PNWD-3960
 

Evaluation of Proposed Ecotoxicological Testing Guidelines Using 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
 

Marine Research Operations
 

1529 West Sequim Bay Road
 

Sequim, Washington 98382
 

Prepared For
 

Rosemary Hall
 

Task Order Project Officer
 

U.S. Environmental Protection Agency
 

Endocrine Disruptor Screening Program
 

Washington, D.C
 

July 10, 2008
 
Quality Assurance Statement 

This report, "Evaluation of Proposed Ecotoxicological Testing Guidelines Using Mysids to 

Determine Endocrine Disruptive Effects from Chemical Exposure to Prochloraz, Flutamide, 

Ketoconazole, and 4-tert-Octylphenol" has been reviewed by Quality Assurance. The following 

audit was conducted. 

Type of Audit Dates Conducted Date Reported to Date Reported to 

Battelle Program Battelle Facility 

Manager Management 

Audit of Data QuaJity, May 11,25,26,29 & June 27, 2008 June 27, 2008 

Summary report June 2, 13, 2008 , 

The final report has been reviewed for accuracy. 

~U(1(OI l-OOf. 

Date .. .. 

Task Order7 Summary Report 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 of Methods Described in the Guideline 2
 

3.1 Self-Starting Siphon	 3
 

3.2 Feeding Regime	 3
 

3.3 Initial Test Population	 5
 

3.4 Mysid Pairing	 5
 

3.5 Endpoint and Statistical Clarification	 5
 

4.0	 Chemical Exposure Characterization 6
 

5.0	 Dose-Response and Endpoint Evaluation 10
 

Statistical Methods 10
 

6.0	 Results 11
 

6.1 Block Effect	 11
 

6.2 Sensitivity and Responsiveness	 14
 

6.3 Statistically Significant Effects	 19
 

6.4 Power Analysis	 30
 

7.0	 Discussion 31
 

7.1 Summary of Individual Studies	 31
 

7.2 Summary of Endpoint 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 Test Population 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 of Variation, and
 

Percentage of Nominal Dose for Each Dose Level Tested During Task Order 7 7
 

2.	 Upper Triangle Spearman Correlation Matrix of Fa-Generation Non-vehicle Control Mean
 

Response 12
 

3.	 Upper Triangle Spearman Correlation Matrix ofF,-Generation Non-vehicle Control Mean
 

Response 12
 

4.	 Spearman Correlation Matrix of Fa- and F1-Generation Non-vehicle Control Mean Response 13
 

5.	 on-vehicle Control Mean Response and Coefficient of Variation Across Testing Periods
 

(Chemicals) by Generation 15
 

6.	 Signal-to-Noise Ratio by Chemical and Generation 17
 

7.	 Percentage ofincrease (+) or Decrease (-) of Response of Mysids Exposed to Concentrations
 

of Prochloraz Relative to the Control Response 21
 

8.	 Analysis of Variance for the Natural-Logarithm-Transformed Fa-Generation 7-Day Weight
 

for Prochloraz 22
 

9.	 Percentage ofincrease (+) or Decrease (-) of Response ofMysids Exposed to Concentrations
 

of Flutamide Relative to the Control Response 23
 

10.	 Analysis of Variance for the Natural-Logarithm-Transformed Fa-Generation 7-Day Weight
 

for Flutamide 24
 

11.	 Percentage ofincrease (+) or Decrease (-) of Response ofMysids Exposed to Concentrations
 

of Ketoconazole Relative to the Control Response 26
 

12.	 Analysis of Variance for the Natural-Logarithm-Transformed Fa-Generation 7-Day Weight
 

for Ketoconazole 27
 

13.	 Percentage ofincrease (+) or Decrease (-) in Response ofMysids Exposed to Concentrations
 

of 4-tert-Octylphenol Relative to the Control Response 28
 

14.	 Analysis of Variance for the Natural-Logarithm-Transformed Fa-Generation Total Number of
 

Young for 4-tert-Octylpheno1. 29
 

15.	 Power with the Achieved Maximum Percentage of Difference, Sigma, and Number of
 

Replicates 30
 

16.	 Sample Size Required with Achieved Maximum Percentage of Difference, Sigma, and 80%
 

Power 30
 

17.	 Maximum Percentage of Difference from the Observed Control Response (NaturalLogarithm-Transformed Data) 31
 

Task Order 7 Summary Report	 IV 
Figures 

1.	 Mean Number of days to first and second broods produced by Control-treatment Fomysids
 

during each Task Order 7 study 4
 

2.	 Mean Number of young produced by Control-treatment Fo-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 of flutamide 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 of the block effect based on the Kruskal-Wallis test on four test
 

exposures 14
 

8.	 Power of a one-sided independent-sample t-test as a function of the percentage of difference
 

detected between the test and reference means with three replicates per treatment (ex = 0.05) 16
 

9.	 Fo- and F[-generation 14-day female length (top) and weight (bottom) as a function of the
 

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 of the mean concentration ofketoconazole
 

with signal-to-noise ratios of 6.10 and 1.29, respectively 18
 

11.	 Variables with an absolute signal-to-noise ratio >2, suggesting responsiveness 19
 

12.	 Inconsistent block effect on the Fo-generation 7-day weight for each concentration of
 

prochloraz 22
 

13.	 Inconsistent block effect on the Fo-generation 7-day weight for each concentration of
 

flutamide 24
 

14.	 Inconsistent block effect on the Fo-generation 7-day weight for each concentration of
 

ketoconazole 27
 

15.	 Inconsistent block effect on the Fo-generation total number of young for each concentration
 

of 4-tert-octylphenol. 29
 

Task Order 7 Summary Report	 v 
1.0 Introduction 

The U.S. Environmental Protection Agency (U.S. EPA) has established the Endocrine Disruptor 

Screening Program (EDSP) to assess the potential of chemical substances to affect endocrine processes 

in humans and wildlife. Battelle has been a principal contractor in support of this 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 of the 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, which was published in draft form in 2005 and finalized in 2006 

(OECD 2006). Interest in the potential effects of EDCs began to develop within the OECD in the late 

1970s and progressed to the development of a five-level Conceptual Framework for Endocrine 

Disrupters Testing and Assessment in 2002 (Gourmelon and Ahtiainen 2007). The five levels represent 

steps of increasing biological complexity and are not intended to function as the tiers of a multi-step 

testing approach. LevelS represents the highest level of complexity and incorporates paI1ial and full 

life-cycle testing in several animal groups, including invertebrates (Gourmelon and Ahtiainen 2007). 

One of the goals of the OECD testing program is to develop guidelines that will protect sensitive 

populations and not just serve to detect potential EDCs (Gourmelon and Ahtiainen 2007). The mysid 

two-generation test is one of several such tests being developed in concert with the OECD program. 

The demonstration study (Task Order 7) is one of the first steps in the evaluation of this proposed 

guideline for use in examining adverse responses in mysids, Americamysis bahia, exposed to ve11ebraterelevant EDCs. Four chemicals with a range of endocrine-disrupting modes of action 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 of survival, development, sexual maturation, reproduction, and growth were made in 

the Fo- and Ft-mysid generations when possible. Specific measurements were made of development, 

growth, gender ratio, and reproduction (i.e., percentage of actively reproducing females, number of young 

per female in first brood, number of young per female in second brood, time to first- and second-brood 

releases, interbrood duration, replicate mean number of young produced per female in each brood, and 

number of total young per female per replicate), which should be indicative of functional 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 

of the control or vehicle-control treatments. 

The purpose of this report is to summarize the results of each 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. It also will 

review some new endpoints that have recently been suggested as possible tools in EDC-related mysid 

research and evaluate whether or not they should be included in the proposed guideline. Finally, some 

recommendations for modifying the guideline are proposed. 

Task Order 7 Summary Report 
2.0 Quality Assurance 

The ABC Laboratories Quality Assurance (QA) persormel monitored the conduct of the studies as 

outlined in the QAPP and their standard operating procedures. The results of the audits (technical 

systems audits and audits of data 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 audit reports 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 of the 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 Study Director for one chemical and 

transferred from the technical staff to the QA unit 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. EPA as 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 summary report, because recommendations were made that could affect 

revisions to the test protocol. 

An audit of data 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 of Methods Described in the Guideline 

The ABC protocols for all four studies differed from the proposed guidelines in four ways. The mysids 

for each study were fed with Artemia nauplii hatched from cysts that were not obtained from a reference 

source (sensu Leger and Sorgeloos 1984), but were from a reliable source that has been 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 of the nearest 0.1 ~g. The draft 

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 of these modifications is likely to have had an adverse effect on the studies 

and they are not discussed further. Two major adjustments, use of a self-starting siphon and the feeding 

regime, were made after the conduct of the 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 Summary Report	 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 of water by the inverted U-shaped, selfstaliing siphon pumps. During the flutamide definitive study, the use of this siphon was discontinued 

when the Fo adults 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. Data from 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 of young produced in the second brood (5.2 per female) 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 larger than 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 FI mysids in retention baskets was increased similarly. During 

the study, it was observed that very few Artemia were observed at any time other than when the mysids in 

retention baskets were just fed. It was also noticed that the brine shrimp nauplii were observed outside ofthe 

mesh baskets, suggesting that they may be pulled through the mesh during the draw-down procedures. These 

observations, coupled with the apparent loss of Artemia 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 of the first brood in the control 

treatment was longer (-23 days) than that observed during the prochloraz study. The number of young 

produced by both control-treatment broods (-2.8 per female) was very small. Only 67% of controltreatment produced at least one brood, which was less than the guideline specification that 70% produce 

one brood. The poor reproduction during the Fo exposures prevented testing the F I generation for 

prochloraz and flutamide. 

These observations, in addition to previous experience at ABC, led to another modification of the feeding 

strategies for the ketoconazole and 4-tert-octylphenol studies. For these two studies, mysids were fed 

Artemia nauplii ad libitum with three feedings per day. Fo mysids were fed an average of about 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 of three commercially available supplements: 

Lifeline Natural Health Enrichment HUFA, AlgaMac-3000, and AlgaMac-Enhance. 

The effects of these feeding regime alterations were examined statistically by comparing the control 

responses for time to first and second brood, number of young 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 that the flutamide feeding ration had 

the longest time 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 most young for each brood (degrees of freedom [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 

30 - --- a-- -- --­

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 of days to first and second broods produced by Control-treatment F0 

mysids during each Task Order 7 study. 

12 

10 

OJ 8 - C 

:J • 0 • -

>-

0 

6 -- - - -------­ Q; 

.c 

Z 

E 

:J • 4 

2- - -- - •----------- ­

0 

Prochloraz	 Flutanide Ketoconazole 4-lert-Oclylphenol 

,. Tolal Young First Brood _ Total Young Second Brood I 

Figure 2.	 Mean Number of young produced by Control-treatment Fo-generation mysids in the 

first and second broods during each Task Order 7 study. 

An analysis of variance (ANOYA) with a generalized linear model was used to examine the main effects 

of brood (1 versus 2), feeding regime, and their interaction on the number of young produced per brood. 

The hypotheses tested were that neither main effect influenced the number of young per brood. The first 

step in the analyses examined the potential interaction between the two main effects, specifically asking 

whether there was a different brood effect under each feeding regime. This interaction was 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 of brood number (l or 2) on brood size (F = 0.39, df= l,p = 0.538). However, there 

was a significant effect of feeding regime on the number of young per brood (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 of Kruskal-Wallis analyses was used to evaluate the effects of diet 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 weight measured 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 mm versus 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 Test Population 

Consistent establishment of brood pairs often was difficult. During the ketoconazole and 4-tertoctylphenol studies, enough F1 males or ovigerous females were not always available in the reproduction 

retention chamber to obtain the desired seven brood pairs per replicate. Therefore, it was necessary to use 

mature F1 mysids from the growth retention 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 of the 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 of the 

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 of the low numbers of mysids available, as described above, the FImysids comprising 

the pairs were 12 to 14 days of age at the time of pairing, depending on the replicate. Observations 

began on these mysid pairs the day after pairing, which was when the mysids were from 13 to 15 days 

old. It was necessary to establish a single starting day to track all FI-brood pairs and "Study Day 13" was 

selected for both studies. 

3.5 Endpoint and Statistical Clarification 

Some endpoint determination procedures in the guideline were not clear. For example, dead young were 

observed in the control-, vehicle-control, and some test-substance treatments for the ketoconazole and 

Task Order 7 Summary Report 5 
4-tert-octylphenol studies; however, this occurrence did not appear to be related to chemical dose. The 

draft guidance document does not describe how the laboratory should evaluate the occurrence of dead 

young or how dead young should be accounted for in the statistical analyses of the number of young per 

brood. For the ketoconazole and 4-tert-octylphenol studies, the developmental stage of the dead young 

was determined and the dead young were included in the counts of the number of young per brood. 

The dry weight endpoint could use clarification. The proposed guideline states simply that "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 of males 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't necessarily test related 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 provided by the 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 lack of guidance allowed for some initial confusion over whether or not to use one-tailed or 

two-tailed tests and whether the treatment response should be compared with the dilution-water or vehicle 

controls for tests that employed both. 

4.0 Chemical Exposure Characterization 

During each of the 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 which to 

evaluate the biological responses (Table 1). The coefficient of variation (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 of prochloraz, flutamide, 

ketoconazole, and 4-tert-octylphenol during the mysid defmitive study. 

The concentration patterns shown for each of the four chemicals varied considerably. The mean 

prochloraz and flutamide concentrations for each dose level were relatively close to the nominal 

concentrations, ranging from 88% to 97% of the 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 

of 6% and 7% (Table 1). Ketoconazole and 4-tert-octylphenol, the two chemicals that have low solubility 

in water, had measured concentrations that were much less than the nominal concentrations, ranging from 

64% to 69% and 66% to 82%, respectively. Within-study variation was moderate for ketoconazole, with 

CVs ranging from 13% to 25%. Lower doses appeared somewhat more variable than higher doses. 

Concentrations of 4-tert-octylphenol at the two lowest dose levels varied considerably, with CVs of 52% 

and 28%, whereas concentrations of the three highest doses varied relatively little, with CVs ranging from 

8% to 13%. 

Prochloraz concentrations within each dose level varied during the study such that the highest measured 

concentration within a dose level was higher than the lowest measured concentration of the 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 Order 7 Summary Report 6 
level 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 of each 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 of Variation, and 

Percentage of Nominal 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 Exposure Period 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 Exposure Period Levell Level 2 Level 3 Level 4 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 of Nominal 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% 

Task Order 7 Summary Report	 7 
140 

::J 120 

01 

::l. 

100 c: -+--Level1

a 

;; - - -D- _. Level 2 111

-

...

c: 80 --.-- Level 3 Ql 

U 

c: ---<> _. Level 4

a 60 ­ (J ----.- Level 5 N
 

111


... ---Nominal 

a 40 .c 

u 

a... 

Q. 20 

-5 o 5 10 15 20 25 30 35 40 45 50 55 

Study Day 

0+---.-----,----,----,------,-------,--.,..------,-----,-----.----.,.------, 

Figure 3. Nominal and measured concentrations of prochloraz during the mysid definitive study. 

-+--Level1 

. - -0- .. Level 2 

--.-- Level 3 

---<> -­ Level 4 

----.- Level 5 

---Nominal 

45 

• 

35 40

• 

30 

• 

25 

• 

o 5 10 15 20

o-.-.<:y.:...- -o-~._._. ­ -~- -­ '. ·0- -' 

-j--'~---- -­--­

2.125 

2 

1.875 

::J 1.75 

01 1.625 

.s 1.5 

c: 1.375 a 1.25 

-= c: 1.125 Ql 

u 1 c: 

a 0.875

(J 

Ql 0.75

"C 0.625 . E 

!!! 0.5 ::J 0.375 LL. 

0.25 

0.125 

0 

-5 

Study Day 

Figure 4. Nominal and measured concentrations of flutamide during the mysid definitive study. 

Task Order 7 Summary Report 8 
60 ....---------------------------, 

...J 

01 50 +-------------------------1 :::L 

o 

c: 40 -1----------------."L-----.....--------j ~Level1

:;:: 

..

III . _.0- .. Level 2 ...

c: 

QI --A--- Level 3

g 30 -f--I-----~---/"-----------~.

o . - -¢' - - Level 4

U _____ Level 5 QI 

"0 20 .-' -:O-~.-----=-----___l N I---Nominal ,,<>---'--­ III 

c: .0----'0-·.	 -'0------0

o <y' '---p-" o 

10 t-:-::~==~=_-_:_7.....-.1k==='lIIF==:::A"""""-:::::::::::____:_-__.:-_f lI:: 

o ~~~:5=!~'=-~..~~".~~~~.~-..~.-~.-=".~.,,~.. ':"::-- $!:"':-'"=:'­~!==~-J

-5 o 5 10 15 20 25 30 35 40 45 50 

Study Day 

Figure 5. Nominal and measured concentrations of ketoconazole during the mysid definitive 

study. 

60 ....---------------------------, 

...J 

~50 -----_~----------------- c: 

o ~Level1 40 

. - -0- - - Level 2 c: 

QI 

o --A--- Level 3 g 30 

u - . -¢' .. Level 4 

"0 

c: __.__Level5

20 I---Nominal Co <>- .0- -... -<>- --.... :0:= ------ .~. ·----o-~O_ . .. __ . <> 

g 

t 10 QI 

O+--..;--,--,---,---~-,------,r-~~:::::::=;:=~L-___1

-5 o 5 10 15 20 25 30 35 40 45 50 

Study Day 

Figure 6.	 Nominal and measured concentrations of 4-tert-octylphenol during the mysid definitive 

study. 

Task Order 7 Summary Report	 9 
5.0 Endpoint Evaluation 

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 of the dilution-water control data was conducted to evaluate 

the block effect 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 Fo measurements were calculated as the difference between the tank response and the mean of all 

three tanks divided by the standard deviation of the three responses. Because of the small sample size (n :::; 

10) a Kruskal-Wallis test was used to determine the significance of the block effect using each 

measurement's standardized residual as a replicate measurement of the block effect. The Spearman 

correlation coefficient (based on ranks) was calculated, and only those measurements that had an absolute 

correlation of less than 0.8 were used in the analysis. 

A sensitivity analysis, ignoring blocks, of the 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 of within-class noise 

defined as [maximum response - minimum response] / 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 

of the maximum percentage of difference between the control and the treatment response and the average 

within-dose maximum relative difference (a signal-to-noise ratio) 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 ofa parametric test because of the small sample size (n:::; 3) for each treatment 

group. Tests of normality and homogeneity of variance that rely on the chi-square distribution require 

sample sizes of at least 30 or the number of treatment groups times five (Snedecor and Cochran 1980; 

Daniel 1978). Chemical-dosing regimes were considered classifications of fixed effects (i.e., control = 0, 

low dose, mid doses, and high dose specific to each chemical). Box plots were used to visually 

characterize the effect of each 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 a Type 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 Summary Report 10 
needed to achieve 80% power was calculated, given the achieved maximum mean difference. Data were 

transformed to the natural logarithm to maintain homogeneity of the within-class variances for the power 

analysis (Snedecor and Cochran 1980). 

6.0 Results 

6.1 Block Effect 

The Spearman correlation of each 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 block effect based on the non-vehicle control 

data was evaluated by a Kruskal-Wallis test on the standardized residuals of measurements. Only those 

measurements for which each block was 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). The pattern 

of correlation observed in the Fa generation was not the pattern observed in the F[ generation (Table 3). 

The F1 female 14-day lengths and weights were highly correlated with the brood-related measurements. 

Note, however, most of the F[ measurements only had 6 observations because of the 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 F1 mysids were 

O-day weights, 7-day lengths, 14-day male weights, and interbrood duration. The measurements were 

standardized and used as unscaled estimates of the block effect. 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 of the 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's test) and after removing the block effect (Dunnett's test). 

Task Order 7 Summary Report II 
>-J Table 2. Upper Triangle Spearman Correlation Matrix of Fo-Generation Non-vehicle Co

en 

o"" 

..., 

0... 

--.J 

\/).

s:: 

:3 

:3 

Q 

'"0 

o 

::+ 

Measurement 7-d 

Wei~ht

14-d Male 

LeneJh 

14-d Female 

Leneth 

Day to 1st 

Brood 

Day to 

2nd Brood 

Interbrood 

Duration 

Tota

1st 

7-d Length 0.50 0.84 0.45 -0.17 -0.18 -0.19 0

7-d Weight 0.27 0.21 0.42 0.48 0.60 -0

14-d Male Length 0.73 -0.35 -0.38 -0.32 0

14-d Female Length -0.56 -0.41 -0.18 0

Day To 1st Brood 0.93 0.78 -0

Day To 2nd Brood 0.92 -0

Interbrood Duration -0

Total Young 1st Brood 

Total Young 2nd Brood 

(Note: Yellow highlighted cells have an absolute correlation >0.8.)
 

Table 3. Upper Triangle Spearman Correlation Matrix of Fj-Generation Non-vehicle Co

........
 

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 D

O-d Length 0.72 -0.07 -0.10 -0.58 -0.37 0.69 0.46 -0.45 -0.35 

O-d Weight 0.18 0.30 0.01 0.14 0.80 0.84 -0.83 -0.84 

7-d Length 0.75 0.64 0.88 0.83 0.89 -0.73 -0.66 

7-d Weight 0.48 0.73 0.61 0.86 -0.73 -0.59 

14-d Length, Male 0.88 0.43 0.49 -0.36 -0.77 

14-d Length, Female 0.83 0.94 -0.83 -0.94 

14-d Weight, Male 0.89 -0.83 -0.89 

14-d Weight, Female -0.93 -0.89 

Day to 1st Brood 0.83 

Day to 2nd Brood 

Interbrood Duration 

Total Young, 1st 

Brood 

Total Young, 2nd 

Brood 

(Note: Yellow higWighted cells have an absolute correlation >0.8.) 
-l Table 4. Spearman Correlation Matrix of F0- aDd F,-Generation NOD-vehicle CODtro

Pl 

(f) 

7';"' 

Q 

0­

(t) ..., 

-..l 

ifJ 

C 

a 

a 

Q 

'"Cl 

§. 

w 

7-d 14-d Day To Inter- 

7-d H-d Male Day To 1st brood 

Measurement Length Fo Weight Length Fo Female Brood Fo 2nd Brood Duration 

Fo Length Fo Fo Fo 

O-d Length F l -0.23 -0.42 -0.30 -0.72 0.81 0.60 0.18 

O-d Weight F[ 0.11 -0.31 0.06 -0.29 0.26 0.08 -0.30 

7-d Length F [ 0.74 0.12 0.94 0.57 -0.53 -0.69 -0.60 

7-d Weight Fj 0.97 0.44 0.95 0.51 -0.49 -0.48 -0.61 

14-d Male Length FI 0.30 -0.06 0.70 0.81 -0.81 -0.86 -0.62 

14-d Female Length F, 0.66 0.14 0.88 0.66 -0.73 -0.91 -0.79 

14-d Male Weight F[ 0.44 -0.31 0.72 -0.19 0.15 -0.60 -0.90 

14-d Female Weight F1 0.75 0.04 0.86 0.07 -0.26 -0.71 -0.90 

Day To 1st Brood FI -0.71 -0.18 -0.66 0.14 0.25 0.83 0.88 

Day To 2nd Brood Fj -0.37 0.34 -0.61 -0.11 0.26 0.83 0.98 

Interbrood Duration F) 0.58 -0.10 0.95 0.13 -0.09 -0.52 -0.73 

Total Young, 1st Brood F[ 0.52 0.38 -0.07 0.45 -0.90 -0.66 -0.44 

Total Young, 2nd Brood F[ -0.44 0.31 -0.72 0.19 -0.15 0.60 0.90 

Total Young, Fj -0.19 -0.15 -0.33 0.62 -0.79 -0.37 -0.13 

(Note: Yellow higWighted cells have an absolute correlation >0.8.) 
Kruskal-Wallis Test on Test1 Kruskal-Wallis Test on 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 on Test4 

Block Median Ave Rank Z Block Median Ave Rank 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 (adjusted for ties) 

Figure 7. Output from the evaluation of the block effect based on the Kruskal-Wallis test on four 

test exposures. Test! = 4-tert-octylphenol, Test2 = ketoconazole, Test3 = flutamide, 

Test4 = prochloraz. 

6.2 Sensitivity and Responsiveness 

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 of days 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 of young during each brood. Controls (non-vehicle) from the individual 

testing periods were combined and used to evaluate the natural variation of each of the 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 testing period 

(chemical exposure) was genera e same, except for the total number of young 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 of offspring in each brood) and 10 of the 11 

F, variables had CVs <30%. 

Task Order 7 Summary Report 14 
Table 5. Non-vebicle Control Mean Response and Coefficient of Variation Across Testing Periods 

(Chemicals) by Generation 

Non-Vehicle Control 

Fo 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 4.92 10.8% 5.27 7.40% 

14-d Female Length 4.85 7.60% 5.47 10.3% 

14-d Male Weight 0.68 26.4% 0.76 16.4% 

14-d Female Weight 0.74 18.5% 1.09 14.1% 

First Brood 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 1st Brood 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%) 

Task Order 7 Summary Report 15 
10% 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% I0 --0-- CV = 30% '­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 of a one-sided independent-sample t-test as a function of the percentage of 

difference detected between the test and reference means with three replicates per 

treatment (ex = 0.05). 

The signal-to-noise ratio (based on the maximum percentage of difference 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 of the 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 Fo length and weight variables 

were the most responsive variables, even though weight tended to have a greater non-vehicle control CV 

than many of the brood-related variables. Flutamide non-vehicle control data for 7-day weight, however, 

were greater than twice the average of all other non-vehicle control data and may not be representative 

(Table A.I). Thus, this CV may be overestimated. Only one of the F1 variables (total number of young 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 FI variables were nonresponsive in terms of producing a change in 

response greater than twice the within-dose noise. However, the lack of offspring in the high doses of 4­

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 of the signal-to-noise ratio <2, thereby 

suggesting a nonresponsive variable) were slightly larger than, but consistent with, the Fo responses at the 

doses measured. The Fo measurement was only responsive at the highest dose (average concentration = 

41 flg/L; Figure 9). 

Task Order 7 Summary Report	 16 
• •

Table 6. Signal-to-Noise Ratio by Chemical and Generation 

Si nal-to-Noise Ratio 

F1 

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 

First Brood 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 [ - ~------­

.s 0 .-­

.; o o g' [ o o .3 5.0 n ~-O- --0 - ---------- - CIl 

to 

E o CIl 

u. o 

-0 4.5 ­ [J­ "<t 

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

o 5 10 15 20 25 30 35 40 45 

Mean Concentration 4-tert-Octylphenol (J.lg/L) 

Task Order 7 Summary Report 17 
1,2,-----------------------------, 

~~

1,0 -1---._- -.~-- .­

CiE 

0 8 - -~'--nr:t-' • O. -0--

• .-----.-- -------­ , [ D. 0 Q 

	 0,6~ - --0 Q) 

iii o E 

	 0.4 _.- 0-­

-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) 

Figure 9.	 Fo- and F,-generation 14-day female length (top) and weight (bottom) as a function of 

the 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. 

For ketoconazole, however, enough FI young were produced, but were nonresponsive at the 

concentrations tested except for the number of young in the second brood. The variable, 7-day weight 

produced a signal-to-noise ratio of 6.10 for the Fa generation but only 1.29 in the FI generation (Figure 

10. ). Interestingly, the F1 generation had heavier weights and greater variability than did the 

Fo generation. The pattern ofresponse for those variables considered responsive was just as 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-..	 -I!I-­ Cl 

'Qj • Id

3: 0,3 - --Er- ---_._----..---­

E 0 ..:.	 0,2 -8- ---._--- _. -­

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 ofthe mean concentration of 

ketoconazole with signal-to-noise ratios of 6.10 and 1.29, respectively. 

Task Order 7 Summary Report	 18 
0.6 

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, suggesting responsiveness. 

6.3 Statistically Significant Effects 

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. 

For the 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 different based on the nonparametric 

Kruskal-Wallis test (p < 0.05). Of those, none of the treatment concentrations produced results that were 

significantly different from the control based on Dunn's test (p > 0.05). Four of the 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. Of those variables, only the 

Fa total young had a significant response relative to the control response under the three highest multiple 

exposure concentrations (32.1, 53.9, and 93 ~g/L).

The difference in these results was that a block effect was removed from the error term for the Dunnett's 

analysis. Thus, more significant results were detected. The largest percentage of difference that was 

Task Order 7 Summary Report 19 
detected 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 of the 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 of blocking to increase the precision of the experiment over a completely randomized 

design was calculated. Effectiveness was based on the ratio of the estimated error variances for the two 

designs and Fisher's adjustment for the increase in the df error (ORB and OCR for the randomized block 

design and completely random 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)s 2 

(J =--'------'---'-:,--------,-----'--­

CR (U-l) 

where s~ = the block mean square from the randomized block ANOVA 

i = the number of treatment levels, and 

j = the number of blocks (Snedecor and Cochran 1980). 

For the 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 of significant responses. 

Task Order 7 Surmnary Report 20 
Table 7. Percentage ofIncrease (+) or Decrease (-) of Response of Mysids Exposed to
 

Concentrations of Procbloraz 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-day Weight 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-day Female Length Fe 2.4% 6.1% 2.1% 2.9% 4.8% 

10.8% 49.0% 96.9% 21.0% 53.7% 

Time to First Brood Fe -0.4% 0.2% 8.6% 5.7% 3.1% 

Time to Second Brood Fe 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 1st Brood Fe -24.0% -13.9% -36.4% -40.8% -37.5% 

Total Young 2nd Brood Fe -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 Weight F 1 -39.5% -22.5% -48.6% 26.3% -31.5% 

7-day Length F) 2 - 3.3% 2 - 2 - 2 -

7-day Weight FI - -11.7% - - -

14-day Male Length F 1 - 0.0% - - -

14-day Female Length F1 - 0.8% - - -

14-day Weight F1 - -30.3% - - -

I Based on Day 0 through Day 28 of the exposure 

2 The number of F [ 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) 

Task Order 7 Summary Report 21 
0.8 

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. Inconsistent block effect on the Fo-generation 7-day weight for each concentration of 

prochloraz. 

Table 8.	 Analysis of Variance Table for the Natural-Logarithm-Transformed Fo-Generation 

7-Day Weight for Prochloraz 

Source of 

Variation 

Degrees of 

Freedom 

Type III 

Sum of 

Squares 

Type III 

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 Simultaneous Tests 

Comparisons with the Non-Vehicle Control 

Exposure 

Level 

Difference 

of Means 

SEof 

Difference T-Value Adjusted P-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: yellow highlighted cell is a significant result by Dunnett's test.) 

Task Order 7 Summary Report	 22 
For 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 treatment concentrations with
 

mean responses that were significant reductions from the control mean response based on the Dunnett's
 

test and natural-logarithm-transformed data.
 

The largest percentage of difference 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 not have a consistent effect on the results of the weight (Figure 13).
 

The effectiveness of blocking to increase the precision of the experiment over a completely randomized
 

design was calculated. For the 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 1 indicate that the blocking did
 

not increase the precision over the completely randomized design. Therefore, Dunnett's analysis
 

probably overestimated the number of significant responses.
 

Table 9.	 Percentage oflncrease (+) or Decrease (-) of Response of Mysids Exposed to 

Concentrations of Flutamide Relative to the Control Response 

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 First Brood 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 significantly different from the control by 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 ------4+.---.	 ---..----1 - 0 

0.452--- - ~- ­

0.4 -+-0­

...:.. 0.35 f-----

0 ------------...,0:::---­0 __ -_-8-­ o 

0.3 _ .._------------- ------ ­

+ 

0.25 

0.2 -I---,....--------r-----,-----,---,---,.---,---,----.---------' 

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 block effect on the Fo-generation 7-day weight for each concentration of 

flutamide. 

Table to. Analysis of Variance Table for the Natural-Logarithm-Transformed Fo-Generation 7-Day 

Weight for 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 Simultaneous Tests 

Comparisons with the Non-Vehicle Control 

Exposure 

Level 

Difference 

of Means 

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 result by Dunnett's test.) 

Task Order 7 Summary Report 24 
For the ketoconazole exposure (Table 11), the Fa-generation 7-day dry weight (mg) and number of 

offspring and the F,-generation number of offspring in the second brood had a significant Kruskal-Wallis 

test (p < 0.05). Of those, only the F1 generation of the number of offspring in the second brood 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 of offspring produced 

and the F} number of offspring 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 of the 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 of difference 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 of the weight (Figure 14). The effectiveness of 

blocking to increase the precision of the experiment over a completely randomized 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 

completely randomized design. To get the same level of precision (or variance of a treatment mean) 

achieved by the randomized block design, the completely randomized design would require 

n =j (o-%~) or 3.6 replicates (not quite a whole number increase; Snedecor and Cochran 1980). 

Therefore, Dunnett's analysis probably overestimated the number of significant responses. 

Task Order 7 Summary Report 25 
Table II. Percentage oflncrease (+) or Decrease (-) of Response of Mysids Exposed to 

Concentrations of Ketoconazole Relative to the Control Response 

Variable Ketoconazole Concentration (1:I:g!L) 

2.15 4.19 8.64 17.3 33.7 

7-day Length Fo 0.2% 6.0% 2.6% 3.8% 1.9% 

-10.2% 1.3% 14.8% 55.5% 94.7% 

14-day Male Length Fo -1.5% 1.0% -3.2% 0.5% -1.4% 

14-day Female Length Fo 4.3% 1.1% 0.9% 2.5% 1.3% 

14-day Male Weight Fo 0.6% 1.4% 4.2% 18.0% 3.4% 

14-day Female Weight Fo -4.2% -1.4% -0.8% 4.4% 9.6% 

Time to First Brood Fo -1.8% 0.0% -1.6% -0.4% 3.4% 

Time to Second Brood Fo -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 FI 4.2% 1.1% 2.0% 7.2% 2.8% 

O-day Weight FI 20.8% 14.6% 43.8% 1.4% -6.0% 

7-day Length F1 2.1% 2.7% 3.6% 6.0% 4.7% 

7-day Weight FI 2.5% 41.0% 47.6% 21.3% 3.5% 

14-day Male Length FI -4.0% -4.3% -2.1% 3.4% 0.9% 

14-day Female Length FI -4.3% -1.6% 0.0% 3.3% 3.1% 

14-day Male Weight I -15.6% 1.4% -3.1% -1.7% -9.4% 

14-day Female Weight FI -14.3% -6.5% -10.4% -7.8% -5.7% 

Time to First Brood F1 -1.8% 8.3% 9.9% -7.7% 4.2% 

Time to Second Brood FI 1.8% 7.7% 7.5% 1.2% 7.2% 

Interbrood Duration F1 18.7% 3.0% -0.1% 24.1% 32.3% 

Total Young 1st Brood FI -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 or boxed cells are significantly different from the control by Dunnett's 

or Dunn's test, respectively; green highlighted variables have a significant Kruskal-Wallis test) 

Task Order 7 Summary Report 26 
0.6 ..,-------------------------=-------, 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 of Variance 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 

Comparisons with 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	 27 
For 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 of the 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. Of those 

vaIiables, all had a reduction from the control response. 

The Fo-generation total number of young for the 41-)..l.glL-exposure concentration had the largest 

percentage of difference 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 of the production of offspring (Figure 15). 

The effectiveness of blocking to increase the precision of the expeIiment over a completely randomized 

design was calculated. For the Fo-generation total number of young, 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 of significant responses. 

Table 13.	 Percentage oflncrease (+) or Decrease (-) in Response of Mysids Exposed to 

Concentrations of 4-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 Fo -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 Fo -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 Fo -0.1% 3.2% -2.4% 2.8% 12.7% 

Interbrood Duration Fo 0.7% 4.4% -7.8% 6.9% 36.3% 

Total Young 1st Brood Fo -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 Fo -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 Weight F[ -13.0% 4.0% 6.3% -3.4% 

14-day Male Length F1 3.9% -1.0% 4.9% 1.8% 

14-day Female Length F, 3.6% 0.2% 2.4% 0.7% -

14-day Male Weight F, 3.0% -6.7% 8.2% -4.9% -

14-day Female Weight F1 -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 Order 7 Summary Report 28 
25 

? 

¢ 

0 

• 

0 

Cl 20 

c: 

;:, 

0 

>­

-0 15 ... 

CII 

.0 

E 

;:, 

z 10

co-0 

I0 

LI.. 5

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 of young for each 

concentration of 4-tert-octylphenol. 

Table 14.	 Analysis of Variance Table for the Natural-Logarithm-Transformed 

Fo-Generation Total Number of Young 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 

Comparisons with the Non-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.) 

Task Order 7 Summary Report	 29 
6.4 Power Analysis 

The power analysis was conducted on the natural-logarithm-transformed responses, ignoring blocks. 

Appendix Tables 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 between means, and the 

required sample size needed to achieve 80% power for each endpoint measured. For ketoconazole and 

4-tert-octylphenol, a comparison of the Fa and F1 achieved power for each ofthe endpoints was made 

(Table 15). For both exposures, the Fa responses generally had greater achieved power. The highest 

power was obtained for the Fa 7-day lengths and weights, the number of offspring from the first brood, 

and total number of young. A similar result can be obtained by looking at the sample size required to 

achieve 80% power (Table 16). 

Table 15.	 Power with the Achieved Maximum Percentage of Difference, Sigma, and umber of 

Replicates 

Variable Prochloraz Flutamide Ketoconazole 4-tert-Oet vlphenol 

Fo Fo Fo F1 Fo 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% 

First Brood 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 of Difference, Sigma, and 

80% Power 

Variable Prochloraz Flutamide Ketoconazole 4-terl-Octylpbenol 

Fe Fe Fo F1 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 

Second Brood 17 10 7 57 15 9 

Interbrood 53 14 13 22 26 27 

Total Young, 1st brood 9 8 3 21 3 40 

Total Young, 2nd brood 5 8 5 2 5 72 

Total Young 4 5 3 5 3 49 

Task Order 7 Summary Report	 30 
The maximum percentage of difference observed between the dose and the control was different for each 

chemical tested (Table 17). For ketoconazole and 4-tert-octylphenol, the difference was usually greater in 

the Fo-generation response than in that of the Fl. 

Table 17.	 Maximum Percentage of Difference from the Observed Control Response (NaturalLogarithm-Transformed Data) 

Variable 

Prochloraz 

Fo 

Flutamide 

Fo 

Ketoconazole 

Fo 

Ketoconazole 

F1 

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-d Weight 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, 1st brood 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 of Individual Studies 

Prochloraz-Analytical measurements of prochloraz made during the study varied and showed 

occasional overlap of exposure concentrations during the study. Reasons for this variation in 

concentrations within dose levels are not known. About 75% of the control pairs produced at least one 

brood, which exceeds the test acceptance criterion (Prochloraz Study Report). However, the numbers of 

F I-generation mysids were not sufficient to allow complete evaluation of variables for that generation. 

All but one of the 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 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 7). However, because the blocking was 

determined to be ineffective, removing the block effect reduced the error for testing. This likely 

overestimated the number of significant responses. Thus, the responses of mysids 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 of flutamide made during the study were stable and there was no 

overlap among exposure concentrations during the study. Average measured concentrations were ::::90% 

of the nominal values for each dose level. Only 67% of the control pairs produced at least one brood, 

which does not meet the test acceptance criterion (Flutamide Study Report). The numbers of 

F I-generation mysids were not sufficient to allow evaluation of variables for that generation. Three 

variables evaluated during the flutamide study, 7-day length and 14-day male and female length, were 

Task Order 7 Summary Report	 31 
determined 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 of the 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 blocking was determined to be 

ineffective, removing the block effect reduced the error for testing. This likely overestimated the number 

of significant responses. Thus, the responses of mysids to flutamide probably should be considered as 

generally inconclusive. The lack of response 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% of the 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 of young, were determined to be responsive. One 

FI-generation variable, number of total young in the second brood, was determined to be responsive. 

Ignoring the block effect, statistical analyses of the endpoints based on the Kruskal-Wallis and Dunn's 

tests showed that the FI-generation number of young 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 after removing the block effect, indicated that the FI-generation number of young in the second 

brood for the Level 3, Level 4, and LevelS doses were significantly different from that in the control 

treatment. These results 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 of young in the first and second broods and total number 

of young, showed significant differences at the highest dose level. The fourth, Fo-generation 7-day 

weight, showed significant differences at dose Levels 4 and 5. Blocking was determined to be effective 

(i.e., blocking increased precision versus a completely randomized design); the Dunnett's test may have 

slightly overestimated the number of significant responses. Thus, dose-dependent response to 

ketoconazole exposure may have occurred for five endpoints, four of which measured reproductive 

responses. 

4-tert-Octylphenol-Analytical measurements of 4-tert-octylphenol made during the study were highly 

variable. There was some overlap among exposure concentrations during the study, but this occurred 

only between the two lowest dose levels. Average concentrations were much less than the nominal values 

for each dose level. More than 94% of the control and vehicle-control pairs produced 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 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 nine endpoints showed significant differences 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 

of significant responses. Thus, the responses of mysids to 4-tert-octylphenol generally were inconclusive. 

Task Order 7 Summary Report 32 
7.2 Summary of Endpoint Evaluations and Protocol Procedures 

Endpoint Evaluations-The results of this study, for which the sensitivity and responsiveness of the 

testing regime were examined and a power analysis for each of the variables and generation responses 

was conducted, should not be considered definitive. Only two of the chemicals, ketoconazole and 4-tertoctylphenol, met the criteria to evaluate an FI-generation response. The difficulties caused by the use of a 

self-starting siphon and the inadequate feeding regimes caused poor reproduction in the prochloraz and 

flutamide studies such that the F1 generation could not be 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 prochloraz were observed in the 7-day weight, for flutamide in the 

number of young in the second brood, for ketoconazole in the Fa 7-day weight, and for 4-tert-octylphenol 

in the total young produced. These differences were detectable even though weight was recorded to the 

nearest 1 Ilg during these studies, not the 0.1 Ilg recommended by the proposed guideline. Reproductive 

variables were not very responsive, possibly because they tended to be very variable. 

In general, the Fa-generation length and weight measurements and number of young produced had the 

most statistical power. These variables also had the smallest number ofreplicates required to achieve 

80% power and the larger signal-to-noise ratios. For some of the chemicals, these variables showed an 

increase over the control, whereas others showed a decrease from the control. A two-sided test should be 

used if both types of change are of interest. 

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 Fo measurements required less replication and had greater 

power than did the F I parameters. 

Protocol Procedures-The use of the self-starting siphon is not required by the proposed guideline but is 

listed as an example of a method to help ensure good water exchange between test chambers. However, 

use of the 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 of mysids 

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 ofFo mysids that the guideline recommends be available to initiate the testing may be 

inadequate and makes consistent establishment of brood pairs difficult. Small initial population sizes may 

render some statistical results equivocal because ofreduced numbers ofreplicates or individuals per 

replicate. 

All F1 mysids used for pairing were 12 to 14 days of age, depending on the replicate. Because it was 

necessary to establish a single starting day to track all F I-brood pairs, "Study Day 13" was selected. This 

meant that pairs often differed in age. Had pairs of equal 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. For example, 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 of young per brood. The primary focus 

of the testing is on the number of live young produced and including dead young in the tally of young 

produced could be misleading because dead young can be overlooked or consumed by other mysids in the 

Task Order 7 Summary Report 33 
replicate. Because the guidance is unclear, laboratories conducting the test may treat dead young 

differently. Exclusion of dead young from the analyses is supported by the similar exclusion of mortality 

that results from impingement or other causes not related to test treatments. The analysis of dry weight 

by gender might reduce confounding of that endpoint by reducing the potential effects of gender-related 

size differences. Separate analyses might also help identify effects that might occur with 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 Study Design and Statistical Guidance 

The design of the exposure experiment should be a completely randomized design unless blocking is 

required. In the case of the latter, blocks should be characterized by the environmental qualities that 

make them different and relevant to the experiment. Blocking makes the analysis of the data more 

difficult, especially when a nonparametric analysis is required, and provides a potentially underestimated 

variance for testing when the blocks are artificial. A completely randomized design allows statistical 

comparisons to be run easily, whereas a block design requires the fitting of a 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 of the treatment tanks. 

Either the Kruskal-Wallis, followed by the Dunn's test, or the Dunnett's test is appropriate to test the 

treatment responses under this proposed protocol. One advantage of using 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 them to the vehicle-control responses. 

8.2 Self-Starting Siphon 

The ketoconazole and 4-tert-octylphenol studies, for which the use of the self-starting siphon was 

discontinued, were completed successfully with no deterioration of water quality. Thus, a simple 

overflow drain or similar system that reduces the likelihood of impingement is recommended as a 

modification of the proposed guideline. Ifreference to a self-starting siphon remains in the guideline, a 

note of caution that the siphon likely will increase impingement should be added. 

8.3 Feeding 

The comparisons of the control responses under the different feeding regimes strongly suggest that 

mysids should be fed brine shrimp nauplii ad libitum. 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 of which are relatively poor in natural strains of Artemia (Hanaee et al. 2005). Ranaee et al. 

(2005) found that enriching Artemia 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 of diet 

on reproduction and survival in Mysidopsis almyra. One of the diets was Artemia nauplii that had been 

enriched with HUFA. They found no significant difference in larval production and adult survival aft r 30 

Task Order 7 Summary Report 34 
days between mysids fed enriched Artemia compared with those fed only Artemia, but did find that larval 

survival in the enriched-Artemia treatment was greater than that in the Artemia-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 Enrichment HUFA, 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 of spray-dried cel1s of Schizochytrium sp., an alga that has been grown in a sterile 

environment (Aquafauna 2008a). AlgaMac-Enhance is a spray-dried formulation comprising the intact 

cel1s of two algae, Schizochytrium and Haematococcus, both of which are non-genetically modified 

organisms (Aquafauna 2008b). The supplement is high in DHA and astaxanthin. It also contains 

inactivated yeast, which serves as an additional source of amino acids, nucleic acids, vitamins, and betaglucans. The addition of these 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 Test Population Size 

Increasing the population of the F I-mysid generation available for pairing may increase the certainty of 

establishing seven brood pairs and may increase confidence in the statistical results. This, of course, 

would require establishing a larger Fa-mysid population at test initiation. It is recommended that the U.S. 

EPA consider increasing the population size of the 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 F1 pairing to occur on the same day, 

criteria for the selection of the first study day for F1 mysids following pairing should be developed. 

8.5 Endpoint Clarification 

The proposed guideline provides considerable detail directing how the weight endpoint is to be obtained, 

which is appropriate. However, it is recommended that the requirement to measure weight to the "nearest 

0.1 /lg" be changed to the "nearest 1 /lg." Most other endpoints 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 of mortality that may arise during the 

testing, but that might not be dose-related. For example, guidance could be included that directs a 

laboratory not to include mortality from impingement in the survival endpoint. Also, direction is needed 

to specify how dead young observed in broods produced by Fa and F1 mysids should be treated. The 

guideline should specify that the occurrence and developmental state of dead young could be determined 

and recorded, but should not be included in counts of young per brood even if well-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. Data analyses 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 of the Saltwater 

Mysid, Americamysis bahia, Conducted under Flow-Through Test Conditions. ABC Laboratories Study 

No. 62295. Final Report Prepared for U.S. EPA. 

Task Order 7 Summary Report 35 
Aufderheide, J. 2008b. Flutamide: Two-Generation Life-Cycle Toxicity Test of the 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 Product Information. 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. 200 la. Effects of Different Food Items on the 

Culture of the Mysid Shrimp Mysidopsis almyra (Crustacea: Pericaridea) in a Static Water System. 

Aquaculture International 9:393-400. 

Domingues, P.M., P.E. Turk, J.P. Andrade, and P.G. Lee. 2001 b. Effects of Enriched Artemia Nauplii on 

Production, Survival and Growth of the Mysid Shrimp Mysidopsis almyra Bowman 1964 (Crustacea: 

Mysidacea). Aquaculture Research 32:599-603. 

Gourmelon, A. and J. Ahtiainen. 2007. Developing Test Guidelines on invertebrate development and 

reproduction for the assessment of chemicals, including potential endocrine active substances-The 

OEeD perspective. Ecotoxicology 16: 161-167. 

Hanaee, J., N. Agh, M. Hanaee, A. Delazar, and S.D. Sarker. 2005. Studies on the 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 of the 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 on Artemia. XXIX. Nutritional evaluation of 

Artemia nauplii from different geographical origin for the marine crustacean Mysidopsis 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 Toxicity Testing 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. Lifeline Natural Health Enrichment HUFA Product 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
 

Data Used in the Statistical Evaluations
 
Table A.l Non-Vehicle Control Mean Response Per Replicate 

>
 

Variable Prochloraz Flutamide Ketoconazole 4-tert-Octylphenol 

A B C A B C A B C A B C 

7 -day Length 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-day Weight FO 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-day Male Length FO 4.18 3.83 4.9 --­ 5.17 4.99 5.3 5.64 5.16 4.95 5.12 

14-day Female 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-day Male Weight FO --­ --­ --­ --­ 0.566 0.592 0.95 0.797 0.839 0.474 0.544 

14-day Female 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 

Interbrood Duration 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-day Length 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-day Male Length F1 4.54 --­ 4.9 --­ --­ --­ 5047 5.78 5.25 5.57 5.35 5.31 

14-day Female Length Fl 4.57 --­ 4.59 --­ --­ --­ 5.92 5.98 5.73 5.79 5.57 5.61 

14-day Male Weight Fl --­ --­ --­ --­ --­ --­ 0.914 0.872 0.792 0.753 0.63 0.607 

14-day Female Weight Fl --­ --­ --­ --­ --­ --­ 1.18 1.28 1.17 1.08 0.89 0.93 

Time to First Brood 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 

Interbrood Duration Fl --­ --­ --­ --­ --­ --­ 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 17.6 
Table A.2 Mean Relative Difference Between the Maximum and Minimum Response Within a Dose Averaged Over Dose 

Variable FO Fl 

Prochloraz Flutamide Ketoconazole 4-tert-Octylphenol Prochloraz Ketoconazole 4-tert-Octylphenol 

7-day Length 6.36% 5.05% 2.94% 5.06% 2.30% 4.72% 6.65% 

7 -day Weight 51.8% 28.2% 15.5% 14.3% 19.4% 37.0% 28.6% 

14-day Male Length 12.9% 6.24% 5.05% 3.95% 7.63% 6.31% 4.60% 

14-day Female Length 6.45% 5.19% 6.16% 6.26% 1.74% 7.46% 2.34% 

14-day Male Weight -­ 15.8% 23.7% 14.4% -­ 28.3% 12.0% 

14-day Female 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% 

Table A.3 Maximum (±) Percentage of Difference from Control Response 

>­

N Variable FO Fl 

Prochloraz Flutamide Ketoconazole 4-tert-Octylphenol Prochloraz Ketoconazole 4-tert-Octylphenol 

7-day Length -6.77% -11.2% 6.02% -13.4% 3.25% 6.00% -4.23% 

7-day Weight 156% -32.8% 94.7% -38.1 % -11.7% 47.6% -13.0% 

14-day Male Length 8.52% -15.4% -3.18% -6.63% 0.00% -4.28% 4.91% 

14-day Female Length 6.12% -13.6% 4.30% -13.6% 0.78% -4.35% 3.61% 

14-day Male Weight -­ -17.6% 18.0% -19.9% -­ -15.6% 8.16% 

14-day Female 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 

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 -day Length FO 0.06 3.66 0.27 0.10 0.99 1.00 1.00 8 

7-day Weight FO 0.19 122.64 0.43 0.32 0.05 0.06 0.10 6 

14-day Male Length FO 0.94 4.17 0.11 0.23 0.48 0.99 1.00 23 

14-day Female Length FO 0.32 2.88 0.16 0.12 0.98 1.00 1.00 14 

14-day Weight 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 

Interbrood Duration FO 0.63 6.89 0.07 0.70 0.10 0.30 0.98 53 

Total Young 1st Brood 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-day Weight F1 0.59 47.90 0.09 0.13 0.05 0.06 0.10 16 
Table A.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 -day Length FO 0.03 6.06 0.83 0.09 1.00 1.00 1.00 3 

7-day Weight FO 0.17 28.61 0.37 0.18 0.08 0.19 0.87 6 

14-day Male Length FO 0.04 7.64 0.64 0.17 0.87 1.00 1.00 3 

14-day Female Length FO 0.03 6.69 0.58 0.15 0.92 1.00 1.00 3 

14-day Male Weight FO 0.10 14.74 0.16 0.18 0.10 0.28 0.96 7 

14-day Female Weight FO 0.03 16.01 0.22 0.16 0.11 0.33 0.98 6 

Time to First Brood FO 0.47 6.26 0.08 1.03 0.12 0.38 0.99 24 

Time to Second Brood FO 0.26 9.08 0.12 1.00 0.14 0.46 1.00 10 

Irrterbrood Duration FO 0.15 25.50 0.10 2.40 0.06 0.08 0.26 14 

Total Young I st 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 
Table A.6 Ketoconazole 

Vl 

Variable 

KruskalWallis 

p-value 

Maximum 

0/0 

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 -day Length FO 0.07 3.01 0.61 0.06 1.00 1.00 1.00 4 

7-day Weight FO 0.01 77.57 0.99 0.11 0.07 0.15 0.74 3 

14-dav Male Length FO 0.44 1.46 0.11 0.09 1.00 1.00 1.00 23 

14-dav Female Length FO 0.82 1.89 0.11 0.11 1.00 1.00 1.00 21 

14-daY Male Weight FO 0.47 18.01 0.15 0.42 0.08 0.18 0.85 15 

14-day Female Weight FO 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-dav Weight Fl 0.81 43.85 0.08 0.17 0.05 0.06 0.09 39 

7-dav Length Fl 0.35 3.01 0.30 0.08 1.00 1.00 1.00 8 

7-day Weight FI 0.29 38.82 0.26 0.23 0.06 0.10 0.42 9 

14-day Male Length Fl 0.30 1.97 0.08 0.18 0.81 1.00 1.00 23 

14-dav Female Length Fl 0.38 4.35 0.10 0.87 0.39 0.96 1.00 26 

14-dav Male Weight Fl 0.80 15.63 0.07 0.84 0.06 0.09 0.34 27 

14-day Female Weight FI 0.61 10.23 0.18 0.20 0.17 0.62 1.00 12 

Time to First Brood FI 0.66 3.30 0.07 0.48 0.34 0.93 1.00 51 

Time to Second Brood FI 0.87 2.31 0.07 0.39 0.59 1.00 1.00 57 

Interbrood Duration F1 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 

Total Young Fl 0.16 29.16 0.58 1.11 0.10 0.29 0.98 5 
Table A.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 

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-day Length FO 0.13 13.40 0.96 0.36 0.77 1.00 1.00 3 

7-day Weight FO 0.09 35.70 0.96 0.06 0.14 0.48 1.00 3 

14-day Male Length FO 0.04 6.63 0.61 0.41 0.95 1.00 1.00 4 

14-day Female Length FO 0.08 13.57 0.42 1.08 0.24 0.77 1.00 4 

14-day Male Weight FO 0.10 16.38 0.36 0.12 0.15 0.52 1.00 7 

14-dav Female Weight FO 0.07 33.01 0.74 0.19 0.11 0.32 0.98 3 

Time to First Brood 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 Length F 1 0.28 6.51 0.14 0.16 0.29 0.85 1.00 8 

O-dav Weight Fl 0.67 40.00 0.07 0.04 0.05 0.06 0.08 29 

7-day Length FI 0.47 1.93 0.06 0.28 0.32 0.89 1.00 74 

7-dav Weight Fl 0.73 11.03 0.06 0.24 0.06 0.08 0.25 73 

14-day Male Length F I 0.44 2.19 0.10 0.15 0.94 1.00 1.00 13 

14-dav Female Length FI 0.34 1.62 0.15 0.08 1.00 1.00 1.00 8 

14-dav Male Weight Fl 0.36 8.16 0.07 0.28 0.09 0.22 0.90 26 

14-dav Female Weight Fl 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