### PNWD-3960



### Evaluation ofProposed EcotoxicologicalTesting GuidelinesUsing Mysids to Determine Endocrine Disruptive Effects from Chemical Exposure to Prochloraz, Flutamide, Ketoconazole, and 4-tert-Octylphenol



### RoyK. Kropp Valerie 1. 

### Cullinan Battelle, Pacific Northwest Division MarineResearch Operations 1529 West SequimBayRoad Sequim, Washington 98382

Prepared For RosemaryHall Task OrderProject Officer U.S. Environmental Protection Agency Endocrine Disruptor ScreeningProgram Washington, D.C

July 10, 2008

### Quality Assurance Statement

Thisreport, "Evaluation ofProposed Ecotoxicological Testing Guidelines Using Mysidsto Determine Endocrine Disruptive Effects from Chemical Exposure to Prochloraz, Flutamide, Ketoconazole,and4-tert-Octylphenol"has beenreviewed by Quality Assurance. The following audit was conducted.

Type ofAudit Dates Conducted Date Reported to Date Reportedto Battelle Program Battelle Facility Manager Management AuditofData QuaJity, May 11,25,26,29 & June 27, 2008 June 27, 2008 Summary report June 2, 13, 2008 , 

### The final reporthas been reviewedfor accuracy.

## ~U(1(OI 

## l-OOf. Date

.. ..

Task Order7 SummaryReport 1

### Signature Page

Submitted by: Battelle, Pacific NOltbwest Division Marine Research Operations 1529 West Sequim Bay Road Sequim, Washington 98382

Prepared by:

Battelle, Pacific Northwest Division Marine Research Operations



# 7/1 0

# /08
Valerie 

### 1. Cullinan, Ph.D. Battelle, Pacific Northwest Division Marine Research Operations

Task Order 7 Summary Report 11

## Contents

Quality Assurance Statement

.i Signature Page ii 1.0 Introduction 1 2.0 Quality Assurance 2 3.0 Primary Modifications ofMethods Described in the Guideline 2 3.1 Self-Starting Siphon 3 3.2 FeedingRegime 3 3.3 Initial TestPopulation 5 3.4 Mysid Pairing 5 3.5 Endpoint and Statistical Clarification 5 4.0 ChemicalExposure Characterization 6 5.0 Dose-Response and Endpoint Evaluation 10 Statistical Methods 10 6.0 Results 11 6.1 BlockEffect 11 6.2 Sensitivity and Responsiveness 14 6.3 Statistically SignificantEffects 19 6.4 Power Analysis 30 7.0 Discussion 31 7.1 Summary ofIndividual Studies 31 7.2 Summary ofEndpoint Evaluations and Protocol Procedures 33 8.0 Recommendations 34 8.1 Study Design and Statistical Guidance 34 8.2 Self-Statting Siphon 34 8.3 Feeding 34 8.4 Initial TestPopulation Size 35 8.5 Endpoint Clarification 35 9.0 References 35 Appendix A A.1

Task Order 7 Summary Repolt III

## Tables

I. Nominal Dose Concentrations, Mean Exposure Dose, Coefficient ofVariation, and Percentage ofNominal Dose for Each Dose Level Tested During Task Order 7 7 2. UpperTriangle Spearman Correlation Matrix ofFa-Generation Non-vehicle Control Mean Response 12 3. Upper Triangle Spearman Correlation Matrix ofF,-GenerationNon-vehicle Control Mean Response 12 4. Spearman Correlation Matrix ofF 

### a - and F1-Generation Non-vehicle Control Mean Response 13 5. on-vehicle Control Mean Response and Coefficient ofVariation Across Testing Periods (Chemicals) by Generation 15 6. Signal-to-Noise Ratio by Chemical and Generation 17 7. Percentage ofincrease (+) or Decrease (-) ofResponse ofMysids Exposed to Concentrations ofProchlorazRelative to the Control Response 21 8. Analysis ofVariance for the Natural-Logarithm-Transformed Fa-Generation 7-Day Weight for Prochloraz 22 9. Percentage ofincrease (+) or Decrease (-) ofResponse ofMysids Exposed to Concentrations ofFlutamide Relative to the Control Response 23 10. Analysis ofVariance for the Natural-Logarithm-Transformed Fa-Generation 7-Day Weight for Flutamide 24 11. Percentage ofincrease (+) or Decrease (-) ofResponse ofMysids Exposed to Concentrations ofKetoconazole Relative to the Control Response 26 12. Analysis ofVariancefor the Natural-Logarithm-Transformed Fa-Generation 7-Day Weight for Ketoconazole 27 13. Percentage ofincrease (+) or Decrease (-) in Response ofMysids Exposed to Concentrations of4-tert-Octylphenol Relative to the Control Response 28 14. Analysis ofVariance for the Natural-Logarithm-Transformed Fa-Generation Total Number of Young for 4-tert-Octylpheno1. 29 15. Power with the Achieved Maximum Percentage ofDifference, Sigma, and Number of Replicates 30 16. Sample Size Required with Achieved Maximum Percentage ofDifference, Sigma, and 80% Power 30 17. Maximum Percentage ofDifference from the Observed Control Response (NaturalLogarithm-Transformed Data) 31

Task Order 7 Summary Report IV

## Figures

1. Mean Number ofdays to first and second broods produced by Control-treatmentFomysids during each TaskOrder 7 study 4 2. Mean Number ofyoung produced by Control-treatmentFo-generation mysids in the first and second broods during each Task Order 7 study



### .4 

### 3. Nominal and measured concentrations ofprochloraz during the mysid definitive study 8 4. Nominal and measured concentrations offlutamide during the mysid definitive study 8 5. Nominal and measured concentrations ofketoconazole during the mysid definitive study 9 6. Nominal and measured concentrations of

### 4-tert-octylphenol during the mysid definitive study 9 7. Output from the evaluation ofthe blockeffect based onthe Kruskal-Wallis test on four test exposures 14 8. Power ofa one-sided independent-sample t-test as a function ofthe percentage ofdifference detected between the test and reference means with three replicates pertreatment (ex = 0.05) 16 9. Fo - and F[-generation 14-day female length (top) and weight (bottom) as a function ofthe mean concentration of

### 4-tert-octylphenol (Ilg/l) with signal-to-noise ratios of-2.17 and 1.54 for length and -2.92 and 0.18 for weight, respectively 18 10. Fo - and F}-generation 7-day weight as a function ofthe mean concentration ofketoconazole with signal-to-noise ratios of6.10 and 1.29, respectively 18 11. Variables with an absolute signal-to-noise ratio >2, suggestingresponsiveness 19 12. Inconsistentblockeffect onthe Fo-generation 7-day weight for each concentration of prochloraz 22 

### 13. Inconsistentblock effect on the Fo-generation 7-day weight for each concentration of flutamide 24 14. Inconsistentblockeffect onthe Fo-generation 7-day weight for each concentration of ketoconazole 27 15. Inconsistent blockeffect on the Fo-generation total number ofyoung for each concentration of

### 4-tert-octylphenol. 29

TaskOrder7 Summary Report 

### v

## 1.0 Introduction
TheU.S. Environmental Protection Agency (U.S. EPA) has established the Endocrine Disruptor Screening Program (EDSP) to assess the potential ofchemical substances to affect endocrine processes in humans and wildlife. Battelle has been a principal contractor in support ofthis program and has provided ecotoxicological testing services including chemical, analytical, statistical, and quality assurance/quality control (QAlQc) support to assist the U.S. EPA in developing, standardizing, and validating certain ecotoxicological tests for identifying and characterizing endocrine effects through exposure to pesticides, industrial chemicals, and environmental contaminants. The studies conducted under contractual Task Order 7 represent an initial evaluation ofthe testing procedures and methods proposed by the U.S. EPA to the Organisation for Economic Co-Operation and Development (OECD) in the draft guideline for a two-generation mysid test to evaluate suspected endocrine-disrupting compounds (EDC). The draft guideline considers recommendations from a detailed review paper prepared for U.S. EPA and OECD, whichwas published in draft form in 2005 and finalized in 2006 (OECD 2006). Interest in the potential effects ofEDCs began to develop within the OECD in the late 1970s and progressed to the development ofa five-level Conceptual Framework for Endocrine DisruptersTesting and Assessment in 2002 (Gourmelon and Ahtiainen 2007). The five levels represent steps ofincreasing biological complexity and are not intended to function as the tiers ofa multi-step testing approach. LevelS represents the highest level ofcomplexity and incorporatespaI1ial and full life-cycle testing in several animal groups, including invertebrates (Gourmelon and Ahtiainen 2007). One ofthe goals ofthe OECD testing program is to develop guidelines that will protect sensitive populations and notjustserve to detect potential EDCs (Gourmelon and Ahtiainen 2007). The mysid two-generation test is one ofseveral such tests being developed in concert with the OECD program. The demonstration study (Task Order 7) is one ofthe first steps in the evaluation ofthis proposed guideline for use in examining adverse responses in mysids,

### Americamysis bahia, exposed to ve11ebraterelevant EDCs. Four chemicals with a range ofendocrine-disrupting modes ofaction were used in the overall study and tested in this sequence: prochloraz, flutamide, ketoconazole, and

### 4-tert-octylphenol. These four chemicals were evaluated during independent studies conducted by ABC Laboratories (ABC), Columbia, Missouri. Definitive testing was conducted from June 2007 through November 2007, during which observations ofsurvival, development, sexual maturation, reproduction, and growth were made in the Fo - and Ft-mysid generations when possible. Specific measurements were made ofdevelopment, growth, gender ratio, and reproduction (i.e., percentage ofactivelyreproducing females, number ofyoung per female in first brood, number ofyoung per female in second brood, time to first- and second-brood releases, interbrood duration, replicate mean number ofyoung produced per female in each brood, and number oftotal youngper female perreplicate), which should be indicative offunctional endocrine regulation for this species. An individual report was prepared for each study. The data generated were used in the individual study reports to statistically determine the no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC) values for the individual biological endpoints. Additionally, the mean responses for the experimental treatments were compared with those ofthe control or vehicle-control treatments. The purpose ofthis report is to summarize the results ofeach individual study (Aufderheide 2008a, 2008b, Hicks 2008a, 2008b), examine the variability ofthe chemical dosing, discuss the main deviations from the proposed guideline, and evaluate the various endpoints for sensitivity and power. 

### Italso will review some new endpoints that have recently been suggested as possible tools in EDC-related mysid research and evaluate whether or not they shouldbe included in the proposed guideline. Finally, some recommendations for modifying the guideline are proposed.

TaskOrder 7 Summary Report

## 2.0 Quality Assurance
The ABC Laboratories Quality Assurance (QA) persormel monitored the conduct ofthe studies as outlined in the QAPP and their standard operating procedures. The results ofthe audits (technical systems audits and audits ofdata quality) were reported to the ABC Study Director and Management as required. Responses to any audit findings were provided by ABC study personnel and were reviewed by QA. Completed audit reports were provided to the Battelle QA for this task order (Windward Company). The auditreports were reviewed for data and study quality issues and were summarized in monthly reports to the U.S. EPA. No data quality issues were identified that would have a significant impact to the conduct, reporting, or conclusions ofthe ABC studies. Each study report details any amendments and/or deviations to the study protocol, including assessing the potential effects on the study. The Quality Assurance Project Plan (QAPP) dated February 2007 was amended once to change the Battelle program manager. Differences from the QAPP are:
• Section 4.3, ABC personnel: Chris Hughes did not serve as StudyDirectorfor one chemical and

transferred from the technical staffto the QAunit within ABC Laboratories.
• Section 7.1: Deviations to assay acceptance criteria are described in detail within each ABC

Laboratories study report.
• Section 9.3.1: The individual ABC study reports were provided to the U.S. EPAas they were

finalized and, therefore, were not included as an appendix to the summary report.
• Section 9.3.1: The revised "Transferable Mysid Two-Generation Test Protocol" was not included

as an appendix to the summaryreport, becauserecommendations were made that could affect revisions to the test protocol. An audit ofdata quality was conducted by Battelle QA on this summary report and has been reported as outlined in the QAPP (see the QA statement).


## 3.0 Primary Modifications ofMethods Described in the Guideline
TheABC protocols for all four studies differed from the proposed guidelines in four ways. The mysids for each study were fed withArtemia nauplii hatched from cysts that were not obtained from a reference source (sensu Leger and Sorgeloos 1984), butwere from areliable source that hasbeen used successfully by ABC in many previous studies. Retention baskets used during the testing were constructed from nylon mesh having a slightly larger mesh opening, 355**~m,** than the 210- to**330-~m** opening stipulated in the guideline. Mysid dry weights were measured to the nearest 1**~g** instead ofthe nearest 0.1**~g.** Thedraft guideline specifies that females be paired with the largest suspected males. However, ABC used a dissecting microscope to examine all mysids, except for ovigerous females, to confirm gender and maturity before pairing. None ofthese modifications is likely to have had an adverse effect on the studies and they are not discussed further. Two major adjustments, use ofa self-starting siphon and the feeding regime, were made after the conduct ofthe prochloraz study to improve the studies and are described below. Two other difficulties and subsequent adjustments that occurred during the testing are also discussed in this section.

Task Order 7 SummaryReport 2

### 3.1 Self-Starting Siphon
The first modification was prompted by the observations made during the prochloraz study that many mysids were becoming impinged during the periodic draw-down ofwater by the inverted U-shaped, selfstaliing siphon pumps. Duringthe flutamide definitive study, the use ofthis siphon was discontinued when the Foadults were paired. For the later two studies, ketoconazole and 4-teli-octylphenol, the selfstarting siphon was discontinued altogether and the tanks were equipped with overflow drains. This modification not only reduced mysid loss from impingement, but also probably reduced loss ofArtemia from the exposure system. Water quality during the ketoconazole and 4-tert-octylphenol studies met guideline-specified criteria even though the self-starting siphon was not used. 

### 3.2 Feeding Regime
Results from the prochloraz study suggested that the feeding rations specified in the draft guidelines were not sufficient. Datafrom the control treatment (i.e., no interference from a test chemical) demonstrated that the time to first brood was longer (-18 days) than previously observed during testing at ABC (-13 to 15 days). Also, the number ofyoung produced in the second brood (5.2 perfemale) was the same as that produced in the first brood (5.1 per female), which was contrary to the usual situation observed during testing at ABC in which the second brood was much largerthan the first. Additionally, generally poor reproduction among all treatments by day 27 suggested that feeding was inadequate. After day 28, the feeding ration was increased to 1,350 nauplii per brood cup by increasing the number offeedings from two to three. The feeding regime for the F I mysids in retention baskets was increased similarly. During the study, it was observed that very fewArtemia were observed at any time otherthan when the mysids in retention baskets werejustfed. Itwas also noticed that the brine shrimp nauplii were observed outside ofthe mesh baskets, suggestingthatthey may be pulled through the mesh during the draw-down procedures. These observations, coupled with the apparent loss ofArtemia nauplii via the self-starting pump operations, led to the hypothesis that the mysids were undernourished and that the recommended feeding regime was inadequate. For the flutamide definitive study, the feeding regime was increased by about 50% more than the draft guideline specifications. The ration adjustment did not appear to be successful, even though the self-starting siphon was discontinued after pairing. The time to release ofthe first brood in the control treatment was longer (-23 days) than that observed during the prochloraz study. The number ofyoung produced by both control-treatment broods (-2.8 perfemale) was very small. Only 67% ofcontroltreatment produced at least one brood, which was less than the guideline specification that 70% produce one brood. The poor reproduction during the Foexposures prevented testing the F I generationfor prochloraz and flutamide.
These observations, in addition to previous experience at ABC, led to another modification ofthe feeding strategies for the ketoconazole and 4-tert-octylphenol studies. For these two studies, mysids were fed Artemia nauplii adlibitum with three feedings per day. Fomysids were fed an average ofabout 6,500 nauplii per day during the ketoconazole study and about 5,200 nauplii per day during the 4-tertoctylphenol study. FJ mysids received a slightly larger ration for each study. Additionally the brine shrimp used each day were enhanced with a mixture ofthree commercially available supplements: Lifeline Natural Health Enrichment HUFA, AlgaMac-3000, and AlgaMac-Enhance.
The effects ofthese feeding regime alterations were examined statistically by comparing the control responses for time to first and second brood, number ofyoung for each brood, 7- and 14-day lengths, and dry weight to three food rations. Three food rations were evaluated: those used for prochloraz, flutamide, and ketoconazole/4-tert-octylphenol. A Kruskal-Wallis test showed thatthe flutamide feeding ration had the longesttime to first and second broods (Figure 1) and the fewest young for the first and second broods (Figure 2); conversely the diet for ketoconazole/4-tert-octylphenoJ had the shortest time to first brood and the mostyoung for each brood (degrees offreedom [dfj = 

## 2for each comparison;p = 

## 0.009, 0,009, 0,011,

Task Order 7 Summary Report 3-- 

### -- - - - - - 

# • •

- -------- - - - ---

----- - --- ---

- ------ - --- --

--- -- - - -- --

-- - - --

---- -- 

### - -- comparison;p = 0.009,0.009,0.011, and 0.037 for each respective comparison). (Note that the analyses were done with the control data for the ketoconazole/4-telt-octylphenol studies combined as a single feeding regime, but the figures show the data for each individual study).

35

- --- 

## a--

-- --­ 30

25

20 

### ,., c 

### '" 

# • 15 

# • •

­
10

5-
----- - - - - -- ­
0 Prochloraz Flutanide Ketoconazole 4-tert-Octylphenol 

# I. Day of First Brood. Day of Second Brood

# I Figure 1. Mean Number ofdays to first and second broods producedby Control-treatmentF0 

### mysids during each Task Order7 study.

12

10

- OJ 8 C :J 

# • 

# •

0 

# - >- 0 6
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### - ------- 

### ­ Q; .c E 

# •

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# • 2-

- -- - ------ ----- ­

0 Prochloraz Flutanide Ketoconazole 4-lert-Oclylphenol

,.Tolal Young First Brood

_ Total Young Second Brood

## I Figure 2. Mean Number ofyoungproduced by Control-treatmentFo-generation mysids in the first and second broodsduring each Task Order7 study.
An analysis ofvariance (ANOYA) with a generalized linear model was used to examine the main effects ofbrood (1 versus 2), feeding regime, and their interaction on the number ofyoung producedperbrood. Thehypotheses tested were that neither main effect influenced the number ofyoung perbrood. The first step in the analyses examined the potential interaction between the two main effects, specifically asking whether there 

## was adifferent

## brood effect

## undereachfeedingregime. 

## This interactionwas not 

## significant

Task Order 7 Summary Report 4(F = 0.39, df= 2, p = 0.680). Thus, the analysis proceeded to examine each main effect. There was no significant effect ofbrood number (l or 2) on brood size (F = 0.39, df= l,p = 0.538). However, there was a significant effect offeeding regime on the number ofyoungperbrood (F = 16.48, df= 2, 

### p < 0.001), with the regime used for the ketoconazole/4-tert-octyphenol studies producing more young than the other two diets. Another set ofKruskal-Wallis analyses was used to evaluate the effects ofdiet on mysid growth. There were no significant differences among diets in mysid length measured at day 7 (H = 3.12, overall df= 12, P= 0.210, adjusted for ties), but there was a significant difference in dry weightmeasured on day 7 (H = 6.38, overall df= 

### 12,p = 0.041). The weights ofmysids measured during the flutamide study (median weight = 0.4570 mg) were greater than those for the other studies (median weights 0.1960 to 0.2035 mg). There is no obvious reason why the mysids in the flutamide study were twice the weight of those in the other studies at day 7. At day 14, the male and female mysids in the prochloraz study were significantly smaller than those in the other three studies (males: H = 5.80, overall df= 

### 10,p = 0.055, median length = 4.2 mm versus**~5.1** mm; females: H = 6.09, overall df= 11, p = 0.048 adjusted for ties, median length = 4.4 mmversus 5.0 mm). Because mysid 14-day weights were not separated by gender for prochloraz, only the latter two feeding regimes (flutamide, ketoconazole/4-tert-octylphenol) were compared. There was no significant difference in male weights between the two groups (H = 0.15, overall df= 7,p = 0.699, median weights = 0.597 and 0.797 mg, respectively). However, females in the flutamide feeding regime were significantly smaller at day 14 that those in the ketoconazole/4-tertoctylphenol feeding regime (H = 4.00, overall df= 8,p = 0.046, median weights = 0.576 and 0.808 mg, respectively). The female mysids in the flutamide study grew very little from day 7 to day 14; whereas the females in the latter two studies approximately quadrupled in weight during that timeframe. 

### 3.3 Initial 

### TestPopulation Consistent establishment ofbrood pairs often was difficult. During the ketoconazole and 4-tertoctylphenol studies, enough F 1males or ovigerous females were not always available in the reproduction retention chamber to obtain the desired seven brood pairs perreplicate. Therefore, it was necessary to use mature F 1mysids from the growthretention baskets to complete the pairing. However, there were often not enough young produced by the Fo-mysid brood pairs to allow the growth retention baskets and reproduction retention baskets for a replicate to be initiated on the same day. Therefore, establishing seven Ft-mysid brood pairs was difficult when not enough 12- to 16-day-old mysids were available for pairing. Some statistical results obtained during the individual chemical studies may be equivocal because ofthe small initial population size. For example, during the ketoconazole and 4-tert-octylphenol studies, several treatments had fewer than three replicates available for evaluation and some ofthe replicates that were available contained as few as two individuals. 

### 3.4 Mysid Pairing During the ketoconazole and 4-tert-octylphenol studies, all pairing was completed on the same day. However, because ofthe low numbers ofmysids available, as described above, the FImysids comprising the pairs were 12 to 14 days ofage at the time ofpairing, depending on the replicate. Observations began on these mysid pairs the day afterpairing, which was when the mysids were from 13 to 15 days old. Itwas necessary to establish a single starting day to track all FI-brood pairs and "Study Day 13" was selected for both studies. 

### 3.5 Endpointand 

### StatisticalClarification
Some endpoint determination procedures in the guideline were not clear. Forexample, dead young were observed in the control-, vehicle-control,

### and some test-substance treatments for the ketoconazole

## and

Task Order 7 Summary Report 54-tert-octylphenol studies; however, this occurrence did not appear to berelated to chemical dose. The draft guidance document does not describe how the laboratory should evaluate the occurrence ofdead young or how dead young should be accounted for in the statistical analyses ofthe number ofyoung per brood. Forthe ketoconazole and 4-tert-octylphenol studies, the developmental stage ofthe dead young was determined and the dead young were included in the counts ofthe number ofyoung per brood.
Thedry weight endpoint could use clarification. The proposed guideline states simplythat "At weekly intervals (Day 7 and Day 14) animals from the growth baskets are subsampled for growth measurements (length and dry weight)." During the studies conducted for Task Order 7, this was interpreted to allow for separate weight measurement ofmales and females. The intent with this approach was to reduce the potential for increased variability that might simply be attributable to gender-related differences in body size. It was thought that the increased variability that wasn'tnecessarily testrelated might compromise comparisons among treatments. To permit the analyses by gender, immature individuals were necessarily excluded, although their weights were recorded and presented in the results tables.
Statistical guidance providedbythe proposed guidelines is minimal, essentially only describing use of ANOVA or Kruskal-Wallis tests and recommending that the laboratory include a statistician in the process. The lackofguidance allowed for some initial confusion over whether or not to use one-tailed or two-tailed tests and whether the treatmentresponse should be compared with the dilution-water or vehicle controls for tests that employed both.


## 4.0 Chemical Exposure Characterization During each ofthe four studies, the chemical concentration was analyzed at least weekly (see Aufderheide 2008a, 2008b, Hicks 2008a, 2008b for specific sampling dates). The mean concentration during the exposure period was determined for each dose level and used as the reference by whichto evaluate the biological responses (Table 1). The coefficient ofvariation (CV) also was determined for each dose measured during the exposure to evaluate how well each dose level was maintained (Table 1). Figures 3 through 6 illustrate nominal and measured concentrations ofprochloraz, flutamide, ketoconazole, and 4-tert-octylphenol during the mysid defmitive study.
The concentrationpatterns shown for each ofthe four chemicals varied considerably. Themean prochloraz and flutamide concentrationsfor each dose level were relatively close to the nominal concentrations, ranging from 88% to 97% ofthe nominal concentrations (Table I). However, the two differed considerably in the within-study variability. Prochloraz concentrations at each dose level were relatively variable throughout the study, with CVs ranging from 19% to 26% (Table I). Conversely, flutamide concentrations at each dose level were maintained fairly constantly during the study, with CVs of6% and 7% (Table 1). Ketoconazole and4-tert-octylphenol, the two chemicalsthat have low solubility in water, had measured concentrations that were muchless than the nominal concentrations, ranging from 64% to 69% and 66% to 82%, respectively. Within-study variation was moderate for ketoconazole, with CVsranging from 13% to 25%. Lower doses appeared somewhat more variable than higher doses. Concentrations of4-tert-octylphenol at the two lowest dose levels varied considerably, with CVs of52% and 28%, whereas concentrations ofthe three highest doses varied relatively little, with CVsranging from 8% to 13%.
Prochloraz concentrations within each dose level varied during the study suchthat the highest measured concentrationwithin a dose level was higherthan the lowest measured concentration ofthe next highest dose level (Figure 3; Table 2 in prochloraz study report). For example, the highest concentration measured for dose level 4 (76.0 Ilg/L) was greater than the two lowest concentrations measured for dose

Task Order7 Summary Report 6level 5 (70.7 flg/L and 72.2 flg/L) and about the same as the third lowest (78.3 flg/L). Thus, there was overlap ofeach dose level at some time during the study. Dose overlap did not occur during the flutamide and ketoconazole studies and occurred only between the two lowest dose levels during the 

### 4-tertoctylphenol study.


### Table 1. 

### Nominal Dose Concentrations, Mean Exposure Dose, Coefficient ofVariation, and Percentage ofNominal Dose for Each Dose Level Tested During Task Order 7 Nominal Units Levell 

### Level 2 Level 3 Level 4 LevelS Prochloraz flg/L 13.0 21.6 36.0 60.0 100 Flutamide mgJ'L 0.125 0.250 0.500 1.00 2.00 Ketoconazole JJ.g/L 3.30 6.50 13.0 25.0 50.0 

### 4-tert-Octylphenol ).1g/L 3.30 6.50 13.0 25.0 50.0


### Mean over ExposurePeriod Units Levell 

### Level 2 Level 3 Level 4 LevelS Prochloraz JJ.glL 11.9 19.0 32.1 54.2 95.0 Flutamide mg/L 0.121 0.225 0.457 0.914 1.80 Ketoconazole IlgiL 2.15 4.19 8.64 17.3 33.7 

### 4-tert-Octylphenol Ilg/L 2.18 4.87 9.15 17.9 41.0 

### CV Over ExposurePeriod Levell 

### Level 2 Level 3 Level4 LevelS Prochloraz 19% 20% 19% 26% 21% Flutamide 6% 7% 7% 7% 7% Ketoconazole 25% 20% 19% 13% 15% 4-tert-Octvlpheno1 52% 28% 11% 8% 13% 

### Percenta2e ofNominal Dose Levell 

### Level 2 Level 3 Level 4 LevelS Prochloraz 92% 88% 89% 90% 95% Flutamide 97% 90% 91% 91% 90% Ketoconazole 65% 64% 66% 69% 67% 

### 4-tert-OctyIphenol 66% 75% 70% 72% 82%

TaskOrder7 Summary Report 7140


### ::J 120 01 ::l. 100 -+--Level1 c: a ;;

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-5 

## o 5 10 15 20 25 30 35 40 45 50 55 Study Day 0+---.-----,----,----,------,-------,--.,..------,-----,-----.----.,.------,



### Figure3. Nominal and measured concentrations ofprochlorazduring the mysid definitive study.

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. E !!! 0.5 ::J 0.375 LL. 0.25 0.125 0 -5 Study Day 

### Figure4. Nominal and measured concentrations offlutamide during the mysid definitive study.

Task Order7 SummaryReport 

### 860 ....---------------------------,

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### Figure 5. Nominal and measured concentrations ofketoconazole during the mysid definitive study.

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### Figure 6. Nominal and measured concentrations of4-tert-octylphenol 

### during the mysid definitive study.

Task Order 7 Summary Report 

### 9

## 5.0 EndpointEvaluation Statistical Methods The design used for the definitive mysid tests was a randomized complete block design. However, only a single diluter and water table were used to conduct each experiment; therefore, even though the randomization was constrained to within a block, blocks were only used to arrange replicate aquaria (tanks) within the water table. An analysis ofthe dilution-water control data was conducted to evaluate the blockeffect and to determine whether it could be ignored for the sensitivity and power analyses. The mean response for a tank was used for the analysis. For each experiment, the standardized residuals for the Fomeasurements were calculated as the difference between the tank response and the mean ofall three tanks divided by the standard deviation ofthe three responses. Because ofthe small sample size (n :::; 10) a Kruskal-Wallis test was used to determine the significance ofthe block effect using each measurement's standardized residual as areplicate measurement ofthe blockeffect. The Spearman correlation coefficient (based on ranks) was calculated, and only those measurements that had an absolute correlation ofless than 0.8 were used in the analysis.
A sensitivity analysis, ignoring blocks, ofthe Fo - and FJ-mysid exposure was conducted to determine which variables had consistently small variance and were responsive across exposures. Variables meeting these qualifications would be recommended for collection in the screening test so that the likelihood of detecting small differences, should they exist, would be efficient. The mean response from the tank was used for the analysis. Descriptive statistics included the mean treatment response, the coefficient of variation for each dose, the within-dose maximum relative difference (a measure ofwithin-class noise defined as [maximumresponse - minimumresponse] / mean response), and the maximum percentage of difference between the treatment and control dose (i.e., [dose mean response - control mean response] / control mean response). Variables with a small «10%) within-treatment group CV tend to require less replication to detect biologically meaningful (10%) differences with high power. Likewise, a high ratio ofthe maximum percentage ofdifference between the control and the treatment response and the average within-dose maximumrelative difference (a signal-to-noiseratio) across chemicals indicates a more responsive variable.
For a given chemical, statistical significance for each endpoint was evaluated, ignoring the block effect, with the nonparametric Kruskal-Wallis test to determine whether the median response from at least one dose was significantly different from the rest (i.e., an H-statistic with 5 df), followed by a Dunn's test to compare each treatment with the control when the Kruskal-Wallis was significant. The nonparametric test was used instead ofaparametric test because ofthe small sample size (n:::; 3) for each treatment group. Tests ofnormality and homogeneity ofvariance that rely on the chi-square distribution require sample sizes ofat least 30 or the number oftreatment groups times five (Snedecor and Cochran 1980; Daniel 1978). Chemical-dosingregimes were considered classifications offixed effects (i.e., control = 0, low dose, mid doses, and high dose specific to each chemical). Boxplots were used to visually characterize the effect ofeach treatment. A two-sided Dunnett's test was conducted on the naturallogarithm-transformed mean response from each replicate tank after removing the block effect (i.e., t[a = 0.05, q = 5, \) = 10]).
Power analysis assuming aType I error rate of

### a = 0.05 was used to compare measured endpoints, ignoring the block effect. The minimum detectable difference assuming 80% power for the given sample size and standard deviation achieved during testing was calculated. The achieved power, given the observed maximum difference between means, was also calculated. Finally, the required sample size

Task Order 7 SummaryReport 10needed to achieve 80% powerwas calculated, given the achieved maximum mean difference. Data were transformed to the natural logarithm to maintain homogeneity ofthe within-class variances for the power analysis (Snedecor and Cochran 1980).


## 6.0 Results 6.1 Block Effect The Spearman correlation ofeach measurement for the non-vehicle control data was based on n = 6 to 12 observations. Because the blocks for these experiments were arbitrary (i.e., created from random locations within a single water table; see Section 5.0), the blockeffect based on the non-vehicle control data was evaluated by a Kruskal-Wallis test on the standardizedresiduals ofmeasurements. Only those measurements for which eachblockwas represented for a given test were used in the analysis. For the 

### Fa generation, the brood-related measurements were highly correlated with each other (Table 2). Thepattern ofcorrelation observed in the 

### Fa generation was not the pattern observed in the F[ generation (Table 3). The F 1female 14-day lengths and weights were highly correlated with the brood-related measurements. Note, however, most ofthe F[ measurements only had 6 observations because ofthe low production of offspring in the flutamide and prochloraz testing periods. Except for 7-day weight and total young, the 

### Fa measurements were highly correlated with at least one other F] measurement (Table 4). The measurements chosen for the block analysis for the 

### Fa mysids were 7-day lengths and weights, days until first brood, interbrood duration, and total young produced; measurements for the F 1mysids were O-day weights, 7-day lengths, 14-day male weights, and interbrood duration. The measurements were standardized and used as unscaled estimates ofthe blockeffect. Test exposures 3 and 4 (flutamide and prochloraz) had limited to no F[ measurements. The Kruskal-Wallis analysis was not significant (p > 0.25) for each ofthe test exposures (Figure 7). Thus, blocks were ignored for the sensitivity and power analyses. The statistical comparisons between treatments were conducted by ignoring blocks (KruskalWallis followed by Dunn'stest) and afterremoving the block effect (Dunnett's test).

TaskOrder7 SummaryReport 

### II>-J 

### Table 2. UpperTriangle Spearman CorrelationMatrix ofFo-Generation Non-vehicle ControlMeanResponse en 

# "" o ..., 0...
--.J \/). s:: 

### :3 :3 

## Q

'"0 o ::+ Measurement 7-d**Wei~ht** 14-dMale LeneJh 14-d Female Leneth Day to 1st Brood Day to 2nd Brood Interbrood Duration Total Young 1stBrood Total Young 2ndBrood Total Youne 7-d Length 0.50 0.84 0.45 -0.17 -0.18 -0.19 0.10 0.32 0.25 7-d Weight 0.27 0.21 0.42 0.48 0.60 -0.54 -0.41 -0.36 14-d Male Length 0.73 -0.35 -0.38 -0.32 0.40 0.49 0.37 14-dFemale Length -0.56 -0.41 -0.18 0.43 0.41 0.34 DayTo 1st Brood 0.93 0.78 -0.93 -0.83 -0.85 DayTo 2nd Brood 0.92 -0.98 -0.90 -0.91 Interbrood Duration -0.91 -0.92 -0.89 Total Young 1st Brood 0.91 0.91 Total Young 2nd Brood 0.97 (Note: Yellow highlighted cells have an absolute correlation >0.8.)


### Table3. UpperTriangle Spearman Correlation MatrixofFj-Generation Non-vehicle ControlMean Response

........ N Measurement O-d Weight 7-d Length 7-d Weight 14-d Male Length 14-d Female Length 14-d Male Weight 14-d Female Weight Day to 1st Brood Day to 2nd Brood Interbrood Duration Tot al Young

lst Brood Total Young

2nd Brood Total Young O-d Length 0.72 -0.07 -0.10 -0.58 -0.37 0.69 0.46 -0.45 -0.35 0.75 -0.52 -0.69 -0.80 O-d Weight 0.18 0.30 0.01 0.14 0.80 0.84 -0.83 -0.84 0.97 0.28 -0.80 -0.07 7-d Length 0.75 0.64 0.88 0.83 0.89 -0.73 -0.66 0.79 0.20 -0.83 -0.31 7-d Weight 0.48 0.73 0.61 0.86 -0.73 -0.59 0.67 0.54 -0.61 0.00 14-d Length, Male 0.88 0.43 0.49 -0.36 -0.77 0.39 0.54 -0.43 0.71 14-d Length, Female 0.83 0.94 -0.83 -0.94 0.72 0.60 -0.83 0.26 14-d Weight, Male 0.89 -0.83 -0.89 0.85 0.09 -1.00 -0.26 14-d Weight, Female -0.93 -0.89 0.85 0.49 -0.89 -0.03 Dayto 1st Brood 0.83 -0.81 -0.47 0.83 0.10 Dayto 2nd Brood -0.79 -0.43 0.89 -0.20 Interbrood Duration 0.13 -0.85 -0.26 Total Young, 1st Brood -0.09 0.71 Total Young, 2nd Brood 0.26

(Note: Yellow higWighted cells have an absolute correlation>0.8.)-l 

### Table4. Spearman CorrelationMatrixofF0- 

### aDd F,-Generation NOD-vehicle CODtrol Mean Response Pl (f) 7';"' 

### Q 0­ (t) 

### ..., -..l ifJ C 

# a a Q

'"Cl 

### §.

w 7-d 14-d Day To Inter- Total Total Total 7-d H-d Male Day To 1st brood Young Measurement Length Fo Weight Length Fo Female Brood Fo 2nd Brood Duration YOUJlg 1st 2nd Brood Young Fo Length Fo Fo Fo Brood Fo Fo Fo O-d Length F l -0.23 -0.42 -0.30 -0.72 0.81 0.60 0.18 -0.59 -0.39 -0.55 O-d WeightF[ 0.11 -0.31 0.06 -0.29 0.26 0.08 -0.30 -0.09 0.07 -0.10 7-d Length F [ 0.74 0.12 0.94 0.57 -0.53 -0.69 -0.60 0.66 0.71 0.50 7-d WeightFj 0.97 0.44 0.95 0.51 -0.49 -0.48 -0.61 0.46 0.82 0.57 14-d Male Length F I 0.30 -0.06 0.70 0.81 -0.81 -0.86 -0.62 0.93 0.67 0.60 14-d Female Length F, 0.66 0.14 0.88 0.66 -0.73 -0.91 -0.79 0.91 0.88 0.76 14-d Male WeightF[ 0.44 -0.31 0.72 -0.19 0.15 -0.60 -0.90 0.67 0.82 0.53 14-d Female Weight F1 0.75 0.04 0.86 0.07 -0.26 -0.71 -0.90 0.67 0.94 0.70 DayTo 1st BroodF I -0.71 -0.18 -0.66 0.14 0.25 0.83 0.88 -0.70 -0.85 -0.74 DayTo 2nd BroodFj -0.37 0.34 -0.61 -0.11 0.26 0.83 0.98 -0.92 -0.90 -0.70 Interbrood Duration F) 0.58 -0.10 0.95 0.13 -0.09 -0.52 -0.73 0.58 

## I 0.66 0.28 Total Young, 1stBrood F[ 0.52 0.38 -0.07 0.45 -0.90 -0.66 -0.44 0.49 0.62 0.74 Total Young, 2ndBrood F[ -0.44 0.31 -0.72 0.19 -0.15 0.60 0.90 -0.67 -0.82 -0.53 Total Young, Fj -0.19 -0.15 -0.33 0.62 -0.79 -0.37 -0.13 0.43 0.21 0.37 (Note: Yellow higWighted cells have an absolute correlation>0.8.)Kruskal-WallisTest onTest1 Kruskal-WallisTeston Test3
Block N edian AveRank Z Block N Median A eRank Z 1 9 ..(U713 14.8 0.36 1 5 -0.5401 7.2 -0.49 2 9 -0.4246 13.2 -0.36 2 5 0.2682 9.8 1.10 3 9 -0.4057 14.0 0.00 3 5 -0.6138 7.0 -0.61 Overall 27 14.0 Overall 15 8.0
H=0.17 DF=2 P=0.917 H = 1.2 DF= 2 P = 0.543 H = 0.17 DF= 2 P = 0.917 (adjusted for ties)
KrusL:a1-Wallis Test on Test2 KrusL:a1-WaIlis Test onTest4 Block Median Ave Rank Z Block Median AveRank Z 1 9 -0.440 1 .9 -0.51 1 5 -0.3103 6.6 -0.86 2 9 0.2086 17.4 1.59 1 5 0.1944 8. 0.24 3 9 -0.1325 11.7 -1.08 3 5 0.7193 9.0 0.61 Overall 27 14.0 Overall 15 .0
H = 2.65 DF= 2 P=0.266 H = 0.78 DF= 2 P = 0.677 H = .65 DF= 2 P = O. 66 (adjustedfor ties)


### Figure7. Output from the evaluation oftheblock effect based on the Kruskal-Wallis test on four test exposures. Test! 

### =4-tert-octylphenol, Test2 

### =ketoconazole, Test3 

### =flutamide, Test4 = 

### prochloraz.



### 6.2 Sensitivity andResponsiveness
The mean non-vehicle control response for each variable is presented in Appendix A, Table A.l. In general, the responses for a given variable were the same across generations and testing periods (chemical). The notable exceptions were the number ofdays between the first and second brood (interbrood period), which was between 5 and 8 days for all but one replicate during the flutamide testing (19 days), and the number ofyoung during eachbrood. Controls (non-vehicle) from the individual testing periods were combined and used to evaluate the natural variation ofeach ofthe variables. The variability measured as the CVGF,-generation non-vehicle control response was generally less than the Fa-generation response (Ta e 5). The mean response for each variable averaged over testingperiod (chemical exposure) was genera e same, except for the total number ofyoung and the number of young in the second brood. Variables with CVs >30% (colored orange in Table 5) would have low power «80%) even for detecting differences as great as 70% with three replicates (Figure 8). Seven Fa variables (lengths at 7 and 14 days, weights at 14 days, and the number ofoffspring in each brood) and 10 ofthe 11 F, variables had CVs <30%.

Task Order 7 Summary Report 14

### Table 5. Non-vebicle ControlMeanResponse and Coefficient ofVariationAcross TestingPeriods (Chemicals) by Generation
Non-Vehicle Control 

### F

o F, Variable Mean CV Mean CV 7-d Length 3.45 5.00% 3.71 7.80% 7-d Weight 0.27 50.0% 0.33 40.6% 14-d Male Length 5.27 4.92 10.8% 7.40%
5.47 14-d Female Length 4.85 7.60% 10.3%
14-d MaleWeight 0.76 0.68 26.4% 16.4%
14-dFemale Weight 0.74 18.5% 1.09 14.1% FirstBrood 18.4 15.6% 17.7 13.1% Second Brood 24.6 17.8% 23.5 7.70% Interbrood Duration 7.19 52.6% 5.60 10.9% Total Young 1stBrood 5.75 40.2% 6.22 34.6% Total Young 2nd Brood 6.52 55.4% 11.4 14.4% Total Young 11.6 46.6% 16.7 19.2% (Note: orange highlighted cel1s are CVs >30%)

TaskOrder 7 SummaryReport 1510% 20% 30% 40% 50% 60% 70% 80% 100%**-,---------::::::=~=-O---<:>-"""2::::::::i=a==----1!:::::I==lr_-1**

80%**~CV=5%**

__ CV=10% -t--CV =15%


# - lI) 60%



# - Q) --+-- CV = 20%I--0-- CV = 30% 0 '­ Q) ---I:!r- CV =40% 0 40%
­ a. --0-- CV = 50% ·--.---CV=60%

- - -.- - - CV = 70% 

# . .'

# .
,.,., 

# -. 20% 

# "-,,-.-,-." 

# .-'


# ..,." ' 

### ::t::-·'-""

0% +---f-----!---+----t---+---+----!----I 0% % Difference from Control Mean 

### Figure 8. Power ofa one-sided independent-sample t-test as a function ofthepercentage of difference detected between the test and reference meanswith three replicates per treatment (ex = 

### 0.05).
The signal-to-noiseratio (based on the maximum percentage ofdifference between the dose and control response and the average relative maximum difference for within-dose measurements) is negative when the control response is greater than the treatment response, and positive when the treatment response is greater than the control. A general guideline for interpretation ofthe signal-to-noise ratio is that values >2 indicate a responsive variable, whereas values <2 suggest a nonresponsive variable. The largest Fo ratio was the 7-day weight for ketoconazole (Table 6). In general, the Folength and weight variables were the mostresponsive variables, even though weight tended to have a greaternon-vehicle control CV than many ofthe brood-related variables. Flutamide non-vehicle control data for 7-day weight, however, were greater than twice the average ofall other non-vehicle control data and may not be representative (Table A.I). Thus, this CV may be overestimated. Only one ofthe F1variables (total number ofyoung in the second brood for ketoconazole) had a signal-to-noise ratio with an absolute value greater than 2 (Table 6), which suggests that the F I variables were nonresponsive in terms ofproducing a change in response greater than twice the within-dose noise. However, the lackofoffspring 

### in the high doses of4­ tert-octylphenol and prochloraz and an inability to meet the acceptability criterion for reproduction for flutamide make this second-generation assessment indeterminable. For example, the F j female 14-day length and weight for 4-tert-octylphenol (with a magnitude ofthe signal-to-noise ratio <2, thereby suggesting a nonresponsive variable) were slightly larger than, but consistentwith, the Foresponses at the doses measured. The Fomeasurement was only responsive at the highest dose (average concentration = 41 flg/L; Figure 9).

Task Order 7 SummaryReport 

### 16

# • 

# • Table 6. Signal-to-Noise Ratio by Chemical and Generation
Si nal-to-Noise Ratio F 1 Pro- Keto- 4-tert-OctylVariable chloraz conazole henol 7-d Length 1.41 1.27 -0.64 7-d Weight -0.6 1.29 -0.46 14-d Male Length 0 -0.68 1.07 14-d Female 0.45 -0.58 1.54 Len th 14-d Male Weight -0.55 0.68 14-d Female -0.87 0.18 Weight FirstBrood 0.72 -0.79 1.1 1.38 0.41 -0.72 Second Brood 0.78 -1.18 -1.21 0.95 0.39 -1.19 Interbrood Duration 0.52 -0.85 0.86 0.88 0.99 1.07

otai Young 1st -0.88 -0.75 -1.82 -1.38 -0.62 0.41 Brood Total Young 2nd -1.04 -0.76 -1.29 -0.79 -0.32

rood Total Young -1.66 -0.79 -1.32 -0.38 (Note: green higWighted cells are ratios with absolute values >2)

6.0 ,------------------------------, 

# • 

# . E 5.5 [

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# .-­ .s 0 .; 

### o o g' [ 

### o o .3 5.0 n**~-O-** --0

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### o -0 4.5

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40 +---,----r---r---,----,-----,-----,------,-----j 

### o 5 10 15 20 25 30 35 40 45 Mean Concentration 4-tert-Octylphenol (J.lg/L)



### TaskOrder7 Summary Report 171,2,-----------------------------,**~~** 1,0

# -1---._-**-.~--** 

# .­
Ci E • O. 

# • 0 8 -**-~'--nr:t-'** -0-- .-----.-- ----- --­

, [ D. 0 

### Q

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-g '<t

.... 0,2
- --, -- ---­
0,0 -l----,------,------,----,-----,----,-----r-----r------i 

### o 5 10 15 20 25 30 35 40 45 Mean Concentration 4-tert-octylphenol (llg/L)



### Figure9. Fo - 

### and F,-generation 14-day female length (top) and weight (bottom) as a function of the mean concentration of4-tert-octylphenol (Ilg!l) 

### with signal-to-noise ratios of-2.17 and 1.54 for length and -2.92 and 0.18 for weight, respectively.

Forketoconazole, however, enough F I young wereproduced, butwere nonresponsive at the concentrations tested except for the number ofyoung in the second brood. The variable, 7-day weight produced a signal-to-noiseratio of6.10 for the Fa generationbut only 1.29 in the FI generation (Figure 10. ). Interestingly, the F 1generation had heavier weights and greater variability than did the Fogeneration. The pattern ofresponse for those variables consideredresponsive wasjustas inconsistent as those that were considered nonresponsive (signal-to-noise ratio <2). Figure 11 depicts four variables considered responsive.

07 -,---------------------------, 

# • 0,6 r- 

## --t-

- '---0--­**~0,5-** 

# ----.- Cl 

# • E. 141 .:E 0.4 

## If-

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- 

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### _. 

### -­ 0,1---- ---_. ---- ----,-,­

O-l-----,-----,----,----,----r------,------r------!



### o 5 10 15 20 25 30 35 40 Mean Concentration Ketoconazole (llg/L)

10 FO • F1 I 

### Figure 10. Fo - 

### and F,-generation 7-day weight as a function ofthemean concentration of 

## ketoconazolewithsignal-to-noiseratios of6.10 and 1.29,respectively.

TaskOrder 7 Summary Report 180.6

0.5 0 u. .c 0> Qj 0.4 U r-­ 1U 0.3

0.2 Irl

3.6
3.5
0 3.4 u. .c 

### g, 33 OJ --J 3.2 u r-­ 0 3.1
3.0
29 lrt Boxplots of Ket 7d-W by trt (means are indicated by solid circles)



# 0

GJ

0 ;Z g g <> '" N .,; 0 

### ::

Boxplots of Oct 7d-L by trt (means are indicated by solid circles)



## [J 

# 0 Q [J 

# 0

0 g 0 0 0 N <i "' 

# " '" 

## '" Boxplots of Flut 14d by trt (means are Indicated by solid circles)
5.5

0 u. .c e;, c: OJ --J U. U ;:: '5 u:: 50

4.5 

# 8 0 Q

Q

lrl 0 g <> N <> '" N N <> '" <> m <> 0 0 "'

Boxplots of PR 7d-We by trt

(means are indicated by solid circles)
0.7

0.6 0 u. :E 0.5 0> ·w 0.4 U r-­ oc 0.3 (L 0.2 

# EJ g

0.1 lrt 

### '" 0 ;:: 

### ; m 0 bi 

### Figure 11. Variables with an absolute signal-to-noise ratio>2, suggestingresponsiveness. 

### 6.3 Statistically SignificantEffects
The Kruskal-Wallis test (ignoring the block effect), which tested the null hypothesis, Ho : medians are all equal, verses HI: at least one median differs from the rest, was conducted and followed by the Dunn's test to compare each treatment with the control when the Kruskal-Wallis test was significant. The nonparametric tests were conducted for comparison with the two-sided Dunnett's test (conducted after removing the block effect), which for each dose tested the null hypothesis, Ho: the treatment mean is equal to the control mean, verses HI: the treatment mean is not equal to the control mean. Forthe prochloraz exposure (Table 7), only the Fa-generation 14-day dry weight (mg) and total number of offspring had at least one median response that was significantly differentbased on the nonparametric Kruskal-Wallis test (p 

### < 0.05). Ofthose, none ofthe treatment concentrations producedresults that were significantly different from the control based on Dunn's test (p > 0.05). Fourofthe variables, however, had treatment concenh'ations with mean responses that were significantly different from the control mean response based on the Dunnett's test and natural-logarithm-transformed data. Ofthose variables, only the 

### Fa total young had a significantresponse relative to the control response underthe three highest multiple exposure concentrations (32.1, 53.9, and 93**~g/L).** The difference in these results was that a blockeffectwas removed from the error term for the Dunnett's analysis. Thus, more significantresults were detected. The largest percentage ofdifference 

### thatwas

TaskOrder 7 Summary Report 19detected by Dunnett's test but which was not significant using the Kruskal-Wallis test was the Fo-generation 7-day weight (155.7% increase from the control). The blocks did not have a consistent effect on the results ofthe weight (i.e., Block 3 was not always greater; Figure 12). Further, the Block I result for the highest concentration was the sixth smallest weight. The effectiveness ofblocking to increase the precision ofthe experiment over a completelyrandomized design was calculated. Effectiveness was based on the ratio ofthe estimated error variances for the two designs and Fisher's adjustment for the increase in the dferror (ORB and OCR for the randomized block design and completelyrandom design, respectively) in the completely randomized design:

where**o-~B** equals the error mean square from the randomized block ANOVA (S2) and

A 2 

### (j

-

## I)s 

### +j

## (i

-

## 1)s2 (J 

## =--'------'---'-:,--------,-----'--­

CR 

## (U-l) where**s~** = the blockmean square from the randomizedblock ANOVA i = the number oftreatment levels, and j = the number ofblocks (Snedecor and Cochran 1980).
Forthe Fo-generation 7-day weight,**s~** = 0.1205 and S2 = 0.1179 (Table 8), and thus, the adjusted effectiveness was estimated as 0.95. Values <I indicate that the blocking was not effective. Therefore, Dunnett's analysis probably overestimated the number ofsignificant responses.

Task Order7 Surmnary Report 20

### Table 7. Percentage ofIncrease (+) 

### or Decrease (-) ofResponse ofMysids Exposed to Concentrations ofProcbloraz Relative to the Control Response
Variable 11.1 Average Prochloraz Concentration (I!WL) I 18.9 32.1 53.9 93 7-day Length Fe -1.2% 3.2% -6.8% -3.6% 1.5% 7-dayWeight Fe 30.9% 18.5% 88.2% 66.8% 155.7% 14-day Male Length Fe 5.6% 8.5% 6.0% 7.1% 6.8% 14-dayFemale Length Fe 2.4% 6.1% 2.1% 2.9% 4.8% 10.8% 49.0% 96.9% 21.0% 53.7% Time to FirstBroodFe -0.4% 0.2% 8.6% 5.7% 3.1% Time to Second BroodFe 1.0% -1.4% 10.0% 7.9% 8.7% Interbrood Duration Fe 9.2% -9.8% 7.6% 13.6% 20.1% Total Young 1stBrood Fe -24.0% -13.9% -36.4% -40.8% -37.5% Total Young 2nd BroodFe -46.7% -30.9% -49.8% -68.7% -44.6% -37.4% -20.8% -45.6% -55.0% -41.6% O-day Length F) -0.2% -6.3% 0.9% -8.6% 1.1% O-day WeightF 1 -39.5% -22.5% -48.6% 26.3% -31.5% 7-day LengthF) 2

- 3.3% 2



### - 2



### - 2

- 7-day WeightF I

- -11.7%

- 

### - - 14-dayMale Length F 1 

### - 0.0%

- - - 14-dayFemale Length F 1 - 0.8%
- - 

### - 14-dayWeight F 1 - -30.3%

- 

### - -
I Based on Day 0 through Day 28 ofthe exposure
2 The number ofF [mysids was not sufficient to allow evaluation for this treatment level. (Note: yel10w rughlighted cells are significantly different from the control by Dunnett's test; green higWighted variables have a significant Kruskal-Wallis test)



### TaskOrder 7 SummaryReport 210.8
0.7
0.6 Ol E ::: 0.5 s:; Ol 0.4
>, 

## -9 0.3 r-­ 0.2 0,1 -0

0

- 0 

# • c () 

# • n 

# •**~~**



### o o 10 20 30 40 50 60 70 80 90 100 Average Prochloraz Concentration**(~g/L)** 

# I. Block 1 0 Block 2 0 Block 

### 31 Figure 12. Inconsistentblock effect on the Fo-generation 7-day weight for each concentration of prochloraz.

Table 8. Analysis ofVarianceTablefor the Natural-Logarithm-TransformedFo-Generation 7-Day Weight for Prochloraz
Sourceof Variation Degrees of Freedom TypeIII 

### Sumof Squares TypeIII Mean Square F-statistic P-value
Block 2 0.2410 0.1205 1.02 0.395 Treatment 5 1.4052 0.2810 2.38 0.114 Error 10 1.1788 0.1179 Total 17



### Dunnett's SimultaneousTests Comparisonswith the Non-Vehicle Control Exposure Level Difference ofMeans SEof Difference T-Value AdjustedP-Value ILl 0.2699 0.2803 0.9627 0.7970 18.9 0.1791 0.2803 0.639 0.9469 32.1 0.6036 0.2803 2.1532 0.1879 53.9 0.4915 0.2803 1.7534 0.3348 93 0.8392 0.2803 2.9935 0.0497 (Note: yellowhighlighted cell is a significantresult by Dunnett's test.)

Task Order 7 Summary Report 22For the flutamide exposure (Table 9), the Fa-generation 7- and 14-day lengths (mm) and the 14-day female dry weight (mg) had a significant Kruskal-Wallis test (p < 0.05). Ofthose, none ofthe treatment concentrations produced results that were significantly different from the control based on Dunn's test (p > 0.05). The 7- and 14-day lengths (rnm) and the 7-day weight had treatmentconcentrations with mean responses that were significant reductions from the control meanresponse based on the Dunnett's test and natural-logarithm-transformed data.
The largest percentage ofdifference that was detected by Dunnett's test but which was not significant using the Kruskal-Wallis test was for the Fa-generation 7-day weight for the 0.914-mg/L exposure concentration. Again, the blocks did nothave a consistent effect on the results ofthe weight (Figure 13). The effectiveness ofblocking to increase the precision ofthe experiment over a completelyrandomized design was calculated. Forthe Fa-generation 7-day weight,**s~** 

### =0.03702 and S2 

### = 0.02372 (Table 10), and thus, the adjusted effectiveness was estimated as 1.0

### I. Values equal to 1indicate that the blocking did not increase the precision over the completelyrandomized design. Therefore, Dunnett's analysis probably overestimated the number ofsignificant responses.



### Table 9. Percentage oflncrease (+) 

### or Decrease (-) ofResponse ofMysids 

### Exposed to Concentrations ofFlutamide Relative to the ControlResponse Variable Flutamide Concentration (mgIL) 0.121 0.225 0.457 0.914 1.8 5.4% 2.4% 1.0% -0.7% -11.2% -4.5% -6.3% -7.5% -3 .8% -14.8% -1.8% 2.7% -2.3% -9.5% -15.4% 0.4% 3.9% -1.0% -7.4% -13.6% 2.3% 5.4% 11.2% -2.3% -17.6% 8.2% 20.8% -4.6% 10.1% -9.8% Time to FirstBrood 

### Fa -11.4% -9.9% -6.1% -18.4% 10.1% Time to Second Brood

### Fa 0.6% -8.8% 6.2% -28.5% Interbrood Duration 

### Fa 0.9% -27.0% 31.0% -55.2% Total Young 1st Brood

### Fa 29.1% 8.3% 27.2% -15.4% -70.5% Total Young 2nd Brood 

### Fa 16.8% 14.5% -49.8% -43.6% Total Young 

### Fa 13.0% 22.3% -10.7% -16.6% -84.6% (Note: yellow highlighted cells are significantlydifferent from the controlby Dunnett's test; green highlighted variables have a significant Kruskal-Wallis test)

Task Order 7 Summary Report 23------ ------------ 0.65

## -y-----------------------------,
_.­ 0.6 -8---­ 0.55**~+.--** ..-------. 

### ---- - ---- - ------ Ci 

## g 0.5

-- 

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-

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## o 0.2 0.4 0.6 0.8 1.2 1.4 1.6 1.8 2 Average Flutamide Concentration (mg/L)
\. Block 1 0 Block 2 0 Block 3 : 

### Figure 13. Inconsistent blockeffect on the Fo-generation 7-dayweight for each concentrationof flutamide.

Table

## to.Analysis ofVarianceTable for theNatural-Logarithm-TransformedFo-Generation 7-Day Weightfor Flutamide Source of Variation Degrees of Freedom Type 

### 111 Sum of Squares Type III Mean Square F-statistic P-value
Block 2 0.07404 0.03702 1.56 0.257 Treatment 5 0.30738 0.06148 2.59 0.094 Error 10 0.23717 0.02372 Total 17



### Dunnett's SimultaneousTests Comparisonswith theNon-Vehicle Control Exposure

Level Difference ofMeans SEof Difference T-Value Adjusted P-Value 0.121 -0.0616 0.1257 -0.490 0.9814 0.225 -0.0631 0.1257 -0.502 0.9795 0.457 -0.0718 0.1257 -0.571 0.9654 0.914 -0.3978 0.1257 -3.164 0.0377 1.8 -0.1665 0.1257 -1.324 0.5703 (Note: yellow highlighted

### cell 

### is a

### significant

## resultby

## Dunnett'stest.)

Task Order7 SummaryReport 24Forthe ketoconazole exposure (Table 11), the Fa-generation 7-day dry weight (mg) and number of offspring and the F,-generation number ofoffspring in the second brood had a significant Kruskal-Wallis test (p 

### <0.05). Ofthose, only the F 1generation ofthe number ofoffspring in the secondbrood for the 8.64 ).lg/L exposure concentration was significantly different from the control based on Dunn's test (p 

### <0.01). The Fa-generation 7-day lengths (mm) and weights (mg) and number ofoffspringproduced and the F} number ofoffspring produced in the second brood and total offspring had mean responses that were significantly different from the control mean response based on the Dunnett's test and naturallogarithm-transformed data. All ofthe significant offspring-related variables were reductions from the control response. Again, the Fa-generation 7-day weight for the 33.7-).lg/L-exposure concentration had the largest percentage ofdifference detected by Dunnett's test but was not significant using the Dunn's test. The blocks did not have a consistent effect on the results ofthe weight (Figure 14). The effectiveness of blocking to increase the precision ofthe experiment over a completelyrandomized design was calculated. For the Fa-generation 7-day weight, the adjusted effectiveness was estimated as 1.14**(s~** = 0.02262 and S2 = 0.00838) (Table 12). Values >1 indicate that the blocking did increase the precision over the completelyrandomized design. To get the same level ofprecision (or variance ofa treatment mean) achieved by the randomized block design, the completelyrandomized design would require

n 

## =j

# (o-%~) or 3.6replicates (not quite a whole number increase; Snedecor and Cochran 1980). Therefore, Dunnett's analysisprobably overestimated the number ofsignificant responses.

TaskOrder 7 Summary Report 25

### Table II. Percentage oflncrease (+) 

### orDecrease (-) ofResponse ofMysids Exposed to ConcentrationsofKetoconazole Relative to theControlResponse
Variable Ketoconazole Concentration (1:I:g!L) 

### 2.15 4.19 8.64 17.3 33.7 7-dayLength Fo 0.2% 6.0% 2.6% 3.8% 1.9% -10.2% 1.3% 14.8% 55.5% 94.7% 14-dayMaleLengthFo -1.5% 1.0% -3.2% 0.5% -1.4% 14-dayFemale Length Fo 4.3% 1.1% 0.9% 2.5% 1.3% 14-dayMale Weight Fo 0.6% 1.4% 4.2% 18.0% 3.4% 14-dayFemale Weight Fo -4.2% -1.4% -0.8% 4.4% 9.6% Time to FirstBroodFo -1.8% 0.0% -1.6% -0.4% 3.4% Time to Second BroodFo -1.1% 0.9% -0.6% -3.0% 1.5% Interbrood Duration Fo 6.2% 8.9% 7.3% 3.8% 7.3% -17.5% -3.4% 1.4% -18.7% -31.7%

-0.7% -9.5% 17.4% -8.7% -28.9% -10.9% -6.7% 13.8% -19.4% -38.4% O-day Length F I 4.2% 1.1% 2.0% 7.2% 2.8% O-day Weight F I 20.8% 14.6% 43.8% 1.4% -6.0% 7-dayLength F 1 2.1% 2.7% 3.6% 6.0% 4.7% 7-day WeightF I 2.5% 41.0% 47.6% 21.3% 3.5% 14-dayMaleLength F I -4.0% -4.3% -2.1% 3.4% 0.9% 14-dayFemale LengthF I -4.3% -1.6% 0.0% 3.3% 3.1% 14-dayMale Weight I -15.6% 1.4% -3.1% -1.7% -9.4% 14-dayFemale Weight F I -14.3% -6.5% -10.4% -7.8% -5.7% Time to First Brood F 1 -1.8% 8.3% 9.9% -7.7% 4.2% Time to Second Brood F I 1.8% 7.7% 7.5% 1.2% 7.2% Interbrood Duration F 1 18.7% 3.0% -0.1% 24.1% 32.3% Total Young 1stBroodF I -23.2% -23.2% -36.0% -18.5% -3.3% -31.6% -22.4% -69.1% -48.7% -47.5% -32.2% -32.4% -62.5% -39.4% -35.2% (Note: yellow highlighted orboxed cells are significantly different from the control by Dunnett's or Dunn's test, respectively; green highlighted variables have a significant Kruskal-Wallis test)

TaskOrder 7 SummaryReport 260.6 ..,-------------------------=-------, 

## o

_ 0.5-­ 

# .s m

.l: 

# ­ m 

# o .D 

# • 0.4
>. III i'



### ..... 0.3


### 0 

## 0 0.2 !+----\1l.....J-----r----,------,----,---------,----------,----,--------------I 

## o 5 10 15 20 25 30 35 40 Average Ketoconazole Concentration (j.!g/L) [ • Block 1 0 Block 2 0 Block

## 31 '---------------­ Figure 14. Inconsistent block effect on the Fo-generation 7-day weight for each concentration of ketoconazole.

Table 12. Analysis ofVariance 

### Table for the Natural-Logarithm-Transformed Fo-Generation 7-Day Weight for Ketoconazole

Source of Variation Degrees of Freedom Type III Sum of 

### Squares Type III Mean Square F-statistic P-value
Block 2 0.04524 0.02262 2.7 0.116 

### Treatment 5 1.30348 0.26070 31.11 <0.001 

### Error 10 0.08380 0.00838 

### Total 17


### Dunnett's Simultaneous Tests Comparisonswith the Non-Vehicle Control Exposure

Level Difference of Means SE of Difference T-Value Adjusted P-Value 2.15 -0.1084 0.07475 -1.450 0.4940 4.19 0.0137 0.07475 0.184 0.9998 8.64 0.139 0.07475 1.860 0.2886 17.3 0.4405 0.07475 5.894 0.0006 33.7 0.6515 0.07475 8.716 <0.0001 (Note: yellow higWighted cell is a significant result by Dunnett's test.)



### Task Order 7 Summary Report 27For the 4-tert-octylphenol exposure (Table 13), only the Fo-generation 14-day male length had a significant Kruskal-Wallis test (p < 0.05). None ofthe treatment concentrations for this vaIiable produced results that were significantly different from the control based on Dunn's test (p > 0.05). Nine variables had treatment concentrations with mean responses that were significantly different from the control mean response based on the Dunnett's test and natural-logarithm-transformed data. Ofthose vaIiables, all had a reduction from the control response.
The Fo-generation total number ofyoung for the 41-)..l.glL-exposure concentration had the largest percentage ofdifference detected by Dunnett's test but was not significant using the Kruskal-Wallis test. Again, the blocks did not have a consistent effect on the results ofthe production ofoffspring (Figure 15). Theeffectiveness ofblocking to increase the precision ofthe expeIimentover a completelyrandomized design was calculated. For the Fo-generation total number ofyoung, the adjusted effectiveness was estimated as 0.97**(s~** = 0.1377 and S2 = 0.1208) (Table 14). Values <1 indicate that the blocking was not effective. Therefore, Dunnett's analysis probably overestimated the number ofsignificantresponses. Table 13. Percentage oflncrease (+) orDecrease (-) in Response ofMysids Exposed to Concentrationsof4-tert-Octylphenol Relative to the Control Response
Variable 4-tert-Octvlphenol Concentration**(I!~/L)** 2.18 4.87 9.15 17.9 41 7-day Length Fo -1.5% 0.5% 0.1% -0.7% -l3.4% 7-da Wei ht F o -8.0% 0.2% 1.7% -7.4% -38.1% -0.5% -0.1% -4.3% -2.3% -6.6% 14-day Female Length Fo 0.1% 2.3% -4.7% -1.8% -13.6%

- 14-day Male Weight F o -3.4% 2.0% -5.9% -6.0% -19.9% 14-day Female Weight Fo 8.7% 16.5% 2.2% 7.4% -39.1% Time to First Brood Fo -0.2% 4.0% -1.4% 2.2% 7.7% Time to Second Brood F o -0.1% 3.2% -2.4% 2.8% 12.7% Interbrood Duration F o 0.7% 4.4% -7.8% 6.9% 36.3% Total Young 1st Brood F o -23.5% -9.7% -3.7% -34.0% -71.2% Total Young 2nd Brood Fa -44.5% -8.2% -35.7% -42.0% -76.8% Total Young F o -45.5% -12.8% -27.5% -43.5% -83.9% O-day Length F[ 7.6% 2.9% 1.0% 10.0% 4.4% O-day Weight F1 -9.5% -31.1% 8.7% 4.1% -30.9% 7-day Length F, -4.2% 2.5% -0.5% -1.9%
- 7-day WeightF[ -13.0% 4.0% 6.3% -3.4% 14-dayMale Length F1 3.9% -1.0% 4.9% 1.8% 14-day Female Length F, 3.6% 0.2% 2.4% 0.7%

- 14-dayMale Weight

### F, 3.0% -6.7% 8.2% -4.9%

- 14-day FemaleWeightF1 -1.6% -2.5% 0.4% 4.1%



### - Time to First Brood F1 2.5% -3.2% -7.2% -4.0%

- Time to Second Brood F1 2.1% -4.3% -8.1% 0.5%

- Interbrood Duration F1 2.1% -9.0% -6.7% 16.7%

- Total Young 1st Brood FI -18.0% -1.6% 2.4% 18.1%

- Total Young 2nd Brood F1 -13.9% 10.9% 9.1% -13.2%
- Total Young F1 -16.3% 5.5% 6.0% 1.5%

- (Note: yellow highlighted cells are significantly different from the control by Dunnett's test; green higWighted variables have a significant Kruskal-Wallis test

Task Order7 Summary Report 2825

# ? ¢ 0 

# • 0 Cl 20 c: ;:, 0 

### >­ 

# - 15 0



# ... CII .0 E ;:, 10 

### z 

## co 

# - 0I0 5 LI.. 

### 0



## 0 0

0 

# • 0 0


### o 

## o 5 10 15 20 25 30 35 40 45 Average 4-tert-Octylphenol Concentration (llg/L) 

# I. Block 1 0 Block 2 0 Block 

## 31 Figure 15. Inconsistent block effect on the Fo-generation total number ofyoung for each concentration of4-tert-octylphenol.



### Table 14. Analysis ofVariance

### Table for the Natural-Logarithm-Transformed Fo-Generation Total Number ofYoung 

### for 4-tert-Octylphenol 

### Source of Variation Degrees of Freedom Type ill 

### Sum of 

### Squares Typem Mean Square F-statistic P-value
Block 2 0.2754 0.1377 1.14 0.358 Treatment 5 6.7103 1.3421 11.11 0.001 Error 10 1.2076 0.1208 Total 17


### Dunnett's Simultaneous Tests Comparisonswith theNon-Vehicle Control Exposure Level Difference of Means SE of Difference T-Value Adjusted P-Value 2.18 -0.627 0.2837 -2.209 0.1726 4,87 -0.183 0.2837 -0.646 0.9447 9.15 -0.378 0.2837 -1.331 0.5661 17.9 -0.6 0.2837 -2.115 0.1992 41 -1.886 0.2837 -6.647 0.0002 (Note: yellow highlighted cell is a significant result by Dunnett's test.)

TaskOrder 7 SummaryReport 29

### 6.4 Power

### Analysis
The power analysis was conducted on the natural-logarithm-transformed responses, ignoring blocks. AppendixTables AA, A.5, A.6, and A.7 present the achieved minimum detectable difference assuming 80% power, the achieved power given the observed maximum difference betweenmeans, and the required sample size needed to achieve 80% powerfor each endpointmeasured. For ketoconazole and 4-tert-octylphenol, a comparison ofthe Fa and F 1achieved power for each ofthe endpoints wasmade (Table 15). For both exposures, the Fa responses generally had greater achieved power. The highest powerwas obtained for the Fa 7-day lengths and weights, the number ofoffspring from the first brood, and total number ofyoung. A similarresult can beobtained by looking at the sample size required to achieve 80% power (Table 16).

Table 

### 15. Powerwith the Achieved Maximum Percentage ofDifference, Sigma, and umber of Replicates
Variable Prochloraz Flutamide Ketoconazole 4-tert-Oetvlphenol 

### Fo Fo Fo F 1 F

### o F. 7-d Length 27% 83% 61% 30% 96% 6% 7-d Weight 43% 37% 99% 26% 96% 6% 14-d Length, Males 11% 64% 11% 8% 61% 10% 14-d Length, Females 16% 58% 11% 10% 42% 15% 14-d Weight, Males

- 16% 15% 7% 36% 7% 14-d Weight, Females

- 22% 12% 18% 74% 5% FirstBrood 11% 8% 24% 7% 35% 8% Second Brood 14% 12% 34% 7% 9% 13% Interbrood 7% 10% 17% 11% 7% 7% Total Young, 1st brood 26% 27% 83% 11% 90% 6% Total Young, 2nd brood 49% 29% 48% 100% 24% 6% Total Young 71% 55% 91% 58% 99% 6%

Table 16. Sample Size Required with Achieved Maximum Percentage ofDifference, Sigma, and 

### 80% Power Variable Prochloraz Flutamide Ketoconazole 4-terl-Octylpbenol 

### Fe Fe F o F 1 

### Fo F1 7-d Length 8 3 4 8 3 74 7-d Weight 6 6 3 9 3 73 14-d Length Males 23 3 23 23 4 13 14-d Length, Females 14 3 21 26 4 8 14-d Weight, Males

- 7 15 27 7 26 14-d Weight, Females



## - 6 21 12 3 263 First Brood 24 24 9 51 7 19 SecondBrood 17 10 7 57 15 9 Interbrood 53 14 13 22 26 27 Total Young, 1stbrood 9 8 3 21 3 40 Total Young, 2ndbrood 5 8 5 2 5 72 Total Young 4 5 3 5 3 49

Task Order7 Summary Report 30The maximum percentage ofdifference observed between the dose and the control was different for each chemical tested (Table 17). Forketoconazole and 

### 4-tert-octylphenol, the difference was usually greater in the Fo-generation response than in that ofthe Fl.

Table 17. MaximumPercentage ofDifference from the Observed Control Response (NaturalLogarithm-TransformedData)
Variable Prochloraz Fo Flutamide

Fo Ketoconazole Fo Ketoconazole F 1 4-tertOctylphenol 

### Fo 4-tertOctylphenol 

### F1 7-d Length 3.66 6.06 3.01 3.01 13.4 1.93 7-d Weight 123 28.6 77.6 38.8 35.7 11.0 14-d Length, Males 4.17 7.64 1.46 1.97 6.63 2.19 14-d Length, Females 2.88 6.69 1.89 4.35 13.6 1.62 14-dWeight Males



### - 14.7 18.0 15.6 16.4 8.16 14-d Weight, Females

- 16.0 6.76 10.2 33.0 3.74 First Brood 2.51 6.26 1.11 3.30 2.43 2.35 Second Brood 2.79 9.08 0.94 2.31 3.49 2.45 Interbrood 6.89 25.5 4.03 10.6 12.1 6.19 Total Young, 1stbrood 23.5 70.5 31.7 17.3 47.9 9.12 Total Young, 2nd brood 45.8 lOa 28.9 38.9 53.9 5.43 Total Young 28.1 72.2 38.4 29.2 55.8 5.66


## 7.0 Discussion 7.1 Summary ofIndividual Studies Prochloraz-Analyticalmeasurements ofprochlorazmade during the study varied and showed occasional overlap ofexposure concentrations during the study. Reasons for this variation in concentrations within dose levels are notknown. About 75% ofthe control pairs produced at least one brood, which exceeds the test acceptance criterion (Prochloraz StudyReport). However, the numbers of FI-generation mysids were not sufficient to allow complete evaluation ofvariables for that generation. All but one ofthe variables evaluated during the prochloraz study were nonresponsive as indicated by signal-to-noise ratios <2. Only 7-day weight was responsive. Ignoring the block effect, statistical analyses ofthe endpoints based on the Kruskal-Wallis and Dunn's tests showed that there were no significant differences between treatment mean responses and control mean responses. Dunnett's test, which was conducted afterremoving the blockeffect, indicated that four endpoints showed significant differences between the treatment and control responses (Table 7). However, because the blocking was determined to be ineffective, removing the block effect reduced the error for testing. This likely overestimated the number ofsignificant responses. Thus, the responses ofmysids to prochloraz generally were inconclusive. The lack ofresponse during the prochloraz study may have been partly attributable to the variation in and overlap among doses to which the mysids were exposed. However, the poor feeding regime probably also had an important effect on the study. Flutamide--Analytical measurements offlutamide made during the study were stable and there was no overlap among exposure concentrations during the study. Average measured concentrations were ::::90% ofthe nominal values for each dose level. Only 67% ofthe control pairs produced at least one brood, which does not meet the test acceptance criterion (Flutamide StudyReport). Thenumbers of FI-generation mysids were not sufficient to allow evaluation ofvariables for that generation. Three 

## variablesevaluatedduringthe flutamide study, 7 -daylength and 14-daymale and female length, were

TaskOrder 7 SummaryReport 

### 31determined to be responsive (signal-to-noise ratios >2). Ignoring the block effect, statistical analyses of the endpoints based on the Kruskal-Wallis and Dunn's tests showed that there were no significant differences between treatment mean responses and control mean responses. Dunnett's test, which was conducted after removing the block effect, indicated that four endpoints showed significant differences between the treatment and control responses (Table 9). Three ofthe four treatment responses that were found significantly different from the control response were at the highest dose level (1.8 mg/L flutamide), which could suggest a dose response. However, because the blockingwas determined to be ineffective, removing the block effect reduced the error for testing. This likely overestimated the number ofsignificant responses. Thus, the responses ofmysids to flutamide probably should be considered as generally inconclusive. The lack ofresponse during the flutamide study may have been partly attributable to inadequate diet. Ketoconazole--Analytical measurements ofketoconazole made during the study were relatively variable, but there was no overlap among exposure concentrations during the study. Average concentrations were much less than the nominal values for each dose level. More than 95% ofthe control and vehicle-control pairs produced at least one brood, which exceeds the test acceptance criterion (Ketoconazole Study Report). Three Fo-generation variables evaluated during the ketoconazole study, 7-day length and weight and total number ofyoung, were determined to be responsive. One FI-generation variable, number oftotal young in the second brood, was determined to be responsive. Ignoring the block effect, statistical analyses ofthe endpoints based on the Kruskal-Wallis and Dunn's tests showedthat the FI-generation number ofyoung in the second brood for the Level 3 dose (8.64**~lg/L** ketoconazole) was significantly different from that in the control treatment. Dunnett's test, which was conducted afterremoving the block effect, indicated that the FI-generation number ofyoung in the second brood for the Level 3, Level 4, and LevelS doses were significantly different from that in the control treatment. Theseresults imply that the response may have been dose-dependent. Dunnett's test also indicated that six additional endpoints showed significant differences between the treatment and control responses (Table 11). Three ofthese, Fonumber ofyoung in the first and second broods and total number ofyoung, showed significantdifferences at the highest dose level. The fourth, Fo-generation 7-day weight, showed significant differences at dose Levels 4 and 5. Blockingwas determined to be effective (i.e., blocking increased precision versus a completelyrandomized design); the Dunnett's test may have slightly overestimated the number ofsignificantresponses. Thus, dose-dependentresponse to ketoconazole exposure may have occurred for five endpoints, four ofwhichmeasured reproductive responses. 4-tert-Octylphenol-Analytical measurements of4-tert-octylphenol made during the study were highly variable. There was some overlap among exposure concentrations during the study, but this occurred onlybetween the two lowest dose levels. Average concentrations were much less than the nominal values for each dose level. More than 94% ofthe control and vehicle-control pairsproduced at least one brood, which exceeds the test acceptance criterion (4-tert-Octylphenol Study Report). Four Fo-generation variables evaluated during the 4-tert-octylphenol study, 7-day length and weight and 14-day female length and weight, were determined to be responsive. Ignoring the blockeffect, statistical analyses ofthe endpoints based on the Kruskal-Wallis and Dunn's tests showed that there were no significant differences between treatment mean responses and control mean responses. Dunnett's test, which was conducted after removing the block effect, indicated that nine endpoints showed significantdifferences between the treatment and control responses (Table 13). However, because the blocking was determined to be ineffective, removing the block effect reduced the error for testing. This likely overestimated the number ofsignificantresponses. Thus, the responses ofmysids to 4-tert-octylphenol generally were inconclusive.

Task Order 7 Summary Report 32

### 7.2 SummaryofEndpointEvaluations and Protocol Procedures Endpoint Evaluations-Theresults ofthis study, for which the sensitivity and responsiveness ofthe testing regime were examined and a power analysis for each ofthe variables and generationresponses was conducted, should notbe considered definitive. Only two ofthe chemicals, ketoconazole and 4-tertoctylphenol, met the criteria to evaluate an FI-generation response. Thedifficulties caused by the use ofa self-starting siphon and the inadequate feeding regimes causedpoor reproduction in the prochloraz and flutamide studies such that the F 1generation could notbe evaluated. Given these caveats, the most responsive variables appeared to be basic length and weight measurements. The largest percentages of differences from the control data for prochlorazwere observed in the 7-day weight, for flutamide in the number ofyoung in the second brood, for ketoconazole in the Fa 7-day weight, and for 4-tert-octylphenol in the total youngproduced. These differences were detectable even though weight was recorded to the nearest 1Ilg during these studies, not the 0.1 Ilg recommended by the proposed guideline. Reproductive variables were not veryresponsive, possibly because they tended to be very variable.
In general, the Fa-generation length and weight measurements and number ofyoung produced had the most statistical power. These variables also had the smallest number ofreplicatesrequired to achieve 80% power and the larger signal-to-noise ratios. For some ofthe chemicals, these variables showed an increase over the control, whereas others showed a decrease from the control. A two-sided test should be used ifboth types ofchange are ofinterest.
Most variables required more than five replicates to achieve 80% power for the differences obtained in these tests. Either the exposure doses should be increased so that a greater difference can be observed or more replicates should be used. In general, the Fomeasurements required less replication and had greater power than did the F Iparameters. ProtocolProcedures-Theuse ofthe self-starting siphon is notrequired by the proposed guideline but is listed as an example ofa method to help ensure good water exchange between test chambers. However, use ofthe siphon likely increased the impingement ofmysids while the water in the exposure tanks was drawn down. This artificially increased mortality during the test and reduced the number ofmysids available to complete the study requirements.
The recommended feeding regime is very likely inadequate and contributed to the failure in obtaining sufficient F] mysids in the prochloraz and flutamide studies.
The population ofFomysids that the guideline recommendsbe available to initiate the testing may be inadequate and makes consistent establishment ofbrood pairs difficult. Small initial population sizes may render some statistical results equivocal because ofreduced numbers ofreplicates or individuals per replicate.
All F1mysids used for pairing were 12 to 14 days ofage, depending on the replicate. Because it was necessary to establish a single starting day to track all FI-brood pairs, "Study Day 13" was selected. This meant that pairs often differed in age. Had pairs ofequal ages been established, it would have been necessary to track each brood cup individually. This would have increased the study complexity and costs substantially and data evaluation and interpretation would have been more difficult.
Some endpoint detennination procedures in the guideline were not clear. Forexample, dead young were observed in the control-, vehicle-control, and some test-substance treatments for the ketoconazole and 4-tert-octylphenol studies and were included in the total number ofyoungperbrood. Theprimary focus ofthe testing is on the number oflive young produced and including dead young in the tally ofyoung 

## producedcouldbemisleadingbecausedeadyoungcanbeoverlookedorconsumedbyothermysidsinthe

Task Order7 SummaryReport 33replicate. Because the guidance is unclear, laboratories conducting the test may treat dead young differently. Exclusion ofdead young from the analyses is supported by the similar exclusion ofmortality that results from impingement or other causes not related to test treatments. The analysis ofdry weight by gender might reduce confounding ofthat endpoint by reducing the potential effects ofgender-related size differences. Separate analyses might also help identify effects that might occurwith one gender, but not the other. Therefore, analyzing dry weight by each gender is a useful endpoint. However, excluding immature individuals from the weight endpoint could mask potential differences among treatments related to exposure to a chemical being tested. This latter effect is one that should not be ignored.


## 8.0 Recommendations 8.1 StudyDesign and Statistical Guidance The design ofthe exposure experiment should be a completely randomized design unless blocking is required. In the case ofthe latter, blocks should be characterized by the environmental qualities that make them different and relevant to the experiment. Blocking makes the analysis ofthe data more difficult, especially when a nonparametric analysis is required, and provides a potentially underestimated variance for testing when the blocks are artificial. Acompletely randomized design allows statistical comparisons to be run easily, whereas a block design requires the fitting ofa generalized linear model and more statistical expertise. In all cases, it is important to recognize that the replicates are the treatment tanks, not the individual retention baskets within each tank. Thus, the analyses should be conducted on the mean responses ofthe treatment tanks.
Either the Kruskal-Wallis, followed by the Dunn's test, orthe Dunnett's test is appropriate to test the treatmentresponses under this proposed protocol. One advantage ofusing the Kruskal-Wallis is that potential low-dose effects can be evaluated. When a vehicle control is used, it is appropriate to compare the vehicle-control response to the dilution-water control response. However, the dilution-water control is used as a QA step only and is not used to evaluate the treatment responses. The treatment responses should be evaluated by comparing themto the vehicle-control responses.



### 8.2 Self-Starting Siphon
The ketoconazole and 4-tert-octylphenol studies, for which the use ofthe self-starting siphon was discontinued, were completed successfully with no deterioration ofwater quality. Thus, a simple overflow drain or similar system thatreduces the likelihood ofimpingement is recommended as a modification ofthe proposed guideline. Ifreference to a self-starting siphonremains in the guideline, a note ofcaution that the siphon likely will increase impingement should be added. 

### 8.3 Feeding
The comparisons ofthe control responses under the different feeding regimes strongly suggest that mysids should be fed brine shrimp nauplii adlibitum. The comparisons further suggest that the brine shrimp probably should be enhanced by exposing them to supplements that provide additional highly unsaturated fatty acids (HUFA), especially docosahexaenoic acid (DHA), and eicosapentaenoic acid (EpA), both ofwhich are relatively poor in natural strains ofArtemia (Hanaee et al. 2005). Ranaee et al. (2005) found that enrichingArtemia cysts increased the HUFA content about 1.6 times and the DHA and EpA content by 1.2 and 84 times, respectively. Domingues et al. (200Ia, b) evaluated the effects ofdiet on reproduction and survival in Mysidopsis almyra. One ofthe diets wasArtemia nauplii that had been 

## enrichedwithHUFA. Theyfoundno significantdifference inlarvalproductionand adultsurvival aft r30

Task Order7 Summary Report 34days between mysids fed enrichedArtemia compared with those fed onlyArtemia, but did find that larval survival in the enriched-Artemia treatment was greater than that in theArtemia-only treatment. The ketoconazole and 4-tert-octylphenol studies conducted by ABC Laboratories included a diet of Artemia nauplii that had been enriched with Lifeline Natural Health EnrichmentHUFA, AlgaMac-3000, and AlgaMac-Enhance. The Lifeline supplement provides essential marine w 3-HUFA, is especial1y high in DHA and EpA, and contains vitamins C, A, and E (Shrimpfeeder 2008; product information). AlgaMac-3000 consists ofspray-dried cel1s ofSchizochytrium sp., an alga that has been grown in a sterile environment (Aquafauna 2008a). AlgaMac-Enhance is a spray-dried formulation comprising the intact cel1s oftwo algae, Schizochytrium andHaematococcus, both ofwhich are non-genetically modified organisms (Aquafauna 2008b). The supplement is high in DHA and astaxanthin. Italso contains inactivated yeast, which serves as an additional source ofamino acids, nucleic acids, vitamins, and betaglucans. The addition ofthese or similar supplements to the brine shrimp nauplii is a simple and costeffective way to help ensure that the mysid diet is not a confounding factor during the evaluation of potential EDCs. 

### 8.4 Initial TestPopulation Size Increasing the population ofthe FI-mysid generation available for pairing may increase the certainty of establishing seven brood pairs and may increase confidence in the statistical results. This, ofcourse, would require establishing a largerFa-mysid population at test initiation. Itis recommended that the U.S. EPA consider increasing the population size ofthe Fa mysids in each replicate. Ifthe initial Fa population is not increased, or the increased population is not sufficient during some tests (i.e., reproduction is reduced in response to the chemical being studied) to allow all F 1pairing to occur on the same day, criteria for the selection ofthe first study day for F 1 mysids following pairing should be developed. 

### 8.5 Endpoint Clarification
Theproposed guideline provides considerable detail directing how the weightendpoint is to be obtained, which is appropriate. However, it is recommended that the requirement to measure weight to the "nearest 0.1 /lg" be changedto the "nearest 1 /lg." Most otherendpoints are described only by a simple sentence or phrase and may require additional information to allow consistent determinations to be made across testing laboratories. A primary example concerns the treatment ofmortality that may arise during the testing, but that might not be dose-related. Forexample, guidance could be included that directs a laboratory not to includemortality from impingement in the survival endpoint. Also, direction is needed to specify how dead young observed in broods produced by Fa and F 1mysids should be treated. The guideline should specify that the occurrence and developmental state ofdead young could be determined and recorded, but should not be included in counts ofyoung perbrood even ifwell-developed. The guideline instructions for dry weight measurements should be expanded to specify that weights of males, females, and immature individuals should be measured and recorded. Dataanalyses should then be conducted for each gender and for all mysids combined.


## 9.0 References Aufderheide, J. 2008a. Prochloraz: Two-Generation Life-Cycle Toxicity Test ofthe Saltwater Mysid,Americamysis bahia, Conducted underFlow-ThroughTest Conditions. ABC Laboratories Study No. 62295. Final ReportPrepared for U.S. EPA.

TaskOrder7 SummaryReport 

### 35Aufderheide, J. 2008b. Flutamide: Two-Generation Life-Cycle Toxicity Test ofthe Saltwater Mysid,Americamysis bahia, Conducted under Flow-Through Test Conditions. ABC Laboratories Study No. 62300. Final Report Prepared for U.S. EPA. Aquafauna. 2008a. AlgaMac-3000 Product Information. http://www.aquafauna.com!Diets-AlgaMac3000.htm. Accessed May 22,2008. Aquafauna. 2008b. AlgaMac-Enhance ProductInformation. http://www.aquafauna.comiDiets-AlgaMacEnhance.htm. Accessed May 22,2008. Daniel, Wayne W. 1978. Applied Nonparametric Statistics. Houghton Mifflin Company, Boston, Massachusetts,S 10 pages. Domingues, P.M., P.E. Turk, J.P. Andrade, and P.G. Lee. 200la. Effects ofDifferentFood Items on the Culture ofthe Mysid Shrimp Mysidopsis almyra (Crustacea: Pericaridea) in a StaticWater System. Aquaculture International 9:393-400. Domingues, P.M., P.E. Turk, J.P. Andrade, and P.G. Lee. 2001b. Effects ofEnrichedArtemia Nauplii on Production, Survival and Growth ofthe Mysid ShrimpMysidopsis almyra Bowman 1964 (Crustacea: Mysidacea). Aquaculture Research 32:599-603. Gourmelon, A. and J. Ahtiainen. 2007. DevelopingTest Guidelines on invertebrate development and reproduction for the assessment ofchemicals, includingpotential endocrine active substances-The OEeDperspective. Ecotoxicology 16: 161-167. Hanaee, J., N. Agh, M. Hanaee, A. Delazar, and S.D. Sarker. 2005. Studies onthe Enrichment ofArtemia urmiana Cysts for Improving Fish Food Value. Animal Feed Science and Technology 120: 107-112. Hicks, S.L. 2008a. Ketoconazole: Two-Generation Life-Cycle Toxicity Test ofthe Saltwater Mysid,Americamysis bahia, Conducted under Flow-Through Test Conditions. ABC Laboratories Study No. 62304. Final Report Prepared for U.S. EPA. Hicks, S.L. 2008b. 4-tert-Octylphenol: Two-Generation Life-Cycle Toxicity Test ofthe Saltwater Mysid,Americamysis bahia, Conducted under Flow-Through Test Conditions. ABC Laboratories Study No. 62308. Final Report Prepared for U.S. EPA. Leger, P. and P. Sorgeloos. 1984. International study onArtemia. XXIX. Nutritional evaluation of Artemia nauplii from different geographical origin for the marine crustaceanMysidopsis bahia. Marine Ecology Progress Series 15:307-309.
OECD (Organisation for Economic Co-Operation and Development). 2006. Detailed Review Paper on Aquatic Arthropods in Life Cycle ToxicityTesting with an Emphasis on Developmental, Reproductive and Endocrine Disruptive Effects. OECD Environmental Health and Safety Publications, Series on Testing and Assessment No. 55. Organisation for Economic Co-operation and Development, Paris. Shrimpfeeder. 2008. LifelineNatural Health EnrichmentHUFAProduct Information. http://www.shrimpfeeder.comlhufa.html. Accessed May 22, 2008. Snedecor, G.W. and W.G. Cochran. 1980. Statistical Methods, 6th Edition. The Iowa State University Press. Ames, Iowa. 593 pages.

Task Order 

### 7 Summary Report 36

## Appendix A DataUsed in the StatisticalEvaluations

### Table A.l 

### Non-Vehicle Control MeanResponse Per Replicate



## > Variable Prochloraz Flutamide Ketoconazole 4-tert-Octylphenol A B C A B C A B C A B C 7-dayLength FO 3.28 3.25 3.37 3.43 3.5 3.55 3.5 3.78 3.65 3.32 3.2 3.53 7-dayWeightFO 0.15 0.196 0.221 0.562 0.457 0.434 0.161 0.219 0.249 0.188 0.174 0.237 14-dayMale Length FO 4.18 3.83 4.9

--­ 5.17 4.99 5.3 5.64 5.16 4.95 5.12 14-dayFemale Length FO 4.38 4.35 4.51 5.11 4.8

--­ 4.9 5.51 4.6 4.9 5.07 5.25 14-dayMale Weight FO

--­ --­ --­ --­ 0.566 0.592 0.95 0.797 0.839 0.474 0.544 14-dayFemale Weight FO

--­ --­ --­ 0.562 0.59

--­ 0.882 0.896 0.737 0.634 0.717 0.879 Time to First Brood FO 18 19 17.4 21.7 23.3 23.5 16.9 15.9 16.3 16.1 16.3 16.1 Time to Second Brood FO 23.9 25 23.2 26.5 37 27.3 22.2 21.1 21.9 21.6 2204 22.8 InterbroodDuration FO 5.9 6.7 5.8 7.5 19 7 5.3 5.3 5.6 5.4 6.1 6.7 Total Young 1st Brood FO 4.86 4 6.57 3.57 3.17 1.5 7.86 9 6.71 8.43 6.71 6.57 Total Young 2nd Brood FO 6.4 2.3 6.8 3.5 3 2 10.8 12.1 10.1 10.4 4.1 6.7 Total Young FO 11.3 5.5 1204 6.6 5.7 3.5 15.9 18.7 17.3 18.9 10.9 12.3 O-day Length Fl 1.51 1.5

--­ --­ --­ --­ 1045 1.36 1.38 1.31 1.36 1.32 O-day Weight Fl 0.081 0.0514



### --­ --­ --­ --­ 0.0414 0.1618 0.0348 0.0102 0.0224 0.0052 7-dayLength Fl 3.39

--­ 3.37

--­ --­ --­ 4.05 4 3.95 3.68 3041 3.84 7-day Weight Fl 0.235

--­ 0.305

--­ --­ --­ 00401 0.595

--­ 0.277 0.189 0.313 14-dayMale Length F1 4.54

--­ 4.9

--­ --­ --­ 5047 5.78 5.25 5.57 5.35 5.31 14-dayFemale Length Fl 4.57

--­ 4.59

--­ --­ --­ 5.92 5.98 5.73 5.79 5.57 5.61 14-dayMale Weight Fl

--­ --­ --­ --­ --­ --­ 0.914 0.872 0.792 0.753 0.63 0.607 14-dayFemale Weight Fl

--­ --­ --­ --­ --­ --­ 1.18 1.28 1.17 1.08 0.89 0.93 Time to FirstBrood Fl

--­ --­ --­ --­ --­ --­ 16.7 1404 16.5 18 20.3 20.3 Time to Second Brood F1

--­ --­ --­ --­ --­ --­ 22.8 20.9 23.3 23 25.5 25.7 InterbroodDurationFl

--­ --­ --­ --­ --­ --­ 6.2 604 5.8 5 5.3 5 Total Young 1st Brood F1

--­ --­ 

### --­ --­ --­ --­ 4.67 9043 5.17 8 3.75 6.29 Total Young 2nd Brood Fl --,­ --­ --­ --­ --­ --­ 9.7 9.9 10.3 12.7 12.8 13.2 Total Young Fl

--­ --­ --­ --­ --­ --­ 14.3 19.3 12 20.7 16.5 17.6Table A.2 MeanRelative Difference Between the Maximum and Minimum Response Within a Dose Averaged OverDose
Variable FO Fl Prochloraz Flutamide Ketoconazole 4-tert-Octylphenol Prochloraz Ketoconazole 4-tert-Octylphenol 7-dayLength 6.36% 5.05% 2.94% 5.06% 2.30% 4.72% 6.65% 7-dayWeight 51.8% 28.2% 15.5% 14.3% 19.4% 37.0% 28.6% 14-dayMale Length 12.9% 6.24% 5.05% 3.95% 7.63% 6.31% 4.60% 14-dayFemale Length 6.45% 5.19% 6.16% 6.26% 1.74% 7.46% 2.34% 14-dayMale Weight

-­ 15.8% 23.7% 14.4%

-­ 28.3% 12.0% 14-dayFemale Weight

-­ 14.0% 12.7% 13.4%

-­ 16.4% 23.0% Time to First Brood 12.0% 23.4% 3.10% 5.58%

-­ 24.2% 9.96% Time to Second Brood 12.8% 24.1% 2.52% 13.3%

-­ 19.5% 6.75% Interbrood Duration 38.5% 65.2% 10.3% 41.1%

-­ 32.5% 15.5% Total Young 1st Brood 46.2% 94.2% 17.4% 51.5%

-­ 57.7% 44.3% Total Young 2nd Brood 65.8% 131% 22.4% 97.5%

-­ 33.2% 43.7% Total Young 33.1% 107% 18.5% 63.5%

-­ 48.4% 43.0%

TableA.3 Maximum (±) Percentage ofDifference from Control Response 

## >­ N Variable FO Fl Prochloraz Flutamide Ketoconazole 4-tert-Octylphenol Prochloraz Ketoconazole 4-tert-Octylphenol 7-dayLength -6.77% -11.2% 6.02% -13.4% 3.25% 6.00% -4.23% 7-dayWeight 156% -32.8% 94.7% -38.1% -11.7% 47.6% -13.0% 14-dayMale Length 8.52% -15.4% -3.18% -6.63% 0.00% -4.28% 4.91% 14-dayFemale Length 6.12% -13.6% 4.30% -13.6% 0.78% -4.35% 3.61% 14-dayMale Weight

-­ -17.6% 18.0% -19.9%

-­ -15.6% 8.16% 14-dayFemale Weight

-­ 20.8% 9.60% -39.1%

-­ -14.3% 4.08% Time to First Brood 8.64% -18.4% 3.41% 7.69%

-­ 9.90% -7.20% Tjrne to Second Brood 9.99% -28.5% -3.03% 12.7%

-­ 7.69% -8.05% Interbrood Duration 20.1% -55.2% 8.88% 36.3%



### -­ 32.3% 16.7% Total Young 1st Brood -40.8% -70.5% -31.7% -71.2%

-­ -36.0% 18.1% Total Young 2nd Brood -68.7% -100% -28.9% -76.8%

-­ -69.1% -13.9% Total Young -55.0% -84.6% -38.4% -83.9%

-­ -62.5% -16.3%

### Table A.4 Prochloraz

;J> w Variable KruskalWallis p-value Maximum % Difference Observed Achieved Powerwith given MPD, sigma, and n Detectable Difference at 80% Power with given sigma and n Achieved Power with given sigma,

and nand 10% delta Achieved Powerwith given sigma,

and nand 20% delta Achieved Powerwith given sigma, and nand 50% delta Sample Size Required with given MPD and

sigma at 80% Power 7-dayLengthFO 0.06 3.66 0.27 0.10 0.99 1.00 1.00 8 7-dayWeightFO 0.19 122.64 0.43 0.32 0.05 0.06 0.10 6 14-dayMale Length FO 0.94 4.17 0.11 0.23 0.48 0.99 1.00 23 14-dayFemale Length FO 0.32 2.88 0.16 0.12 0.98 1.00 1.00 14 14-dayWeight FO 0.05 65.02 0.58 0.36 0.06 0.09 0.35 5 Time to First Brood FO 0.51 2.51 0.11 0.25 0.92 1.00 1.00 24 Time to Second Brood FO 0.25 2.79 0.14 0.25 0.96 1.00 1.00 17 InterbroodDurationFO 0.63 6.89 0.07 0.70 0.10 0.30 0.98 53 Total Young 1stBrood FO 0.19 23.48 0.26 0.81 0.08 0.20 0.88 9 T()tal Young 2nd Brood FO 0.14 45.77 0.49 1.11 0.07 0.12 0.57 5 T()tal Young FO 0.03 28.08 0.71 0.72 0.12 0.40 1.00 4 O-day Length F1 0.21 5.81 0.14 0.15 0.36 0.93 1.00 8 O-dayWeightF1 0.59 47.90 0.09 0.13 0.05 0.06 0.10 16

### TableA.5 Flutamide



## >­ Variable KruskalWaJlis p-value Maximum % Difference Observed Achieved Power with given MPD, sigma, and n Detectable Difference at 80% Power with given sigma and n Achieved Power with given sigma,

and nand 10% delta Achieved Power with given sigma,

and nand 20% delta Achieved Power with given sigma, and nand 50% delta Sample Size Required with given MPD and sigma at 80% Power 7-dayLength FO 0.03 6.06 0.83 0.09 1.00 1.00 1.00 3 7-dayWeightFO 0.17 28.61 0.37 0.18 0.08 0.19 0.87 6 14-dayMale Length FO 0.04 7.64 0.64 0.17 0.87 1.00 1.00 3 14-dayFemale Length FO 0.03 6.69 0.58 0.15 0.92 1.00 1.00 3 14-dayMale Weight FO 0.10 14.74 0.16 0.18 0.10 0.28 0.96 7 14-dayFemale WeightFO 0.03 16.01 0.22 0.16 0.11 0.33 0.98 6 Time to FirstBroodFO 0.47 6.26 0.08 1.03 0.12 0.38 0.99 24 Time to Second BroodFO 0.26 9.08 0.12 1.00 0.14 0.46 1.00 10 IrrterbroodDuration FO 0.15 25.50 0.10 2.40 0.06 0.08 0.26 14 Total Young Ist Brood FO 0.16 70.51 0.27 3.69 0.05 0.06 0.15 8 Total Young 2nd Brood F() 0.14 100.00 0.29 5.14 0.05 0.06 0.10 8 Total Young FO 0.18 72.17 0.55 1.69 0.06 0.08 0.27 5

### TableA.6 Ketoconazole

Vl Variable KruskalWallis p-value Maximum 0/0 Difference Observed Achieved Powerwith given MPD, sigma, and n Detectable Difference at 80% Power with given sigma and n Achieved Powerwith given sigma,

and nand 10% delta Achieved Powerwith given sigma,

and nand 20% delta Achieved Powerwith given sigma, and nand 50% delta Sample Size Required with given MPD and

sigma at 80% Power 7-dayLength FO 0.07 3.01 0.61 0.06 1.00 1.00 1.00 4 7-dayWeightFO 0.01 77.57 0.99 0.11 0.07 0.15 0.74 3 14-davMale Length FO 0.44 1.46 0.11 0.09 1.00 1.00 1.00 23 14-davFemale Length FO 0.82 1.89 0.11 0.11 1.00 1.00 1.00 21 14-daYMale Weight FO 0.47 18.01 0.15 0.42 0.08 0.18 0.85 15 14-dayFemale WeightFO 0.50 6.76 0.12 0.15 0.21 0.73 1.00 21 Time to First Brood FO 0.15 1.11 0.24 0.06 1.00 1.00 1.00 9 Time to Second Brood FO 0.07 0.94 0.34 0.05 1.00 1.00 1.00 7 Interbrood Duration FO 0.74 4.03 0.17 0.18 0.80 1.00 1.00 13 Total Young 1st Brood FO 0.02 31.74 0.83 2.16 0.12 0.40 1.00 3 Total Young 2nd Brood 

### FO 0.05 28.91 0.48 3.99 0.09 0.24 0.95 5 Total Young FO 0.01 38.40 0.91 5.45 0.11 0.35 0.99 3 O-daY Length FI 0.44 4.64 0.23 0.08 0.84 1.00 1.00 10 O-davWeight Fl 0.81 43.85 0.08 0.17 0.05 0.06 0.09 39 7-davLength Fl 0.35 3.01 0.30 0.08 1.00 1.00 1.00 8 7-dayWeightFI 0.29 38.82 0.26 0.23 0.06 0.10 0.42 9 14-dayMale LengthFl 0.30 1.97 0.08 0.18 0.81 1.00 1.00 23 14-davFemale Length Fl 0.38 4.35 0.10 0.87 0.39 0.96 1.00 26 14-davMale WeightFl 0.80 15.63 0.07 0.84 0.06 0.09 0.34 27 14-dayFemale WeightFI 0.61 10.23 0.18 0.20 0.17 0.62 1.00 12 Time to FirstBrood FI 0.66 3.30 0.07 0.48 0.34 0.93 1.00 51 Timeto Second Brood FI 0.87 2.31 0.07 0.39 0.59 1.00 1.00 57 Interbrood DurationF1 0.75 10.62 0.11 0.66 0.10 0.32 0.99 22 Total Young 1st Brood FI 0.54 17.34 0.11 1.22 0.07 0.14 0.70 21 Total Young 2nd Brood 

### Fl 0.01 38.92 1.00 0.61 0.19 0.65 1.00 2 TotalYoung Fl 0.16 29.16 0.58 1.11 0.10 0.29 0.98 5

### TableA.7 4-tert-Octylphenol



### ?> 0'1 Variable KruskalWallis p-value Maximum
0/0 Difference Observed Achieved Power with given MPD, sigma, and n Detectable Difference at 80% Power with given sivna and n Achieved Powerwith given sigma,

and nand 10% delta Achieved Powerwith given sigma,

and nand 20% delta Achieved Powerwith given sigma, and nand 50% delta Sample Size Required with given MPD and

sigma at 80% Power 7-dayLengthFO 0.13 13.40 0.96 0.36 0.77 1.00 1.00 3 7-dayWeight FO 0.09 35.70 0.96 0.06 0.14 0.48 1.00 3 14-dayMale Length FO 0.04 6.63 0.61 0.41 0.95 1.00 1.00 4 14-dayFemale Length FO 0.08 13.57 0.42 1.08 0.24 0.77 1.00 4 14-dayMale Weight FO 0.10 16.38 0.36 0.12 0.15 0.52 1.00 7 14-davFemale WeightFO 0.07 33.01 0.74 0.19 0.11 0.32 0.98 3 Time to FirstBrood FO 0.09 2.43 0.35 0.12 1.00 1.00 1.00 7 Time to Second Brood FO 0.74 3.49 0.09 0.44 0.50 0.98 1.00 15 Interbrood Duration FO 0.78 12.06 0.07 1.28 0.07 0.12 0.56 26 Total Young 1st Brood FO 0.05 47.87 0.90 0.91 0.09 0.22 0.92 3 Total Young 2nd Brood FO 0.22 53.87 0.24 2.55 0.06 0.07 0.21 5 Total Young FO 0.05 55.84 0.99 1.10 0.09 0.26 0.96 3 O-dav LengthF 1 0.28 6.51 0.14 0.16 0.29 0.85 1.00 8 O-dav WeightFl 0.67 40.00 0.07 0.04 0.05 0.06 0.08 29 7-dayLength FI 0.47 1.93 0.06 0.28 0.32 0.89 1.00 74 7-davWeightFl 0.73 11.03 0.06 0.24 0.06 0.08 0.25 73 14-dayMale Length F I 0.44 2.19 0.10 0.15 0.94 1.00 1.00 13 14-davFemale Length FI 0.34 1.62 0.15 0.08 1.00 1.00 1.00 8 14-davMale Weight Fl 0.36 8.16 0.07 0.28 0.09 0.22 0.90 26 14-davFemale WeightFl 0.96 3.74 0.05 0.42 0.07 0.12 0.55 263 Time to First Brood Fl 0.29 2.35 0.08 0.32 0.75 1.00 1.00 19 Time to Second Brood Fl 0.27 2.45 0.13 0.24 0.97 1.00 1.00 9 Interbrood Duration Fl 0.19 6.19 0.07 0.65 0.11 0.35 0.99 27 Total Young 1st Brood Fl 0.65 9.12 0.06 1.31 0.07 0.13 0.61 40 Total Young 2nd Brood FI 0.58 5.43 0.06 1.24 0.08 0.18 0.81 72 Total YoungFI 0.74 5.66 0.06 1.26 0.09 0.23 0.91 49