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

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# Intra-Laboratory Analysis Report for Mysid: UNT 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 ........................................................................................................ .. 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 .......................................................... 10 3.2 Comparisons of the F0 and F1 generations ................................................................... 11
3.2.1 Outlier screens ................................................................................................... 12 3.2.2 Normality ........................................................................................................... 12
3.2.3 Transformation of data ...................................................................................... 13 3.2.4 Heterogeneou s variances ................................................................................... 13 3.2.5 Monotonicity Test .............................................................................................. 13 3.2.6 F0 generation vers us F1 generation ................................................................... 13
4. Results .................................................................................................................... ....... 13

Tables

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

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

## .......................................................................................................................... 16 Table 1C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F0 ............................................................................................. . 17

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

iv Table 1D. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F1 ............................................................................................. . 18 Table 1E. Comparisons between Two Genera tions for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane. ................................................................................................. ... 19 Table 2A. Summary of Growth Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F0 1,2,3,4,5 

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

## ........................................................................................................................ 21 Table 2C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F0 ............................................................................................. . 22 Table 2D. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F1 ............................................................................................. . 23 Table 2E. Comparisons between Two Generati ons for Growth Endpoints. Laboratory: UNT, Test Chemical: Lindane. ....................................................................................................... ......... 24 Table 3A. Summary of Survival Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F0 1,2,3 

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

## .......................................................................................................................... 26 Table 3C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F0 ............................................................................................. . 27 Table 3D. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F1 ............................................................................................. . 28 Table 3E. Comparisons between Two Genera tion Survival Endpoints. Laboratory: UNT, Test Chemical: Lindane. ....................................................................................................... ......... 29

Figures
Figure 1. Time to Mature By Generation and Group For Chemical Lindane From Lab UNT. .................................................................................................... 30 Figure 2. Number Offspring (brood 1) By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................................... 30 Figure 3. Number Offspring (brood 2) By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................................... 31 Figure 4. Number Offspring (two broods) By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................................... 31 Figure 5. Days to Brood 1 By Generation and Group For Chemical Lindane From Lab UNT. .................................................................................................... 32

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

v Figure 6. Days to Brood 2 By Generation and Group For Chemical Lindane From Lab UNT. .................................................................................................... 32 Figure 7. Days Between Broods 1 and 2 By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................................... 33 Figure 8. Percent of Females Productivel y Active By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................... 33 Figure 9. Sex Ratio By Generation and Group For Chemical Lindane From Lab UNT. ..... 34 Figure 10. Dry Weight (mg) (day 0) By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................................... 34 Figure 11. Dry Weight (mg) (day 7) By Ge neration and Group For Chemical Lindane From Lab UNT. .................................................................................................... 36 Figure 12. Dry Weight (mg) (day 14) By Ge neration and Group For Chemical Lindane From Lab UNT. .................................................................................................... 36 Figure 13. Dry Weight (mg) (Termination) By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................................... 37 Figure 14. Length (mm) (day 0) By Gene ration and Group For Chemical Lindane From Lab UNT. .................................................................................................... 37 Figure 15. Length (mm) (day 7) By Gene ration and Group For Chemical Lindane From Lab UNT. .................................................................................................... 38 Figure 16. Length (mm) (day 14) By Gene ration and Group For Chemical Lindane From Lab UNT. .................................................................................................... 38 Figure 17. Length (mm) (Termination) By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................................... 40 Figure 18. Reproduction Survival Rate (%) (day 7) By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................... 40 Figure 19. Reproduction Survival Rate (%) (day 14) By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................... 41 Figure 20. Reproduction Survival Rate (% ) (Termination) By Generation and Group For Chemical Lindane From Lab UNT. .................................................... 41 Figure 21. Total Survival Rate (%) (day 7) By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................................... 43 Figure 22. Total Survival Rate (%) (day 14) By Generation and Group For Chemical Lindane From Lab UNT. ...................................................................................... 43

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# Intra-Laboratory Analysis Report for Mysid: UNT 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 UNT 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, 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 basket s. At termination (around Day 29 (F0) and Day 30 (F1) for UNT), mysids from the reproduction basket s are also measured for weights and lengths.

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

2 Intra-laboratory statistical analysis of the UNT 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) 1 

## b. Survival rate for all animals including both th e 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. Survival was not determined due to the absence of appropriate data. Total survival at termination was not determined due to the absence of appropriate data.

1 

## The reproduction termination survival rates were based only on the14 adult reproduction mysids in the seven brood cups starting on day 14, as shown in the survival and reproduction Excel data. Mortality before day 14 was not accounted for in the su rvival rates.

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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. Grow th measurements were de termined for mysids on day 0 before they were assigned to growth and reprod uction baskets, for mysids from the growth baskets at day 7 (6 per replicate tank) and day 14 (all surviving mysids on day 14), and for the surviving reproduction animals at termination. For the weight da ta, there are a total of 9 composites (5 mysids per composite) for the F0 generation and a total of 54 co mposites (5 mysids per composite, 9 composites per concentration group among the six concentration groups) for the F1 generation on day 0. On day 7, there are 3 composite (2 mysids per composite) weight data per replicate and three replicates per concentration group.

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

2.2 Data Issues and Data Handling



# Data were received in Excel 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.

2 

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

## Weight and length results were reported pooled across replicate tanks within control or test concentration groups. Tank identities were not provided in the datasheets.

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

4 Table 0. Data information for UNT Lindane
Data Files and data sheets Endpoints Comments Original - Lindane UNT Excel Spreadsheet.xlsx:

− Time to First Brood Release for F0 and F1 generations

− Time to Second Brood Release for F0 and F1 generations

− Interbrood Duration

− Time to Maturation

− Reproduction Fo

− Reproduction F1

− Sex Ratio Fo

− Sex Ratio F1 Reformatted –

 UNTlindanefecunity.x lsx:

- One datasheet per endpoint per generation

 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

 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 females died before the first or second brood, the 0 brood sizes were recorded in the original data files. Per EPA suggestion, such data are changed as: zero if female is still living, and missing for a brood (1 st 

## or 2 nd 

## brood) if female died without releasing the brood. The total offspring for the first 2 broods are therefore recalculated and displayed in the reformatted data. % of female reproductively active, and sex ratios are reported per tank. The other reproductive endpoints are reported per mating pair.

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

 Sex ratios were defined as % of females over all animals in the original data file. They were redefined as #males/#females in the reformatted data. Original- Lindane UNT Excel Spreadsheet.xlsx:

− Fo,F1 Lengths Day 0

− Fo Lengths Day 7

− Fo Lengths Day 14

− Fo Lengths Termination

− F1 Lengths Day 7

− F1 Lengths Day 14

− F1 Lengths Termination

− Fo,F1 Weight Day 0

− Fo Weight Day 7

− Fo Weight Day 14

− Fo Weight Termination

− F1 Weight Day 7

− F1 Weight Day 14

− F1 Weight Termination Reformatted- UNT lindane Growth.xls One sheet for each generation and each of days 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.

 Lengths are reported per mysid.

 Weights are reported by mysid composites per replicate tank. For F0, day 0 9 composites (5 mysids per composite). For F1, day 0 nine composites per concentration group (, 5 mysids per composite).For day 7 three composites per replicate and two mysids per composite), and per mysid for day 14 and for termination.

 Lengths are measured per mysid at all times, from day 0 to termination.

 Replicate (basket) ID’s are not available, thus we are not able to include a random replicate effect into the two-way ANOVA model (when comparing the two generations in STATCHARRMS).

 No gender information is provided for day 14 and termination. We do not analyze the data by gender. Original- Survival and Reproduction Lindane.xlsx

- F0 Survival and Repro.

- F1 Survival and Repro.  Survival (reproduction) (day 7, day 14 and termination)

 Survival (total) (day 7, day  Reformatted survival data were hand entered using the information from the original files. The original files have survival data from day 0 to day 14.

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5 Data Files and data sheets Endpoints Comments Reformatted- UNT Lindane Survival

- F0Survival

- F1Survival 14)

 Survival (total) at termination is not available 4



## . 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 never be more than 20 mysids in a tank. Sometimes there are more than 20 mysids reported alive on day 7, we treated them as 20.

 

## Sometimes more mysids survived at a later date than at a previous date. We will determine the number based on the information. For example, on day 7, the number of survivals was recorded as 19 but there were 20 survivals on day 14. We will assume survival number on day 7 as 20 instead of 19.

 There are no surv ival records for mysids, that were not assigned to the brood chambers after day 14. The survival for termination is determined using the animals from the brood chambers only. This is reproduction survival.

 If there is a male death, which is assumed if number of animals goesfrom 2 to 1 and then back to 2, one addin 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 the data for the two animals in a breeding pairs 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 statis tical procedures for analyses within the F0 and F1 generations as compared to comparisons between the F0 and F1 generations.

4 

## The survival worksheet in the Lindane Excel spreadsheet is problematic to use for termination survival. The numerator values are inconsistent with survival inform ation in the Survival and Re production spreadsheet. The denominator values are not adjusted for non-treatment relate d 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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# 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 procedur e. In the one-way ANOVA procedure, STATCHARRMS pools the results across test or control group. The one-way ANOVA (parametric or nonparametric forms) was applie d to the one generati on data and there are no random replicate effects. For the comp arison between the F0 and F1 generations STATCHARRMS utilizes a two-way mixed effect s analysis of variance, with replicate tanks within treatment groups treated as random effects. For some endpoints, if the replicate information is not available the ra ndom effect was not included in the model. The objectives of the analyses differ. The an alyses within generations compare the test group results to the control gr oup results. The analyses betw een generations compare the results between generations, eith er 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 AVOVA 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 analyses (two-way ANOVA). The statistical potential outliers are screened values base d on the statistical model applied, and they may not be in fact bi ologically important. The potential outliers from both one-way and two-way ANOVA’s s hould be jointly considered. EPA will determine whether the flagged values should be included in the subs equent 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

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# analyses. The screened values however were enumerated in the STATCHARRMS outputs and will be sent to EPA for review.

 Survival for the reproduction an imals at termination is calc ulated based on the animals assigned to or later added in to the brood chambers. This accounts for lethality only within the seven breedin g pairs from day 14 to termination day. It does not account for lethality among the reproductive mysid prio r to being paired at day 14.

 Total survival at termination is not determined. The F0 survival and F1 survival worksheets in the Lindane UNT Excel spreadsheet are not used in the analysis because they are inconsistent with lethality show n in the Survival and Reproduction Lindane spreadsheet. The number of dead mysids per tank is not consistent with the number of mortalities shown in the Survival and Repr oduction Lindane spreadsheet, which is taken to be authoritative with respect to surviv al. The denominators are about 40 in the F0 survival worksheets and about 30 for the F1 survival worksheets. These values do not adjust for the numbers of growth or reproducti ve mysids that were removed from the test for reasons that were not test compound rela ted (e.g. growth mysids removed on day 7 or day 14 for weight and length determinations or breeding pair males that were removed from test because the breeding pair was shut down after the female died.) Survival and reproduction spreadsheet survival values ar e based only on reproduction mysids.

 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 liv ing the 0 brood sizes s hould be counted as 0 and included in the brood size averages. If the female died before the recorded zero size is generated, the brood size should be count ed as “NA” (or recorded as a missing). Battelle has changed the data entries in th e columns for brood size 1 and brood size 2 in the Excel spreadsheet according to this suggestion. In the reformatted data, the changes are also made to the affected endpoints: total number of offspring in the first two broods, and the number 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 ti mes for the brood was also recorded as “NA” in the original data file a nd they are treated as missing in this analysis. If the brood size is missing in either brood 1 or brood 2, then the sum of the two broods is also treated as missing. If the brood size is missing or zero in eith er brood 1 or brood 2, the number of reproductive days is recorded as “NA”, and thus the number of offspring per female per reproductive day is treated as missing This applies to endpoints: number of offspring in brood 1, number of offspring in brood 2, number of offspring in broods 1 and 2.

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#  For the UNT Lindane weight and length growth data the way that the data are reported limits the analysis procedures that can be used. The lengths and weights are reported by individual mysids or by mysid composite, however replicate tank information is not included. Thus the test group response averag es are pooled across re plicate tanks within test or control groups and effects of random variation due to replicate tank cannot be separated from the residual variation in th e two-way analysis of variance model.

3. STATISTICAL METHODS 

# Statistical analysis procedures used confor m to the statistical analysis procedures specified in the STATCHARRMS package. Statistical analys es 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 univariat e 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 pe r mysid data without fi rst 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 dependi ng on how the data were reporte d. Preliminary tests were first carried out to detect outliers, departure from normality, heterogene ity 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 depending on the outcomes of the preliminary analyses. All the test procedures described here are based on the default auto option programs in STATCHARRMS.

3.1.1 Outlier screens 

# For each generation, outlier scr eens were carried out using Tukey’s outlier rule on the untransformed data. Outliers were assessed from residuals (results of subtracting treatment group means from individual values) to avoid co nfounding outliers and treatment effects. Any response more than 1.5 times the interquart ile 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 th e results of the statistical outlier are to be documented and reported to EPA for review to de termine if the deviations, although statistically screened, are in fact biologically unimportant. EPA will determine whether the flagged values should be included in the subseque nt analyses or should be treated as outliers. The subsets of

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# potential outliers are response-specific. Due to time constraints, the st atistical summaries and analyses in this draft report were carried out in cluding the flagged values. The list of screened values is included in the STATACHARRM S outputs and will be sent to EPA.

3.1.2 Normality 

# Normality was formally assessed in the ANOVA procedure on the untransformed data using the Shapiro-Wilk test (Shapiro and Wilk, 1965). It was assessed from residuals (results of subtracting treatment group means from individual values). 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 tr ansformation. Under the auto option in STATCHARRMS, the analysis procedure is firs t applied to the untransformed data. If the normality assumption is valid, then no transforma tion is carried out. If the normality assumption is invalid, then normality tests are again performe d on the log and square-root transformed data. If the normality assumption is va lid after either transformation, then further procedures are applied to the transformed data. If the normality assumption is invalid for both the untransformed data and the transformed data, then nonparametric pro cedures are applied. Transformation is not necessary for nonparametr ic procedures as any transformation does not change the rank scores for the data wh en nonparametric procedures are used.

3.1.4 Heterogeneous variances



# If the normality assumption was accepted for either the untransformed or the transformed data heterogeneity of variances amon g the concentration groups was assessed in the ANOVA procedure on the untransformed or the tr ansformed data with Levene’s test with absolute residuals (Levene, 1960). Screened outlie rs were included in the Levene’s tests. If the normality assumption was invalid for both the untransformed and the transformed data heterogeneity of variances among the concentrat ion groups was not assessed, as nonparametric procedures were applied.

3.1.5 Monotonicity of response 

# In STATCHARRMS, a monotonicity test usi ng linear and quadratic contrasts under oneway ANOVA was applied to normalized rank statisti cs (to avoid the complications that can arise from non-normal or heterogeneous data). If the linear contrast was signi ficant but the quadratic contrasts was not significan t, then that was taken as evidence of monotonicity.

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10 3.1.6 Comparisons of test chemical to controls 

# Depending on the results of the test for monotonici ty either ordered alternatives or general alternatives analysis procedures were used. Ordered alternatives procedures assume that the response is monotonically increasin g or monotonically decreasing with increasing test chemical concentration. Ordered alternativ es procedures were always carri ed out on the ranks of the data. If there was significant departur e from monotonicity, general a lternatives procedures were carried out.
If the test for departure from normality was not significant for either the untransformed or the transformed data, normal theory parametric methods with homogeneous or heterogeneous variances were used. If the test for depart ure from normality was significant for both the untransformed and the transformed data nonpara metric methods were used. The analysis procedures fall into three categories:

 Ordered alternatives

− nonparametric

 General alternatives

− parametric

 General alternatives

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

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# General alternatives – parametric procedure
If the hypothesis of monotonicity of res ponse was rejected and the normality assumption was not rejected, parametric general alternat ive procedures were used. If the overall concentration effect with the ANOVA was significant, comparisons of test concentrations to the control were conducted using Dunnett’s test with homogeneous within-tre atment variances and using the Tamhane-Dunnett test with hetero geneous within-treatme nt variances.
General alternatives – Nonparametric procedure
If the hypothesis of monotonicity of res ponse was rejected and the normality assumption was rejected for both the untransformed and the transformed data, nonparametric general alternative procedures were used. The Kruskal-Wallis test was applied to test for the overall concentration effect. If the ove rall concentration effect was signi ficant, the comparisons of test concentration to the control were con ducted using a modified Dunn’s test.

Exact Permutation Tests
For nonparametric procedures, if a large pe rcentage of the responses have the same measured value (ties), specifically in the case of very small samples, say less than three per concentration for most concentrations, it is may no t be possible to assess in a meaningful way the true distribution of responses acr oss test concentration groups. One can either use an assumed distribution or treat this situation in the same wa y as data with massive ties. Exact permutation test methodology is an appropria te tool to use in these ca ses. The ANOVA component of STATCHARRMS automatically uses exact methods when deemed appropriate. In the ANOVA procedure, there are two conditi ons under which exact permutati on methods are used: (1) There are no more than three experimental units in any treatment group and non-parametric procedures are found appropriate. (2) There are “massive ties” in the responses, i.e., 25% or more of the experimental units have the same response or 40 % or more have one of only two response values or 65% or more have one of only three respons e values. An exact version of the JonckheereTerpstra test is used for the step-down trend te st and an exact version of the Mann-Whitney test with Bonferroni-Holm adjusted p-values is used in place of Dunn’s test.

3.2 Comparisons of the F0 and F1 generations 

# Mysid data for the two generation multi-concen tration test were analyzed according to a common procedure for each generation as descri bed above. Analyses 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 wa y mixed effects ANOVA model, which included generation, concentration group, an d the interaction of generati on and concentration as fixed effects and replicate (concentra tion, generation) as a random e ffect, allowing unequal variances

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# between the two generations. The residual va riance was treated as homogeneous between generations if Levene’s test accepts a homogeneous varian ces. Otherwise a heterogeneous residual variance is used. For the length and we ight responses replicate tank information was not provided in the dataset. Theref ore variation due to replicate ta nk could not be separated from residual variation within tanks. Thus repeated measures analyses could not be carried out.
Preliminary tests were carried out to dete ct outliers, departures from normality, and heterogeneity of variance. These analyses were independent of those carried out for the analyses within generations and did not necessarily ar rive at the same conclusions. Subsequent comparisons among groups were carried out us ing procedures based on the outcomes of the preliminary 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 compar isons of two generations were based on the two-way mixed effect model in section 3.2.6. Outliers were 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 interquart ile range above the third quartile (75 th 

# percentile) or below the first quartile (25 th 

# percentile) is considered an out lier 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 determin e if the deviations, although statistically significant, are in fact biologically important. EPA will determine whether the flagged values should be included in the subseque nt analyses or should be treated as outliers. The subsets of potential outliers are response specific. Due to time constraints, the subsequent summaries and statistical analyses in this report were carried out including the flagged values. The list of screened values is presented in the STATAC HARRMS output and will be sent to EPA for review.

3.2.2 Normality 

# Normality was formally assessed in th e mixed effects 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 a nd generation as well as group by generation interaction were fixed effects. The random replicate tank-to-tank variance was assumed by STATCHARRMS to be heterogeneous between ge nerations (for the growth measurements, random effects was not assumed du e to the lack of replicate in formation). Residual variance within replicate tanks was assumed to be homogene ous between generations. Screened potential outliers were included in the tests.

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13 3.2.3 Transformation of data 

# There are three options for data transformation in STAT CHARRMS: log-transformation, square root transformation, and arc-sin square-r oot transformation. Unde r the auto option in STATCHARRMS, the analysis procedures are firs t applied to the untransformed data. If the normality assumption is valid, then no transforma tion is necessary. If the normality assumption is invalid, then normality tests are again perfor med 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 homogene ity of variance analysis. If the normality assumption is invalid for both transformations, then nonparametric proc edures 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 ei ther the untransformed or the transformed data, heterogeneity of variances among the ge nerations and the concentration groups was assessed using the ANOVA procedure and Levene’s test with absolute residuals (Levene, 1960), where the residuals were from the model fit described in the normality test (Section 3.2.2). Outliers were included in the test.

3.2.5 Monotonicity Test 

# A test for monotonicity was not carried out as ordered alte rnatives procedures are not available in the two wa y mixed effects ANOVA.

3.2.6 F0 generation versus F1 generation 

# If the normality assumptions held, a parame tric 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 ra nk scores. If the generation main effect or the in teraction of concentration by ge neration effect was significant, Ftests for significant differences between genera tions, within concentration groups (including the control group), the so-called s lices in the ANOVA table, were carried out to identify the concentration groups for which the two ge nerations were significantly different.

4. RESULTS 

# For each endpoint the summary tables presen t the following statistical analysis for the control group and each concentration group for each generation:

 Number of samples

 Mean

 Standard deviation

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#  CV (%)
These summary statistics were calculated across all samples and all baskets for each concentration group and for each generation. Su mmary results for the two generations are presented in Tables 1A and 1B (reproduction en dpoints), Tables 2A and 2B (growth endpoints), Tables 3A and 3B (survival endpoints). These mean s and standard deviations are also displayed, sided-by-side for the two gene rations in Figures 1 through 25. The summaries and figures are based on untransformed data with no screened ou tliers removed.
Statistical analysis was performed according to the statistical met hods described in the methods section using STATCHARRMS. The resu lts of statistical 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 su mmarized in Tables 1E, 2E and 3E.DRAFTSummary Tables and Figures for Mysid: UNT Lindane Data15Table 1A. Summary of Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, 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)Mean13.235 5.941 6.308 12.077 19.294 23.800 4.900 80.952 1.126StandardDev.2.412 2.727 4.423 5.346 2.392 2.251 0.316 8.248 0.311N17 17 13 13 17 10 10 3 3CV (%)18.22 45.89 70.12 44.26 12.4 9.46 6.45 10.19 27.640.032 ppb(Group 2)Mean12.444 6.550 8.421 14.947 18.389 24.643 6.067 90.476 1.773StandardDev.3.854 2.564 5.092 6.794 3.883 2.307 1.870 16.496 0.253N18 20 19 19 18 14 15 3 3CV(%)30.97 39.15 60.46 45.46 21.11 9.36 30.82 18.23 14.280.08 ppb(Group 3)Mean12.167 5.450 8.684 14.263 18.167 23.750 5.824 90.476 1.706StandardDev.1.295 2.585 4.435 5.108 1.295 1.483 1.879 16.496 0.345N18 20 19 19 18 16 17 3 3CV(%)10.64 47.43 51.07 35.81 7.13 6.25 32.26 18.23 20.210.2 ppb(Group 4)Mean12.333 5.222 8.588 13.765 18.389 23.000 5.125 95.238 1.956StandardDev.1.782 1.957 3.124 2.796 1.819 0.679 1.088 8.248 0.758N18 18 17 17 18 14 16 3 3CV(%)14.45 37.48 36.37 20.31 9.89 2.95 21.23 8.66 38.730.5 ppb(Group 5)Mean11.591 6.000 6.667 12.467 17.591 23.400 5.667 100.000 1.568StandardDev.1.790 2.121 3.958 4.658 1.790 1.075 1.723 0.000 1.154N22 21 15 15 22 10 12 3 3CV(%)15.45 35.36 59.37 37.36 10.18 4.59 30.41 0 73.591.25 ppb(Group 6)Mean10.105 8.100 7.286 16.286 16.421 19.800 5.667 95.238 1.228StandardDev.1.197 2.382 5.648 7.868 1.539 1.483 4.179 8.248 0.842N19 20 7 7 19 5 6 3 3CV(%)11.85 29.41 77.53 48.31 9.37 7.49 73.75 8.66 68.581.Summary statistics are calculated across all replicatesand all samples in theentire concentration group.2.Time to mature, % of females productively active, sex ratio are measured per tank (replicate), Number of offspring, day to broods, days between broods aremeasured per mating pair.3.Summaries are based on untransformed datawith no screened outliers removed .DRAFTSummary Tables and Figures for Mysid: UNT Lindane Data16Table 1B. Summary of Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F11,2,3Concentration Statistics Time to MatureNumberOffspring(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)Mean11.357 4.867 8.143 13.286 17.214 22.818 5.545 83.333 0.737Standard Dev.1.447 3.204 4.435 6.592 1.477 2.040 1.214 28.868 0.328N14 15 14 14 14 11 11 3 3CV(%)12.74 65.84 54.47 49.61 8.58 8.94 21.88 34.64 44.470.032 ppb(Group 2)Mean10.000 5.824 7.667 13.467 16.000 21.500 5.286 100.000 0.807Standard Dev.1.138 1.776 3.266 3.642 1.138 1.092 1.069 0.000 0.167N18 17 15 15 18 14 14 3 3CV(%)11.38 30.5 42.6 27.05 7.11 5.08 20.23 0 20.630.08 ppb(Group 3)Mean11.429 3.867 7.000 11.167 17.500 22.100 4.900 93.333 1.500Standard Dev.1.453 1.407 3.438 3.407 1.401 0.738 0.316 11.547 0.954N14 15 12 12 14 10 10 3 3CV(%)12.71 36.4 49.11 30.51 8 3.34 6.45 12.37 63.60.2 ppb(Group 4)Mean11.350 4.800 7.842 12.474 17.350 23.000 5.611 90.476 0.992Standard Dev.1.137 1.399 4.311 4.563 1.137 1.749 1.650 8.248 0.310N20 20 19 19 20 18 18 3 3CV(%)10.02 29.15 54.97 36.58 6.55 7.6 29.4 9.12 31.240.5 ppb(Group 5)Mean10.882 5.294 7.917 12.833 16.882 21.125 4.700 89.683 1.183Standard Dev.1.965 3.312 4.010 5.024 1.965 0.835 0.675 9.014 0.708N17 17 12 12 17 8 10 3 3CV(%)18.05 62.56 50.66 39.15 11.64 3.95 14.36 10.05 59.811.25 ppb(Group 6)Mean10.000 8.375 7.667 17.000 16.091 20.333 5.333 83.333 0.933Standard Dev.1.673 2.825 4.041 4.359 1.700 1.155 0.577 28.868 0.353N11 8 3 3 11 3 3 3 3CV(%)16.73 33.73 52.71 25.64 10.57 5.68 10.83 34.64 37.81.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 Lindane Data

17 Table 1C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, 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 Non-Normal NA Monotonic Decreasing trend for the two highest concentration groups Yes NA

Number Offspring (Brood 1) Normal Homogeneous NonMonotonic NA Yes Group 6

Number Offspring (Brood 2) Non-Normal NA NonMonotonic NA No No

Number Offspring (Two Broods) Non-Normal NA NonMonotonic NA No NA

Days to Brood 1 Non-Normal NA Monotonic Decreasing trend for the two highest concentration groups Yes NA

Days to Brood 2 Normal 8 Heterogeneous NonMonotonic NA Yes Group 6 Days Between Broods 1 and 2 Non-Normal NA NonMonotonic NA No NA
% of Females Productively Active Non-Normal NA NonMonotonic NA No NA

Sex Ratio Normal Homogeneous NonMonotonic NA No NA
1. Shapiro-Wilk test for normality. 2. LeveneTest for Homogeneity of Variances. 3. Monotonicity is tested using linear and quadratic contrasts under one-way ANOVA as applied to normalized rank statistics. 4. Concentration Trends are tested using a step -down Jonckheere-Terpstra test procedure. 5. If normality assumption is valid one way ANOVA is applied to the data and F-test is us ed to determine the concentration effect. If normality assumption is invalid, Kruskal-Wallis test is applied to test for the overall concentration effect. 6. Control is group 1 and test concentration groups are group 2 to 6. 7. If overall concentration of ANOVA is significant and monotonicity is rejected, comparisons of test concentration to the control are conducted:

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

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Summary Tables and Figures for Mysid: UNT Lindane Data

18 Table 1D. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F1

Parameters Normality Test 1 Homogeneous 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 Yes No Number Offspring (Brood 1) Normal Heterogeneous Non-Monotonic NA Yes No Number Offspring (Brood 2) NonNormal NA Non-Monotonic NA No NA Number Offspring

(Two Broods) Normal Homogeneous Non-Monotonic NA No NA
Days to Brood 1 NonNormal NA Non-Monotonic NA Yes No
Days to Brood 2 NonNormal NA Monotonic NA Yes NA Days Between Broods 1 and 2 NonNormal NA Non-Monotonic NA No NA
% 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. LeveneTest for Homogeneity of Variances. 3. Monotonicity is tested using linear and quadratic contrasts under one-way ANOVA as applied to normalized rank statistics. 4. Concentration Trends are tested using a step -down Jonckheere-Terpstra test procedure. 5. If normality assumption is valid one way ANOVA is applied to the data and F-test is us ed to determine the concentration effect. If normality assumption is invalid, Kruskal-Wallis test is applied to test for the overall concentration effect. 6. Control is group 1 and test concentration groups are group 2 to 6. 7. If overall concentration of ANOVA is significant and monotonicity is rejected, comparisons of test concentration to the control are conducted:

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

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Summary Tables and Figures for Mysid: UNT Lindane Data

19 Table 1E. Comparisons between Two Generati ons for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane.

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

Number Offspring (Brood 2) Normal Homogeneous Parametric No No No NA

Number Offspring (Two Broods) Normal Homogeneous Parametric No No No NA
Days to Brood 1 Normal Homogeneous Parametric No Yes No Group 2 Days to Brood 2 Normal 4 Homogeneous Parametric Yes Yes Yes Groups 2 and 5 Days Between Broods 1 and 2 Normal Homogeneous Parametric No No No NA

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

Sex Ratio Normal Homogeneous Parametric No Yes No No 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. Normal when log-transformed.

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Summary Tables and Figures for Mysid: UNT Lindane Data

20 Table 2A. Summary of Growth Endpoints. Laboratory: UNT, Test Chemical: Lindane, 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 0.045 0.119 0.573 0.905 1.638 3.144 4.680 6.311

Standard Dev. 0.009 0.046 0.145 0.149 0.176 0.415 0.444 0.172

N 9 9 35 36 45 18 35 36
CV(%) 19.61 38.1 25.21 16.43 10.76 13.19 9.48 2.73

0.032 ppb (Group 2) Mean 0.108 0.472 1.014 3.200 4.710 6.344

Standard Dev. 0.053 0.128 0.223 0.329 0.483 0.268

N 9 29 42 18 29 43
CV(%) 49.38 27.09 22.04 10.28 10.25 4.23

0.08 ppb (Group 3) Mean 0.094 0.485 1.057 3.272 4.905 6.405

Standard Dev. 0.036 0.166 0.218 0.304 0.589 0.282

N 9 36 41 18 37 41
CV(%) 38.58 34.14 20.61 9.31 12.01 4.4

0.2 ppb (Group 4) Mean 0.072 0.481 1.048 3.383 5.211 6.239

Standard Dev. 0.026 0.138 0.255 0.287 0.634 0.376

N 9 27 41 18 27 41
CV(%) 36.42 28.75 24.37 8.5 12.16 6.03

0.5 ppb (Group 5) Mean 0.149 0.563 0.961 3.744 5.195 6.313

Standard Dev. 0.051 0.161 0.278 0.357 0.579 0.313

N 9 38 25 18 38 24
CV(%) 34.57 28.52 28.92 9.53 11.15 4.95

1.25 ppb (Group 6) Mean 0.222 0.595 1.027 4.039 5.294 6.455

Standard Dev. 0.084 0.178 0.251 0.595 0.436 0.284

N 9 35 11 18 35 11
CV(%) 38.02 29.88 24.46 14.74 8.23 4.4 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. 5. Replicate tank within group information is not provided for the growth endpoints.

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Summary Tables and Figures for Mysid: UNT Lindane Data

21 Table 2B. Summary of Growth Endpoints. Laboratory: UNT, Test Chemical: Lindane, Generation: F1 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 0.022 0.247 0.719 1.030 1.482 3.956 5.641 6.273
Standard

Dev. 0.003 0.041 0.125 0.245 0.101 0.340 0.197 0.303

N 9 9 22 27 45 18 22 26 CV(%) 12.94 16.56 17.42 23.78 6.79 8.59 3.49 4.83

0.032 ppb (Group 2) Mean 0.023 0.234 0.716 0.972 1.482 3.772 5.574 6.110
Standard

Dev. 0.006 0.030 0.127 0.231 0.115 0.232 0.300 0.248

N 9 9 23 39 45 18 23 39 CV(%) 24.96 12.77 17.79 23.78 7.78 6.15 5.39 4.06

0.08 ppb (Group 3) Mean 0.023 0.265 0.633 1.031 1.469 4.074 5.347 6.092
Standard

Dev. 0.007 0.071 0.101 0.200 0.114 0.376 0.257 0.277

N 9 9 19 27 45 17 19 26 CV(%) 29.6 26.87 16.04 19.39 7.79 9.23 4.8 4.55

0.2 ppb (Group 4) Mean 0.017 0.237 0.705 0.995 1.427 4.100 5.463 6.128
Standard

Dev. 0.005 0.040 0.104 0.202 0.116 0.428 0.279 0.266

N 9 9 19 29 45 18 19 29 CV(%) 31.6 16.93 14.82 20.29 8.1 10.45 5.11 4.34

0.5 ppb (Group 5) Mean 0.023 0.256 0.672 0.993 1.467 4.194 5.585 6.352
Standard

Dev. 0.007 0.038 0.089 0.192 0.138 0.335 0.208 0.333

N 9 9 13 21 45 18 13 21 CV(%) 29.99 14.7 13.31 19.29 9.42 7.99 3.72 5.24

1.25 ppb (Group 6) Mean 0.022 0.291 0.618 1.007 1.462 4.150 5.525 6.711
Standard

Dev. 0.006 0.134 0.133 0.131 0.185 0.338 0.238 0.203

N 9 9 8 9 45 18 8 9 CV(%) 25.75 46.16 21.5 13.02 12.65 8.15 4.3 3.02 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. 4. Replicate tank within group information is not provided for the growth endpoints.

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

22 Table 2C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, 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 Non-Monotonic NA Yes Group 6 Dry Weight(mg) (day 14) Normal Homogeneous Non-Monotonic NA Yes No Dry Weight (mg)

(Termination) Non-Normal NA Non-Monotonic NA No NA Length (mm) (day 7) Non-Normal NA Non-Monotonic NA Yes Groups 5 and 6
Length (mm)

(day 14) Non-Normal NA Monotonic Increasing trends for the four highest groups Yes NA
Length (mm) (Termination) Non-Normal NA Non-Monotonic NA No NA
1. Shapiro-Wilk test for normality. 2. LeveneTest for Homogeneity of Variances. 3. Monotonicity is tested using linear and quadratic contrasts under one-way ANOVA as applied to normalized rank statistics. 4. Concentration Trends are tested using a step -down Jonckheere-Terpstra test procedure. 5. If normality assumption is valid one way ANOVA is applied to the data and F-test is us ed to determine the concentration effect. If normality assumption is invalid, Kruskal-Wallis test is applied to test for the overall concentration effect. 6. Control is group 1 and test concentration groups are group 2 to 6. 7. If overall concentration of ANOVA is significant and monotonicity is rejected, comparisons of test concentration to the control are conducted:

a. using Dunnett’s test with homogeneous within-treatment varian ces and using Tamhane-Dunnett testwith heterogeneous withintreatment variances for normal distributed data b. using a modified (nonparametric) Dunn’s test if data are not normally distributed. 8. Normal when square root-transformed.

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

23 Table 2D. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, 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 Homogeneous Non-Monotonic NA No NA Dry Weight (mg) (day 7) Non-Normal NA Non-Monotonic NA No NA Dry Weight (mg) (day 14) Normal Homogeneous Non-Monotonic NA No NA Dry Weight (mg) (Termination) Non-Normal NA Non-Monotonic NA No NA

Length (mm)

(day 0) Non-Normal NA Non-Monotonic NA No NA

Length (mm)

(day 7) Normal Homogeneous Monotonic Increasing concentration trends for the two highest

concentration

groups. Yes NA

Length (mm)

(day 14) Normal Homogeneous Non-Monotonic NA Yes Group 3 Length (mm) (Termination) Normal Homogeneous Non-Monotonic NA Yes Group 6
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 Lindane Data

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

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 7) NonNormal NA Nonparametric Yes No Yes Group 6 Dry Weight (mg) (day 14) Normal Homogeneous Parametric Yes Yes Yes Groups 1, 2, 3, 4 and 5 Dry Weight (mg) (Termination) NonNormal NA Nonparametric No No No NA
Length (mm)

(day 7) Normal Homogeneous Parametric Yes Yes Yes Groups 1, 2, 3, 4 and 5 Length (mm)

(day 14) NonNormal NA Nonparametric Yes No Yes Groups 1, 2 and 4 Length (mm) (Termination) NonNormal NA Nonparametric Yes No Yes Groups 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 Lindane Data

25 Table 3A. Summary of Survival Endpoints. Laboratory: UNT, 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)

Control (Group 1) Mean 96.667 78.333 77.302 96.667 80.392

Standard

Dev. 2.887 2.887 10.060 3.819 11.135

N 3 3 3 3 3 CV(%) 2.99 3.69 13.01 3.95 13.85

0.032 ppb (Group 2) Mean 98.333 86.667 83.056 96.667 79.412

Standard

Dev. 2.887 2.887 6.989 3.819 10.605

N 3 3 3 3 3 CV(%) 2.94 3.33 8.41 3.95 13.35

0.08 ppb (Group 3) Mean 98.333 88.333 71.528 99.167 90.196

Standard

Dev. 2.887 7.638 21.700 1.443 6.123

N 3 3 3 3 3 CV(%) 2.94 8.65 30.34 1.46 6.79

0.2 ppb (Group 4) Mean 100.000 95.000 67.703 99.167 86.275

Standard

Dev. 0.000 8.660 10.994 1.443 7.402

N 3 3 3 3 3 CV(%) 0 9.12 16.24 1.46 8.58

0.5 ppb (Group 5) Mean 95.000 80.000 64.128 97.500 86.275

Standard

Dev. 0.000 8.660 7.606 0.000 6.123

N 3 3 3 3 3 CV(%) 0 10.83 11.86 0 7.1

1.25 ppb (Group 6) Mean 100.000 80.000 46.296 98.333 84.314

Standard

Dev. 0.000 18.028 9.601 1.443 8.985

N 3 3 3 3 3 CV(%) 0 22.53 20.74 1.47 10.66
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 Lindane Data

26 Table 3B. Summary of Survival Endpoints. Laboratory: UNT, 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)

Control (Group 1) Mean 97.778 84.444 66.667 98.889 86.111

Standard

Dev. 3.849 13.878 14.434 1.925 6.365

N 3 3 3 3 3 CV(%) 3.94 16.43 21.65 1.95 7.39

0.032 ppb (Group 2) Mean 100.000 95.556 82.165 98.889 91.667

Standard

Dev. 0.000 3.849 16.109 1.925 4.167

N 3 3 3 3 3 CV(%) 0 4.03 19.61 1.95 4.55

0.08 ppb (Group 3) Mean 93.333 88.889 75.354 85.556 75.611

Standard

Dev. 11.547 7.698 7.044 17.105 18.097

N 3 3 3 3 3 CV(%) 12.37 8.66 9.35 19.99 23.93

0.2 ppb (Group 4) Mean 100.000 93.333 70.476 100.000 93.056

Standard

Dev. 0.000 11.547 10.034 0.000 8.674

N 3 3 3 3 3 CV(%) 0 12.37 14.24 0 9.32

0.5 ppb (Group 5) Mean 86.667 82.222 64.545 82.222 70.833

Standard

Dev. 11.547 10.184 18.350 3.849 11.024

N 3 3 3 3 3 CV(%) 13.32 12.39 28.43 4.68 15.56

1.25 ppb (Group 6) Mean 68.889 46.667 58.333 67.778 44.444

Standard

Dev. 13.878 24.037 14.434 8.389 15.775

N 3 3 3 3 3 CV(%) 20.15 51.51 24.74 12.38 35.49
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 Lindane Data

27 Table 3C. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, 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) Non-Normal NA Non-Monotonic NA No NA
Reproduction Survival Rate (%) (day 14) Normal Homogeneous Non-Monotonic NA No NA
Reproduction Survival Rate (%) (Termination) Normal Homogeneous Monotonic Decreasing trend for the highest concentration group No NA

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

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

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 Lindane Data

28 Table 3D. Statistical Analysis Results for Reproduction Endpoints. Laboratory: UNT, Test Chemical: Lindane, 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) Normal Homogeneous Non-Monotonic NA Yes Group 6
Reproduction Survival Rate (%) (day 14) Normal Homogeneous Non-Monotonic NA Yes Group 6
Reproduction Survival Rate (%) (Termination) Normal Homogeneous Non-Monotonic NA No NA

Total Survival Rate (%) (day 7) Non-Normal NA Monotonic Decreasing trend for the highest concentration group Yes NA

Total Survival Rate (%) (day 14) Normal Homogeneous Non-Monotonic NA Yes Group 6

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 Lindane Data

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

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 Yes Group 6

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

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

Total Survival Rate (%) (day 7) NonNormal NA Nonparametric No No No NA

Total Survival Rate (%) (day 14) Normal Homogeneous Parametric Yes Yes Yes Group 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.

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

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

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

9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1Number Offspring (brood 1)

3 4 5 6 7 8 9 10

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

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

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

3 4 5 6 7 8 9 10 11 12

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1Number Offspring (two broods)

8 10 12 14 16 18 20 22

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

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

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

15 16 17 18 19 20 21 22

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1Days to Brood 2

19 20 21 22 23 24 25 26 27

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

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

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

3 4 5 6 7 8

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1% of Females Productively Active

60 65 70 75 80 85 90 95 100

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

34 Figure 9. Sex Ratio By Generation and Group For Chemical Lindane From Lab UNT.

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

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1Dry Weight (mg)(day 0)

0.01 0.02 0.03 0.04 0.05

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

35

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

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

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

0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28 0.30 0.32 0.34

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1Dry Weight (mg)(day 14)

0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

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

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

0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1Length (mm)(day 0)

1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

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

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

2.5 3.0 3.5 4.0 4.5 5.0

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1Length (mm)(day 14)

4.0 4.5 5.0 5.5 6.0

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

39

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

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

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

5.8 6.0 6.2 6.4 6.6 6.8 7.0

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1Reproduction Survival Rate (%)(day 7)

60 70 80 90 100

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

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

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

30 40 50 60 70 80 90 100

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1Reproduction Survival Rate (%)(Termination)

30 40 50 60 70 80 90 100

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

42

# DRAFT
Summary Tables and Figures for Mysid: UNT Lindane Data

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

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

60 65 70 75 80 85 90 95 100

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1Total Survival Rate (%)(day 14)

30 40 50 60 70 80 90 100

Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb Control 0.032 ppb 0.08 ppb 0.2 ppb 0.5 ppb 1.25 ppb


## Group Generation 0 Generation 1