the reproduction data, but that has not been done yet.

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

a. Survival rate for reproduction animals at days 7, 14 and termination (reproduction survival, per basket) 1 

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

For the growth basket mysids the number remove d for weight and length determinations were adjusted for (i.e. were omitted from both the numerator and denominator) in the survival rate calculations. Reproductive survival was determined among the animal s assigned to the brood chambers. Total survival at termination was not determined due to the absence of appropriate data.

1 

### The reproduction termination survival rate s were based only on the 14 adult repro duction mysids in the seven brood cups starting on day 14, as shown in the survival and reproduction Excel data. Mortality be fore day 14 was not accounted for in the survival rates.

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

3 (iii) Growth (F0, F1) (8) 2, 3



## 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).

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. Le ngth and weight measurements were determined for mysids on day 0 before they were assigned to gr owth and reproduction baskets, for mysids from the growth baskets at day 7 (6 per replicate tank) and day 14 (all surviving mysids on day 14), and for the surviving reproduction mysids at termination. For the weight data, there were seven composites for the F0 generation (all in the control) and a total of 53 com posites (9 composites per concentration groups (8 for the highest concentration group)) for the F1 genera tion on day 0. There were no replicate tank identity designations in the growth data on day 0. On day 7, there were 3 composite weights per replicate for each of the three replicates per concentration group. Lengt h and weight measurements were not collected for F1 on day 7 in the Excel data sheets.

Each laboratory ran a concurrent control and five targeted test concentration levels. The actual exposure concentration levels in ge neral differed from the targeted levels. The statistical analysis procedures do not utilize the actual concentration levels. The concentrations are labeled by their targeted levels but the labels are not actually used in the analyses.

2.2 Data Issues and Data Handling

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

2 

### Day 14 and terminal weights and le ngths were not reported by gender.
3 

### Weight and length results were repor ted pooled across replicate tanks within control or test concentration groups

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

4 Table 0. Data information for UNT Vinclozolin

Data Files and data sh eets Endpoints Comments Original - Vinlozolin Spreadsheet UNT.xlsx:

− Time to 1st Brood (per matching pair)

− Time to 2nd Brood (per matching pair)

− F0 and F1 Interbrood Duration (per matching pair

− % Reproducing F0 and F1 (per replicate tank)

− % Reproducing F0 and F1 (per replicate tank)

− % Females with 1st+2nd Brood (per concentration, for each of the two broods)

− Time to Maturation (per replicate tank)

− Sex Ratio (per replicate tank)

 Mysid Vinclozolin Reproduction.xlsx # of offspring in each brood and total over two broods: one data sheet for a chemical concentration per generation. Reformatted –

“ UNTVinReproCharrms.xlsx”

- One datasheet per endpoint per generation  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 variables are not provided:

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

 Total reproductively active days for the first two broods  If a mysid in a matching pair died before the first or second brood, the 0 brood sizes were recorded in the original data files. Per EPA suggestion, brood size values were changed as: zero if female survived, and missing for a brood (1 st 

## or 2 nd 

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

 The total offspring for the first two broods was recorded as the sum of the two broods in the original data sheet treating the missing as zero. Thus it needed to be recalculated such that a missing value is assigned if there is a missing brood size in either brood 1 or brood 2.

 All changes described above are recorded in the reformatted data files.

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

 Replicate tank IDs are given (labeled as 1, 2, 3).

 Sex ratios were originally presented as % of females over all animals in the original data file. They were redefined as #males/#females in the reformatted data. Vinlozolin Spreadsheet UNT.xlsx:

− F0 Day 0 Mysid Lgth and Wt.;

− F0 Day 7 Mysid Lgth and Wt.;

− F0 Day 14 Mysid Lgth and Wt.;

− F0 to Term Mysid Lgth and Wt.

− F1 Day 0 Mysid Lgth and Wt.;

− F1 Day 14 Mysid Lgth and Weight (dry weight) and length

Weight (dry weight) and length for F1 Day 7 are not collected  Lengths were measured per mysid from day 0 to termination.

 Weights were measured per composite of 5 mysids on day 0 and per composite of 2 mysids day 7 (on day 7, per pair on smv), per mysid on day 14 and termination.

 Replicate (basket) ID are not available on day 0. Replicate (basket) ID are available (replicate ID given on day 7) or color-coded on day 14 and at termination).

 There are no growth data for day 7

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

5 Data Files and data sh eets Endpoints Comments

Wt.;

− F1 to Term Mysid Lgth and Wt. for F1 generation.

 Sex information for day 14 and termination are given. Data are not analyze by sex. Original- Survival and Reproduction Vinclozolin.xlsx

- F0 Survival and Repro.

- F1 Survival and Repro. Reformatted- UNT Vinclozolin Survival

- F0Survival

- F1Survival  Survival (reproduction) (day 7, day 14 and termination)

 Survival (total) (day 7, day 14)

 Survival (total) at termination is not available 4



## .  Reformatted survival data were hand entered using the information from the original files. The original files have survival data for both growth and reproduction mysids from day 0 to day 14. Thus the survival data can be determined using the information on days 7 and 14.

 The initial numbers of mysids are 20 for F0 and 15 for F1 per tank. Thus there should not be more than 20 mysids in a tank. if there are more than 20 mysids alive on day 7, we treated them as 20.

 Sometimes more mysids survived at a later date than at a previous date. For example, if on day 7, the number alive was recorded as 19 but 20 were alive on day 14, we assume survival number on day 7 as 20 instead of 19.

 There are no survival records for mysids that were not assigned to the brood chambers after day 14. The reproduction mysid survival for termination is determined using the animals from the brood chambers only. This didn’t adjust for the mortality rate before days 14, when they were paired and assigned to the brood chamber as we could not determine the survival status of mysids that were not assigned to the brood chambers. This survival rate could be overestimated.

 On the reproductive basket, there are fewer than 14 living mysids in some replicates on day14, however there are 14 or more than 14 mysids

4 

### The survival worksheet in the Vinclozolin Excel spreadsheet is problematic to use for termination survival. The numerator values are inconsistent with survival in formation in the Survival and Reproduction sp readsheet. The denominator values are not adjusted for non-treatment related mortality , in particular for the growth mysids that are removed at day 7 and day 14 for determination of lengths and weights.

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

6 Data Files and data sh eets Endpoints Comments (7 pairs of mysids in the brood chambers for the replicates). The data are used as they are.

 If there is a male death, which is assumed if number of animals goes from 2 to 1 and then back to 2, one add-in animal should be included in the survival pool. If there is a female death, which is assumed if number of animals goes from 2 to 1 and then stops (study for the brood chamber was terminated), one dead and one live are counted.

 If survival/reproduction data for two animals in a brood pair ended at the same time before the terminal day, they are assumed to be dead

 Survival endpoints are reported per replicate tank.

2.3 Analysis Considerations

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

 STATCHARRMS uses totally independent statistical procedures for analyses within the F0 and F1 generations as compared to comparisons betw een the F0 and F1 generations. Monotonicity tests and concentration trend tests are only made available under the one-way ANOVA in STATCHARRMS. Thus for the analyses within ge nerations it was elected to use the one-way ANOVA procedure. In the one-way ANOVA procedure, STATCHARRMS pools the results across the test or control groups. The one-wa y ANOVA (parametric or nonparametric forms) was applied to the one generati on data and there are no random replicate effects. For the comparison between the F0 and F1 genera tions STATCHARRMS utilizes a two-way mixed effects analysis of variance, with replicate tanks within treatment groups treated as random effects. The objectives of the analyses differ. The anal yses within generations compare the test group results to the control group results. The an alyses 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 variance, and monotonicity were carried out independently for the analyses within the F0 generation, the analyses within the F1 generation, and the analyses between the F0 and F1 generations. There is no test for monotonicity within the two-way AVOVA for compar isons between generati ons. The results of the preliminary tests may differ among the F0 anal ysis, the F1 analysis, and the analysis for comparison between F0 and F1. Thus the a pproaches for the principal tests used are independently determined among these analyses. For example, for a particular endpoint some of

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

7 the analyses may be parametric for one-generation and nonparametric for th e other generation, or they may be based on different transf ormations of the endpoint (or none).

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

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

 Survival for the reproduction animals at termina tion is calculated based on the animals assigned to and later added into the brood chambers, as showed in the Survival and Reproduction Vinclozolin Excel file. This accounts for letha lity only within the seven or less breeding pairs from day 14 to termination day. It does not account for lethality among the reproductive mysid prior to being paired at day 14.

 Total survival at termination is not determined. The Pre and Post Paring Survival worksheet in the Vinclozolin UNT Excel spreadsh eet are not used in the analysis because they are inconsistent with lethality shown in the Survival and Reproduc tion Vinclozolin Excel spreadsheet.

 Survival for both reproduction and growth anim als on day 7 and day 14 are determined based on the data in the Survival and Reproduction Vinclozolin Excel file.

 For the reproductive response when 0 is reported as a brood size the question arises as to whether or not to treat this as a missing value or as a 0 numerical value in the analysis. EPA has suggested that if the female is living the 0 br ood sizes should be counted as 0 and included in the brood size averages. If the female died before th e recorded 0 size is generated, the brood size should be counted as “NA” (or recorded as a missing) . Battelle has changed the data entries in the columns for brood size 1 and brood size 2 in the Ex cel spreadsheet according to this suggestion. In the reformatted data, the changes are also ma de to the affected endpoints: 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 0 or “NA”, the times for the brood was also recorded as “NA” in the original da ta 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

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

8 is also treated as missing. This applies to e ndpoints: number of offspring in brood 1, number of offspring in brood 2, number of offspring in broods 1 and 2, and per cent reproductive females.

 Total reproduction days in the first two broods (per breeding pair) and number of young per female per reproductive day in the first two brood (per breeding pair) are not available for the UNT vinclozolin study.

3. STATISTICAL METHODS
Statistical analysis procedures used conform to th e statistical analysis procedures specified in the STATCHARRMS package. Statistical analyses include two components: comparisons of test chemical with the control within each generation and comparis on of the F0 and F1 generations. The statistical analysis was carried out in a univariate fashion independently for each endpoint.

Statistical summaries and statistical analyses were applied to the data as reported without first summarizing within baskets. For example if the data were reported on a per mysid basis then the analysis procedures were applied to per mysid data without fi rst averaging across the mysids in the same 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 in the one ge neration analyses 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 avoi d confounding outliers and treatment effects. 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.

The statistical analysis plan states that the r esults of the statistical outlier screens are 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 included in the STATACHARRMS outputs and will be sent to EPA.

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

9 3.1.2 Normality

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

3.1.3 Transformation of data

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

3.1.4 Heterogeneous variances

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

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

10  Ordered alternatives

− nonparametric

 General alternatives

− parametric

 General alternatives

− nonparametric
Ordered alternatives – step down procedure

If the hypothesis of monotonicity of response w as accepted, ordered alternatives procedures were used. Step down multiple comparison procedures ar e particularly useful for ordered alternatives situations. The test is carried out as long as there are significant effects, starting from all concentration groups (including the control), successively omitting the highest concentration group. If at any stage the comparison is nonsignificant, the process stops and a ll means in that concentration group and lower are declared to be compatible with the null hypothesis of equality to the control group mean. No adjustment for simultaneity is required. In STATCHARRMS, the nonparametric Jonckheere-Terpstra (Jonckheere (1954)) test of trend was used on the rank data.

Additionally, global overall concentration effects were tested. These were based on the parametric analysis of variance F-test if the norma lity assumption held or the Kruskal-Wallis test if the normal assumption did not hold for both the untransfo rmed and the transformed data. See the general 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.

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

11 Exact Permutation Tests

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

3.2 Comparisons of the F0 and F1 generations

Mysid data for the two generation multi-concentr ation test were analyzed according to a common procedure for each generation as described above. Analy ses were also carried out compare the results in the F0 and F1 generations. Analyses were carried out using data reported on a per mysid, per breeding pair, or per basket basis. The comparisons of the F0 and F1 generations were performed with a two way mixed effects ANOVA model, which included generation, concentration group, and the interaction of generation and concentration as fixed effects and replicate (concentration, generation) as a random effect, allowing unequal variances between the two genera tions. The residual variance was treated as homogeneous or heterogene ous between generations basen on the results of Levene’s test.

Preliminary tests were carried out to detect outliers, departures from nor mality, and heterogeneity of variance. These analyses were independent of tho se carried out for the analy ses within generations and did not necessarily arrive at the same conclusions. Subsequent comparisons among groups were carried out using procedures based on the outcome of the prelim inary tests. All test procedures described in this report are based on the default auto option programs in STATCHARRMS

3.2.1 Outlier screens

In STATCHARMS, the outliers for the comparisons of two generations were based on the twoway mixed effect model in section 3.2.6.Outliers we re detected based on the residuals from the mixed effects model fit using Tukey’s outlier rule on the untra nsformed data. 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. 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 biol ogically 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 are response specific. Due to time constraints, the subsequent summaries and

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

12 statistical analyses in this report were carried out including the flagged values. The list of screened values is presented in the STATACHARRMS output.

3.2.2 Normality

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

3.2.3 Transformation of data

There are three options for data transformati on in STATCHARRMS: log-transformation, square root transformation, and arc-sin square-root transf ormation. Under the auto option in STATCHARRMS, the analysis procedures are first applied to the unt ransformed data. If the normality assumption is valid, then no transformation is necessary. If the normality assumption is invalid, then normality tests are again performed on the log and the square-root 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), where the residuals were from the model fit described in the normality test (S ection 3.2.2). 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 in STATCHARRMS.

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.

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# Intra-Laboratory Analysis Report fo r Mysid: UNT Vinclozolin Data

13 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). These means and standard deviations are also displaye d, sided-by-side for the two generations in Figures 1 through 22. 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, Tables 2C and 2D, and Tables 3C and 3D. Comparisons between the F0 and F1 generations are summarized in Tables 1E, 2E and 3E.DRAFTSummary Tables and Figures for Mysid: UNT Vinclozolin Data14Table 1A. Summary of Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F01,2,3Concentration StatisticsTime toMature(Females)NumberOffspring(Brood 1)NumberOffspring(Brood 2)NumberOffspring(TwoBroods)Days toBrood 1Days toBrood 2DaysBetweenBroods 1and 2% of FemalesProductivelyActiveSex Ratio(males/females)Control(Group 1)Mean11.333 3.800 4.900 8.700 17.800 25.412 7.588 95.233 1.472StandardDev.0.577 2.191 3.432 4.692 2.745 3.759 3.429 8.256 0.284N3 20 20 20 20 17 17 3 3CV (%)5.09 57.66 70.04 53.93 15.42 14.79 45.19 8.67 19.3125 ppb(Group 2)Mean12.000 4.714 4.400 8.933 17.750 24.769 6.769 95.233 1.241StandardDev.1.000 2.217 3.680 4.559 2.049 2.522 2.166 8.256 0.409N3 21 15 15 20 13 13 3 3CV(%)8.33 47.02 83.64 51.03 11.54 10.18 32.00 8.67 32.9850 ppb(Group 3)Mean10.000 3.000 3.714 6.714 19.389 24.182 5.455 85.700 1.336StandardDev.0.000 1.495 3.024 2.525 3.775 3.311 2.382 14.300 0.463N3 18 14 14 18 11 11 3 3CV(%)0.00 49.84 81.41 37.60 19.47 13.69 43.67 16.69 34.62100 ppb(Group 4)Mean10.333 5.095 3.533 9.133 18.571 23.000 5.625 100.000 2.248StandardDev.1.155 3.506 4.373 4.051 3.310 2.268 1.768 0.000 0.801N3 21 15 15 21 8 8 3 3CV(%)11.17 68.80 123.77 44.35 17.82 9.86 31.43 0.00 35.62200 ppb(Group 5)Mean9.667 4.789 5.400 10.067 18.789 26.214 7.357 90.467 2.354StandardDev.0.577 1.653 3.460 4.234 2.394 3.093 2.818 8.256 0.781N3 19 15 15 19 14 14 3 3CV(%)5.97 34.50 64.07 42.06 12.74 11.80 38.30 9.13 33.17600 ppb(Group 6)Mean10.667 2.769 3.400 6.100 21.000 26.200 5.200 65.567 3.922StandardDev.0.577 1.787 3.658 4.864 4.000 3.493 0.447 15.032 0.958N3 13 10 10 11 5 5 3 3CV(%)5.41 64.52 107.58 79.73 19.05 13.33 8.60 22.93 24.431.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 .DRAFTSummary Tables and Figures for Mysid: UNT Vinclozolin Data15Table 1B. Summary of Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F11,2,3Concentration StatisticsTime to Mature(Females)NumberOffspring(Brood 1)NumberOffspring(Brood 2)NumberOffspring(Two Broods)Days toBrood 1Days toBrood 2 Days Between Broods 1 and 2% of FemalesProductivelyActive Sex RatioControl(Group 1)Mean9.333 4.143 7.200 11.350 16.095 21.150 5.000 100.000 1.434Standard Dev.2.082 1.931 2.419 3.117 0.700 0.671 0.324 0.000 0.613N3 21 20 20 21 20 20 3 3CV(%)22.30 46.61 33.60 27.46 4.35 3.17 6.49 0.00 42.7825 ppb(Group 2)Mean12.000 5.286 6.889 12.000 18.619 23.625 5.375 100.000 1.403Standard Dev.2.828 2.148 3.359 4.256 4.177 4.380 1.310 0.000 0.350N2 21 18 18 21 16 16 3 3CV(%)23.57 40.64 48.76 35.47 22.43 18.54 24.38 0.00 24.9750 ppb(Group 3)Mean11.000 5.500 7.944 13.556 18.200 23.111 5.000 95.233 1.237Standard Dev.2.000 2.439 3.038 4.655 3.037 3.234 0.485 8.256 0.455N3 20 18 18 20 18 18 3 3CV(%)18.18 44.34 38.25 34.34 16.68 13.99 9.70 8.67 36.74100 ppb(Group 4)Mean8.333 4.381 5.474 9.684 16.524 21.353 4.882 100.000 0.817Standard Dev.1.155 2.312 3.272 3.400 1.289 1.412 0.485 0.000 0.135N3 21 19 19 21 17 17 3 3CV(%)13.86 52.79 59.78 35.11 7.80 6.61 9.94 0.00 16.56200 ppb(Group 5)Mean10.500 5.800 6.333 12.111 17.600 22.625 6.125 92.850 0.537Standard Dev.0.707 4.417 4.387 6.716 3.169 2.973 2.100 10.112 0.424N2 10 9 9 10 8 8 2 3CV(%)6.73 76.16 69.28 55.46 18.01 13.14 34.29 10.89 79.01600 ppb(Group 6)Mean8.333 5.000 3.750 8.800 17.619 25.000 7.000 100.000 1.729Standard Dev.2.517 2.258 3.307 3.365 2.037 3.588 3.000 0.000 0.444N3 21 20 20 21 17 17 3 3CV(%)30.20 45.17 88.18 38.24 11.56 14.35 42.86 0.00 25.661.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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# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

16 Table 1C. Statistical Analysis Results for Re production Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, 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 Normal Homogeneous Monotonic No Yes NA Number Offspring (Brood 1) NonNormal NA NonMonotonic NA Yes No

Number Offspring (Brood 2) NonNormal NA NonMonotonic NA No NA

Number Offspring (Two Broods) Normal Homogeneous NonMonotonic NA No NA

Days to Brood 1 NonNormal NA Monotonic Increasing trend for the two highest dose

groups Yes NA

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

% of Females Productively Active NonNormal NA Monotonic Decreasing trend for the highest

dose group Yes NA

Sex Ratio Normal Homogeneous NonMonotonic NA Yes Group 6

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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# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

17 Table 1D. Statistical Analysis Results for Re production Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F1

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 Normal Homogeneous NonMonotonic NA No NA Number Offspring (Brood 1) Normal 8 Homogeneous NonMonotonic NA No NA Number Offspring (Brood 2) Normal Homogeneous Monotonic Decreasing trend for the highest dose group Yes NA Number Offspring

(Two Broods) Normal 9 Homogeneous Monotonic Decreasing trend for the highest dose group Yes NA

Days to Brood 1 NonNormal NA NonMonotonic NA No NA

Days to Brood 2 NonNormal NA Monotonic Increasing trend for the highest dose group Yes NA Days Between Broods 1 and 2 Normal NA NonMonotonic NA Yes Group 6

% of Females Reproductively Active NonNormal NA NonMonotonic NA No NA

Sex Ratio 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. 8. Normal when square root-transformed. 9. Normal when log-transformed.

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# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

18 Table 1E. Comparisons between Two Generation s for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin.

Parameters Normality Test 1 Homogeneous of Variance Test 2 Parameteric/ Nonparametric Concentration Main Effect Generation Main Effect Concentration by Generation

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

Number Offspring (Brood 1 Normal Homogeneous Parametric No Yes No Groups 3 and 6

Number Offspring (Brood 2) Normal Homogeneous Parametric No Yes No Group 3

Number Offspring (Two Broods) Normal Homogeneous Parametric No Yes No Groups 2 and 3
Days to Brood 1 NonNormal NA Nonparametric Yes No No NA
Days to Brood 2 Normal Homogeneous Parametric Yes Yes No Group 5
Days Between Broods 1 and 2 NonNormal NA Nonparametric Yes No No NA

% of Females Productively Active NonNormal NA Nonparametric Yes No Yes Group 6

Sex Ratio Normal Homogeneous Parametric Yes Yes Yes Group 4, 5 and 6 1. Shapiro-Wilk test for normality. 2. Levene test for homogeneous of variances. 3. Control is group 1 and test concentration groups are group 2 to 6. 4.

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# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

19 Table 2A. Summary of Growth Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F0 1,2,3,4,5

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 0.033 0.200 0.522 1.043 1.641 3.572 4.914 6.225

Standard Dev. 0.004 0.030 0.132 0.266 0.189 0.364 0.384 0.410

N 7 9 36 32 34 18 36 32

CV(%) 13.55 15.21 25.38 25.53 11.53 10.20 7.82 6.58

25 ppb (Group 2) Mean 0.175 0.565 0.904 3.489 5.119 6.218

Standard Dev. 0.049 0.181 0.180 0.395 0.565 0.347

N 9 31 34 18 31 34

CV(%) 28.07 32.02 19.90 11.33 11.03 5.58

50 ppb (Group 3) Mean 0.191 0.541 0.953 3.567 5.015 6.039

Standard Dev. 0.085 0.104 0.202 0.376 0.249 0.346

N 9 34 27 18 34 28

CV(%) 44.43 19.17 21.19 10.53 4.96 5.72

100 ppb (Group 4) Mean 0.186 0.487 0.944 3.528 5.179 6.241

Standard Dev. 0.030 0.166 0.228 0.276 0.598 0.359

N 9 24 39 18 24 39

CV(%) 15.99 34.10 24.11 7.83 11.54 5.75

200 ppb (Group 5) Mean 0.194 0.476 0.885 3.456 5.080 6.230

Standard Dev. 0.112 0.133 0.235 0.419 0.574 0.370

N 9 25 34 18 25 33

CV(%) 57.52 27.96 26.51 12.13 11.31 5.93

600 ppb (Group 6) Mean 0.158 0.386 0.837 3.344 4.726 6.182

Standard Dev. 0.047 0.111 0.173 0.479 0.450 0.438

N 9 23 28 18 23 28

CV(%) 29.62 28.80 20.65 14.33 9.53 7.09
1. Summary statistics are calculated across all replicates and all samples in the entire concentration group. 2. On day 0, all mysids are in control environments. 3. Length is measured per mysid. Weight were measured on averag es per (replicate) tank on day 0, per mating pair on day 7, and pe r mysid on day 14 and termination. 4. Summaries are based on untransformed data with no screened outliers removed. 5. Replicate tank within group information is not provided for the growth endpoints.

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# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

20 Table 2B. Summary of Growth Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F1 1,2,3,4

Concentration Statistics Dry Weight (mg)

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

Control (Group 1) Mean 0.058 0.602 0.931 1.340 5.276 6.100

Standard Dev. 0.050 0.095 0.201 0.123 0.199 0.283

N 9 29 34 45 29 34
CV(%) 87.42 15.73 21.57 9.20 3.78 4.64

25 ppb (Group 2) Mean 0.044 0.574 1.037 1.349 5.292 5.933

Standard Dev. 0.049 0.097 0.251 0.155 0.215 0.405

N 9 12 40 45 12 40
CV(%) 110.41 16.86 24.23 11.47 4.07 6.82

50 ppb (Group 3) Mean 0.064 0.581 1.021 1.402 5.252 6.358

Standard Dev. 0.042 0.133 0.235 0.144 0.198 0.302

N 9 25 39 45 25 38
CV(%) 65.88 22.83 23.00 10.25 3.77 4.75

100 ppb (Group 4) Mean 0.050 0.585 1.041 1.391 5.206 6.292

Standard Dev. 0.053 0.114 0.289 0.135 0.223 0.383

N 9 32 37 45 32 36
CV(%) 105.88 19.57 27.79 9.67 4.28 6.08

200 ppb (Group 5) Mean 0.066 0.654 1.064 1.369 5.200 6.357

Standard Dev. 0.033 0.056 0.269 0.116 0.312 0.287

N 9 8 13 45 8 14
CV(%) 50.43 8.49 25.28 8.51 5.99 4.52

600 ppb (Group 6) Mean 0.068 0.509 1.070 1.402 5.282 6.598

Standard Dev. 0.036 0.114 0.317 0.133 0.429 0.431

N 8 22 42 40 22 42
CV(%) 53.42 22.33 29.63 9.48 8.13 6.53
1. Summary statistics are calculated across all replicates and all samples in the entire concentration group. 2. Length is measured per mysid. Weight were measured on averag es per (replicate) tank on day 0, per mating pair on day 7, and pe r mysid on day 14 and termination. 3. Summaries are based on untransformed data with no screened outliers removed. 4. Replicate tank within group information is not provided for the growth endpoints.

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# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

21 Table 2C. Statistical Analysis Results for Re production Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, 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 8 Homogeneous NonMonotonic NA No NA

Dry Weight(mg)

(day 14) Normal Heterogeneous NonMonotonic NA Yes Group 6

Dry Weight (mg) (Termination) Normal Homogeneous Monotonic Decreasing trend for the highest

dose group Yes NA

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

(day 14) NonNormal NA NonMonotonic NA Yes Groups 2 and 4 Length (mm) (Termination) NonNormal NA 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 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. 8. Normal when square root-transformed.

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# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

22 Table 2D. Statistical Analysis Results for Re production Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F1

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 0) Normal 8 Homogeneous NonMonotonic NA No NA

Dry Weight (mg) (day 14) Normal 8 Homogeneous NonMonotonic NA Yes Group 6

Dry Weight (mg) (Termination) NonNormal NA NonMonotonic NA No NA

Length (mm)

(day 0) NonNormal NA Monotonic Increasing trend for the highest

dose group Yes NA

Length (mm)

(day 14) Normal Heterogeneous NonMonotonic NA Yes No

Length (mm) (Termination) Normal Homogeneous Monotonic Increasing trend for the highest four dose groups Yes 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. 8. Normal when log-transformed.

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# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

23 Table 2E. Comparisons between Two Generati ons for Growth Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin.

Parameters Normality Test 1 Homogeneous of Variance Test 2 Parameteric/ Nonparametric Concentration Main Effect Generation Main Effect Concentration by Generation

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

Length (mm)

(day 14) Normal Homogeneous Parametric No Yes No Group 6 Length (mm) (Termination) Normal Homogeneous Parametric Yes No Yes Groups 2, 3 and 6 1. Shapiro-Wilk test for normality. 2. Levene test for homogeneous of variances. 3. Control is group 1 and test concentration groups are group 2 to 6.

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# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

24 Table 3A. Summary of Survival Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, 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)

Control (Group 1) Mean 100.000 85.000 72.309 100.000 88.235

Standard

Dev. 0.000 5.000 7.647 0.000 2.941

N 3 3 3 3 3
CV(%) 0 5.88 10.58 0 3.33

25 ppb (Group 2) Mean 98.333 76.667 70.476 95.833 78.431

Standard

Dev. 2.887 12.583 3.434 3.819 10.329

N 3 3 3 3 3
CV(%) 2.94 16.41 4.87 3.98 13.17

50 ppb (Group 3) Mean 93.333 80.000 64.583 95.833 84.314

Standard

Dev. 7.638 8.660 9.547 2.887 3.396

N 3 3 3 3 3
CV(%) 8.18 10.83 14.78 3.01 4.03

100 ppb (Group 4) Mean 96.667 90.000 67.479 97.500 83.333

Standard

Dev. 2.887 13.229 12.953 2.500 6.123

N 3 3 3 3 3
CV(%) 2.99 14.7 19.2 2.56 7.35

200 ppb (Group 5) Mean 93.333 85.000 65.926 94.167 82.353

Standard

Dev. 7.638 8.660 18.802 5.204 5.094

N 3 3 3 3 3
CV(%) 8.18 10.19 28.52 5.53 6.19

600 ppb (Group 6) Mean 88.333 73.333 67.857 90.000 70.588

Standard

Dev. 2.887 7.638 9.449 5.000 2.941

N 3 3 3 3 3
CV(%) 3.27 10.41 13.93 5.56 4.17
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


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

25 Table 3B. Summary of Survival Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, 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)

Control (Group 1) Mean 97.778 93.333 72.639 96.444 80.889

Standard

Dev. 3.849 6.667 19.792 3.355 22.562

N 3 3 3 3 3
CV(%) 3.94 7.14 27.25 3.48 27.89

25 ppb (Group 2) Mean 86.667 84.444 73.849 86.923 82.479

Standard

Dev. 11.547 13.878 11.616 11.331 16.732

N 3 3 3 3 3
CV(%) 13.32 16.43 15.73 13.04 20.29

50 ppb (Group 3) Mean 100.000 97.778 79.779 96.154 91.197

Standard

Dev. 0.000 3.849 17.536 3.846 4.737

N 3 3 3 3 3
CV(%) 0 3.94 21.98 4 5.19

100 ppb (Group 4) Mean 97.778 95.556 83.929 98.889 96.420

Standard

Dev. 3.849 3.849 11.152 1.925 3.710

N 3 3 3 3 3
CV(%) 3.94 4.03 13.29 1.95 3.85

200 ppb (Group 5) Mean 40.000 22.222 63.889 43.333 31.778

Standard

Dev. 40.000 38.490 19.642 16.667 5.004

N 3 3 2 3 3
CV(%) 100 173.21 30.74 38.46 15.75

600 ppb (Group 6) Mean 86.349 53.016 83.194 91.904 60.716

Standard

Dev. 13.345 46.329 9.320 8.757 41.074

N 3 3 3 3 3
CV(%) 15.45 87.39 11.2 9.53 67.65
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


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

26 Table 3C. Statistical Analysis Results for Re production Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, 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 Survival Rate (%) (day 7) Normal Homogeneous Monotonic Decreasing trend for the highest

dose group 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 Monotonic Decreasing trend for the highest

dose group No NA

Total Survival Rate (%) (day 14) Normal Homogeneous Monotonic Decreasing trend for the highest

dose group Yes 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.

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

27 Table 3D. Statistical Analysis Results for Re production Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin, Generation: F1

Parameters Normality Test 1 Homogeneous of Variance Test 2 Monotonicity Test 3 Concentration Trend Tests 4 Overall Concentration Effect 5 Significant Pairwise Comparisons to Control 6,7 Reproduction Survival Rate (%) (day 7) NonNormal NA NonMonotonic NA No NA

Reproduction Survival Rate (%) (day 14) Normal Homogeneous Monotonic Decreasing trend for the highest

dose group Yes NA

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

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

Total Survival Rate (%) (day 14) Normal Homogeneous NonMonotonic NA Yes Group 5

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.

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

28 Table 3E. Comparisons between Two Generati on Survival Endpoints. Laboratory: UNT, Test Chemical: Vinclozolin.

Parameters Normality Test 1 Homogeneous of Variance Test 2 Parameteric/ Nonparametric Concentration Main Effect Generation Main Effect Concentration by Generation

Interaction Groups with Significant Difference between Generations 3 Reproduction Survival Rate (%) (day 7) NonNormal NA Nonparametric Yes No No NA

Reproduction Survival Rate (%) (day 14) Normal Homogeneous Parametric Yes No Yes Group 5

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

Total Survival Rate (%) (day 7) Normal Homogeneous Parametric Yes Yes Yes Group 5

Total Survival Rate (%) (day 14) Normal Homogeneous Parametric Yes No Yes Group 5

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

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

29 Figure 1. Time to Mature By Generation a nd Group For Chemical Vinclozolin From Lab UNT.

Figure 2. Number Offspring (brood 1) By Generation and Group For Chemical Vinclozolin From Lab UNT.Time To Mature (Days)

6 7 8 9 10 11 12 13 14

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



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

1 2 3 4 5 6 7 8

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

30 Figure 3. Number Offspring (brood 2) By Generation and Group For Chemical Vinclozolin From Lab UNT.

Figure 4. Number Offspring (two broods) By Generation and Group For Chemical Vinclozolin From Lab UNT.Number Offspring (brood 2)

1 2 3 4 5 6 7 8 9

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



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

3 4 5 6 7 8 9 10 11 12 13 14 15

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

31 Figure 5. Days to Brood 1 By Generation and Group For Chemical Vinclozolin From Lab UNT.

Figure 6. Days to Brood 2 By Generation and Group For Chemical Vinclozolin From Lab UNT.Days to Brood 1

15 16 17 18 19 20 21 22 23

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1Days to Brood 2

21 22 23 24 25 26 27 28

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

32 Figure 7. Days Between Broods 1 and 2 By Generation and Group For Chemical Vinclozolin From Lab UNT.

Figure 8. Percent of Females Productively Acti ve By Generation and Group For Chemical Vinclozolin From Lab UNT.Days Between Broods 1 and 2

4 5 6 7 8 9

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



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

50 60 70 80 90 100

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

33 Figure 9. Sex Ratio By Generation and Gro up For Chemical Vinclozolin From Lab UNT.

Figure 10. Dry Weight (mg) (day 0) By Generation and Group For Chemical Vinclozolin From Lab UNT.Sex Ratio

0 1 2 3 4 5

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



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

0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

34 Figure 11. Dry Weight (mg) (day 7) By Generation and Group For Chemical Vinclozolin From Lab UNT.

Figure 12. Dry Weight (mg) (day 14) By Generation and Group For Chemical Vinclozolin From Lab UNT.Dry Weight (mg)(day 7)

0.14 0.15 0.16 0.17 0.18 0.19 0.20 0.21 0.22 0.23 0.24

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



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

0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

35 Figure 13. Dry Weight (mg) (Termination) By Generation and Group For Chemical Vinclozolin From Lab UNT.

Figure 14. Length (mm) (day 0) By Generation and Group For Chemical Vinclozolin From Lab UNT.Dry Weight (mg)(Termination)

0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group GenerationF0 GenerationF1Length (mm)(day 0)

1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

36 Figure 15. Length (mm) (day 7) By Generation and Group For Chemical Vinclozolin From Lab UNT.

Figure 16. Length (mm) (day 14) By Generation and Group For Chemical Vinclozolin From Lab UNT.Length (mm)(day 7)

3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.60 3.65 3.70

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



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

4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

37 Figure 17. Length (mm) (Termination) By Gene ration and Group For Chemical Vinclozolin From Lab UNT.

Figure 18. Reproduction Survival Rate (%) (day 7) By Generation and Group For Chemical Vinclozolin From Lab UNT.Length (mm)(Termination)

5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group GenerationF0 GenerationF1Reproduction Survival Rate (%)(day 7)

10 20 30 40 50 60 70 80 90 100

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

38 Figure 19. Reproduction Survival Rate (%) (day 14) By Generation and Group For Chemical Vinclozolin From Lab UNT.

Figure 20. Reproduction Survival Rate (%) (Termination) By Generation and Group For Chemical Vinclozolin From Lab UNT.Reproduction Survival Rate (%)(day 14)

0 10 20 30 40 50 60 70 80 90 100

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



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

30 40 50 60 70 80 90 100

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1

## DRAFT


# Summary Tables and Figures for Mysid: UNT Vinclozolin Data

39 Figure 21. Total Survival Rate (%) (day 7) By Generation and Group For Chemical Vinclozolin From Lab UNT.

Figure 22. Total Survival Rate (%) (day 14) By Generation and Group For Chemical Vinclozolin From Lab UNT.Total Survival Rate (%)(day 7)

20 30 40 50 60 70 80 90 100

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



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

10 20 30 40 50 60 70 80 90 100

Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb Control 25 ppb 50 ppb 100 ppb 200 ppb 600 ppb



## Group Generation F0 Generation F1