BES Project No. 27568 EPA Contract No. EP-W-06-026 

Study Title 

Effect of Vinclozolin on the Survival, Development, and Reproduction of the Marine 

Copepod Amphiascus Tenuiremis

Block Environmental Services Authors 

David S. Block, Ph.D. 

Nanette Bradbury 

Study Completion Date 

 May 30, 2012 

Submitted To 

RTI International 

3040 East Cornwallis Road 

Research Triangle Park, NC 27709-2194 

Performing Laboratory 

Block Environmental Services, Inc 

2451 Estand Way 

Pleasant Hill, CA 94523 

Laboratory Project ID 

 BES Study No. 27568 

 RTI Study No. 0210114.030 

 EPA Contract No. EP-W-06-026 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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QUALITY ASSURANCE STATEMENT 

The study conduct, raw data and report for “Effect of Vinclozolin on the Survival and 

Reproduction of the Marine Copepod Amphiascus Tenuiremis” was inspected by the Quality 

Systems Director at Block Environmental Services, Inc (BES) to determine adherence with the 

Quality Assurance Project Plan (QAPP) and laboratory standard operating procedures. Dates of 

study inspections, inspection types, and dates reported to the Program Manager are given below. 

Inspection Date Inspection Type Reported to Program Manager 

 27568 (Vinclozolin)

9/26/2011 in-test, setup 9/26/2011 

10/24/2011 in-test, mating 10/24/2011 

11/15/2011 analytical methods verification 11/15/2011 

1/6/2011 chemistry 1/12/2012 

3/30/2011 data, biology spreadsheets 4/4/2012 

 

5/18/2012 draft report 5/29/2012 

 

BLOCK ENVIRONMENTAL SERVICES, INC. 

________________________________________________ ____May 29, 2012____

Ronald M. Block, Ph.D. Date 

Quality Systems Director 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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KEY STUDY PERSONNEL 

The following BES personnel were responsible for the conduct of the work and reporting of the 

study results. 

David S. Block, Ph.D. 

5/30/2012 

 Program Manger/Technical Report Writer Date 

Ronald M. Block, Ph.D Quality Systems Director 

Nanette Bradbury Senior Biologist/Technical Report Writer 

Terence Clohesy Biologist 

Sebastian Castro Biologist 

Amy Ayala Biologist 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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DATA RETENTION 

All records that would be required to reconstruct the study will be maintained. The original raw 

data and the original final report will be maintained indefinitely in the form of a hard copy to 

comply with EPA record keeping regulations. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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TABLE OF CONTENTS 

QUALITY ASSURANCE STATEMENT .................................................................................. 2

KEY STUDY PERSONNEL........................................................................................................ 3

DATA RETENTION .................................................................................................................... 4

TABLE OF CONTENTS ............................................................................................................. 5

ABSTRACT................................................................................................................................... 8

1 INTRODUCTION................................................................................................................... 9

2 MATERIALS AND METHODS ......................................................................................... 10

2.1 QAPP/Protocol.............................................................................................................. 10 

2.2 Culturing test organisms ............................................................................................... 10 

2.2.1 Source of copepods................................................................................................... 10 

2.2.2 Culture medium & diet ............................................................................................. 10 

2.2.3 Laboratory Reagent Water for Culture Media Preparation....................................... 10 

2.2.4 Culture Sediment ...................................................................................................... 11 

2.2.5 Photoperiod............................................................................................................... 11 

2.3 Toxicity test procedure ................................................................................................. 11 

2.3.1 Test chemical ............................................................................................................ 11 

2.3.2 Preparation of test solutions...................................................................................... 12 

2.3.3 Test equipment.......................................................................................................... 12 

2.3.4 Collection of copepod nauplii in preparation for the test ......................................... 13 

2.3.5 Diet during testing..................................................................................................... 14 

2.3.6 Daily observations .................................................................................................... 14 

2.3.7 Test conditions.......................................................................................................... 14 

2.3.8 Vinclozolin concentrations in test solutions ............................................................. 14 

2.3.9 Statistical analyses .................................................................................................... 15 

3 RESULTS .............................................................................................................................. 16

3.1 Physico-chemical properties of test solutions............................................................... 16 

3.2 Measured Vinclozolin concentrations in test solutions ................................................ 16 

3.3 Biological data .............................................................................................................. 16 

3.3.1 Survival on day 16 .................................................................................................... 16 

3.3.2 Survival on day 36 .................................................................................................... 17 

3.3.3 Day 16 Versus Day 36 Survival................................................................................ 17 
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3.3.4 Development – Maturation time between naupliar and copepodite stages............... 17 

3.3.5 Development – Maturation time between copepodite and adult stages.................... 17 

3.3.6 Development – Maturation time between naupliar and adult stages........................ 18 

3.3.7 Sex ratio .................................................................................................................... 18 

3.3.8 Reproduction – time to first egg extrusion ............................................................... 18 

3.3.9 Reproduction – number of hatched nauplii from first brood .................................... 18 

3.3.10 Reproduction – time to second egg extrusion....................................................... 19 

3.3.11 Reproduction – number of hatched nauplii from second brood ........................... 19 

3.3.12 Reproduction – number of nauplii hatched from two successive broods ............. 19 

3.3.13 Reproduction - fertilization success...................................................................... 19 

3.4 Summary of NOEC and LOEC for all endpoints ......................................................... 20 

4 DISCUSSION ........................................................................................................................ 20

4.1 Quality Objectives and criteria ..................................................................................... 20 

4.2 Discussion of results ..................................................................................................... 20 

5 ACKNOWLEDGEMENTS ................................................................................................. 21

6 REFERENCES...................................................................................................................... 21

7 FIGURES............................................................................................................................... 23

Figure 1: Layout of Microplate Test Chamber ................................................................. 23 

8 TABLES................................................................................................................................. 23

Table 1: Method for preparation of Vinclozolin 200 mg/L primary stock solutions........ 23 

Table 2: Method for preparation of Vinclozolin test solutions......................................... 24 

Table 3: Water quality parameters: pH, salinity, dissolved oxygen ................................. 24 

Table 4: Water quality parameter: daily temperature ....................................................... 24 

Table 5: Summary of water quality parameters................................................................ 25 

Table 6: Measured Vinclozolin test concentrations.......................................................... 26 

Table 7: Survival on day 16.............................................................................................. 27 

Table 8: Survival on day 36.............................................................................................. 28 

Table 9: Time to transformation to copepodite stage ....................................................... 29 

Table 10: Time in copepodite stage.................................................................................. 30 

Table 11: Time to transformation to adult stage............................................................... 31 

Table 12: Sex ratio............................................................................................................ 32 

Table 13: Time to first egg extrusion since date of pairing .............................................. 33 

Table 14: Number of nauplii hatched from first brood..................................................... 34 

Table 15: Time to second egg extrusion following hatching of first brood offspring...... 35 
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Table 16: Number of nauplii hatched from second brood ................................................ 36 

Table 17: Total number of nauplii that hatched from two successive broods .................. 37 

Table 18: Fertilization success.......................................................................................... 38 

Table 19: Summary table of NOEC and LOEC for all endpoints .................................... 39 

9 APPENDIX I: QUALITY ASSURANCE PROJECT PLAN (QAPP)............................. 40

10 APPENDIX II: QUALITY OF LABORATORY WATER AT BES ............................... 75

11 APPENDIX III: VINCLOZOLIN CHEMISTRY DATA REPORT ............................... 76

12 APPENDIX IV: STATISTICAL DATA OUTPUTS....................................................... 116

13 APPENDIX V: STUDY PROTOCOL (P27568) .............................................................. 165

14 APPENDIX VI: DEVIATIONS FROM STUDY PROTOCOL ..................................... 177

14.1 List of deviations......................................................................................................... 177 
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ABSTRACT 

The current study assessed the chronic sensitivity of the meiobenthic copepod Amphiascus 

tenuiremis to vinclozolin concentrations of 12.96, 21.6, 36, 60, and 100-μg/L using a microplatebased life-cycle screening assay. Copepod survival in the laboratory control and carrier control 

on Day 16 was 80% and 90%, respectively, with respective survival rates decreasing to 75% and 

84% by Day 36. No Vinclozolin test concentration significantly affected copepod survival on 

Day 16 and Day 36 time points relative to the carrier control response. Vinclozolin did not have 

a significant effect on A. tenuiremis development into either the copepodite or adult life-stage in 

any exposure concentration. Based on the development data obtained during the study, NOEC 

and LOEC values were calculated to be 100μg/L and >100μg/L respectively. None of the 

Vinclozolin concentrations significantly affected the ratio of female:male copepod development. 

Vinclozolin was observed to have a significant effect on copepod reproduction, with an increase 

in the time for virgin females to extrude their first brood in the 21.6 and 36μg/L treatments 

relative to the carrier control; no such delay was observed in the higher vinclozolin exposure 

concentrations. Vinclozolin did not have a significant effect on the total number of viable eggs 

and hatched nauplii, nor the percentage of successful mating pairs compared to the carrier 

control. Based on the mating success data, NOEC and LOEC values were calculated to be 100 

and >100μg/L, respectively. All statistical analyses were performed using nominal values. 
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1 INTRODUCTION 

The U.S. Environmental Protection Agency (EPA) is currently implementing the Endocrine 

Disruptor Screening Program (EDSP). To support this program, EPA has contracted with RTI 

International (RTI) to provide comprehensive toxicological testing services. These services were 

designed to assist the EPA in validating a suite of longer term Tier 2 in vivo, arthropod 

ecotoxicological tests for identifying and characterizing endocrine effects through exposure to 

pesticides, industrial chemicals, and environmental contaminants. The objective of the validation 

studies are two-fold: one, ascertain the value added from a mysid two-generation growth and 

reproduction test as compared to existing mysid life cycle methodologies; and two, evaluate a 

microplate-based copepod development and reproduction test method using the marine 

harpacticoid copepod Amphiascus tenuiremis. In addition to method validation, the results and 

conclusions from these studies will be used to develop appropriate guidance documents for peer 

review of the methods and standardize technical guidance and test guidelines in support of the 

Office of Chemical Safety and Pollution Prevention regulatory programs. These studies were 

conducted under the study protocol, applicable Quality Assurance Project Plans (QAPPs), 

relevant program and facility standard operating procedures (SOPs), and published guidance 

documents. 

The purpose of work to be conducted by Block Environmental Services, Inc. (BES) under this 

assignment is to demonstrate a copepod development and reproduction test based on ASTM E 

2317-04 “Standard guide for conducting renewal microplate-based life-cycle toxicity tests with a 

marine meiobenthic copepod” (ASTM, 2004) and the June 2009 proposed OECD Test 

Guidelines for the Harpacticoid Copepod Development and Reproduction Test with Amphiascus 

tenuiremis (OECD 2009). As indicated above, the test is intended to serve as a higher tier test 

with an aquatic arthropod for collecting definitive concentration-response information on adverse 

effects suitable for use in ecological risk assessment. Specifically, collection of development, 

reproductive and life-table parameter data from this copepod species provides sensitive and 

ecologically relevant measures that can be used to determine the potential chronic toxicity of 

many water-borne contaminants. Endpoints included survival, development, and specific 

reproductive output measures, time to first and second broods, number of offspring, and 

reproductive success. 

The experimental phase of the study was conducted from September 27, 2011 to November 2, 

2011. The original raw data, protocol, and technical report produced during this study are stored 

at the BES laboratory, located in Pleasant Hill, California. 
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2 MATERIALS AND METHODS 

2.1 QAPP/PROTOCOL 

This study was conducted according to the procedures outlined in the Quality Assurance Project 

Plan (QAPP) for Task Order 30 Mysid/Copepod Inter laboratory Test Evaluation. The QAPP can 

be found in Appendix I. The study protocol was designed to assess the effect of Vinclozolin on 

the survival rate, development, mating success and reproductive output of the copepod A. 

Tenuiremis (OECD, 2009). Where deviations occurred they have been listed in and discussed 

where required in Appendix VI. 

2.2 CULTURING TEST ORGANISMS 

2.2.1 Source of copepods 

A gift of A. tenuiremis was received from Professor G.T. Chandler (University of South 

Carolina, School of Public Health, Columbia, South Carolina, USA) on March 19, 2011. 

Information on the original source of this copepod population can be found in ASTM (2004). 

On arrival at the BES Laboratory, copepod cultures were maintained in food grade polyethylene 

(Tupperware®) containers containing approximately 2 liters of culture media and one centimeter 

of 63μm sieved and washed sediment. 

2.2.2 Culture medium & diet 

Care for the copepod culture at BES followed the instructions presented by Ms. Kate Washburn 

with the Chandler Laboratory. Synthetic Instant Ocean® seawater was used to prepare the 

copepod culture medium. The synthetic seawater was prepared by dissolving Instant Ocean® sea 

salt into laboratory grade deionized water in accordance with the manufacturer’s instructions at 

appropriate amounts and volumes necessary to achieve 30 ‰ salinity. Copepod culture media 

was prepared by filtering the synthetic seawater through a 0.2μm hydrophilic polyethersulfone 

membrane (VWR® 47mm glass filter holder; Pall Corporation, Port Washington, USA). The 

quality of the culture media was checked weekly using measurements of dissolved oxygen, pH, 

salinity, and temperature. The culture media was siphoned and replenished two times per week. 

During culture media renewal, the fresh media was amended on an alternating basis with either 

the live alga Dunaliella tertiolecta, a chlorophyte, or Isochrysis galbana, a chrysophyte. The 

algae were added to the copepod culture medium to provide a final density of approximately 

2.8x108 algal cells/L. 

2.2.3 Laboratory Reagent Water for Culture Media Preparation 

Laboratory reagent water is prepared by processing carbon scrubbed municipal water in tandem 

through a reverse osmosis membrane and deionization resin. The quality of the laboratory water 

is checked once annually, at a minimum. The analytical report for the most recent samples, 

which were collected and analyzed in May 2011, is provided in Appendix II. 
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2.2.4 Culture Sediment 

Concentrated sediment previously filtered through a 63μm wire mesh sieve was received as a 

gift from Professor G.T. Chandler and stored in the dark at 4°C prior to use. According to the 

Chandler Laboratory, the sediment was obtained from Bread and Butter Creek, located near 

Charleston, South Carolina. The sediment was prepared in accordance with the Chandler 

Laboratory procedures. Specifically, a tablespoon of concentrated sediment was resuspended in 

one liter of 0.2μm 30 ‰ synthetic seawater using an electric blender (Waring Products, 

Torrington, CT). The sediment slurry was allowed to settle overnight and then decanted. The 

resuspension and settling process was immediately repeated. The final washed sediment was 

added to the copepod cultures when the quantity of in-culture sediment had diminished so that 

the bottom of the polyethylene container was visible. 

2.2.5 Photoperiod 

A 12-h light (min-max: 380-420 ft-c) and 12-h dark photoperiod was maintained throughout the 

culturing of A. tenuiremis. 

2.3 TOXICITY TEST PROCEDURE 

2.3.1 Test chemical 

Three samples of the test chemical were received from the EDSP Chemical Repository, housed 

at Battelle's Pacific Northwest Division, Sequim, Washington. Upon receipt at BES, the test 

substances were stored at room temperature in the original container in a dark ventilated cabinet. 

Concentrations were adjusted for the purity of the test substance and are presented as active 

ingredient (a.i.). The following information was provided: 

Name: Vinclozolin

CAS registry number: 50471-44-8 

Source: Battelle Marine Science Laboratory, Sequim 

WA 98382, U.S.A. 

Lot / Batch number: Lot: 372-152B

Purity: >99.5%

Form White powder 

Date of receipt at BES March 11, 2011 

Location of storage Fume hood in chemical storage room 
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2.3.2 Preparation of test solutions 

The primary stock solution of Vinclozolin was prepared with new test chemical on a weekly 

basis due to concerns of chemical loss by degradation. Each weekly stock solution was stored in 

an amber glass bottle at 4°C in Sample Locker 1 and used for a seven-day period throughout the 

36-day test. For each stock solution, Vinclozolin was weighed using an analytical balance 

(Sartorius Series A 200S) and dissolved into pesticide grade acetone to achieve a stock 

concentration of 200mg/L as detailed in Table 1. The primary stock concentration was selected 

so that the total acetone carrier volume would not exceed 0.05% in any test treatment. Acetone 

was stored at room temperature in a self-closing organic solvent safety cabinet. Acetone was 

measured using a glass graduated cylinder. 

Nominal Vinclozolin test concentrations of 12.96, 21.6, 36, 60, and 100 μg/L were prepared 

from the weekly Vinclozolin primary stock as detailed in Table 2. Test solutions were prepared 

every 3 days, on the day of water change. Calculated amounts of the primary stock solution was 

spiked into 500 mL glass volumetric flasks containing 0.2μm 30 ‰ synthetic Instant Ocean®

seawater (see 2.2.2 and 2.2.3) using Hamilton glass volumetric syringes with stainless-steel 

needles. Test solutions were not prepared by serial dilution, but instead prepared independently 

by pipetting the calculated amount of primary stock solution directly into the designated 

volumetric flask. To prevent bias, the acetone solvent carrier was added to an aliquot of the 

control solution in a volume equivalent to the highest amount added into the Vinclozolin 

treatments. Once prepared, test solutions were sealed, mixed for 1-hr using Teflon® coated stirbars, and stored in the chemical storage room until used. 

2.3.3 Test equipment 

The 36-day microplate test design followed the draft OECD (2009) guideline. The following 

equipment was used: 

 96-well polystyrene microplates (Corning Costar 3474, Lot number 16011004, exp 

6/18/2014); 

 12-well polystyrene microplates (Corning Costar 3477 Netwell® (purchased from VWR, 

USA) with a 74μm polyester mesh that retains gravid females but allows nauplii through 

the mesh to the bottom of the well; 

 Oxygen meter suitable to measure >60mL volumes (YSI 550A); 

 pH meter (Orion 230A); 

 Conductivity meter for salinity measurements (YSI 30); 

 Temperature recorder measuring continuous air temperature within environmental 

chamber (Dixon DT8 Recorder #39585); 

 Alcohol thermometer immersed in a glass beaker containing dilution water located in the 

humidity chamber. Thermometer is calibrated to an NIST certified thermometer every 

three months; 

 Two inverted stereo compound microscopes with dark-field (Two AmScope IN200B 

with No equipment ID); 
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 Two dissection stereomicroscope, with dark-field illumination and LED episcopic 

illumination (Two AmScope SM-1TZ-PL); 

 Temperature and light regulated environmental chamber (Lab-Line Environ-Shaker 

#3597 designated as environmental chamber #3, located in Testing Room #4); 

 One 0.047 m3 plastic container with sealable lid to create humidity chambers for the 

microplates (to minimize evaporative loss of test solutions). A plexiglass platform 

(perforated with 0.3cm3 holes) was supported above standing water inside the sealed 

plastic container; 

 Stainless-steel mesh sieves (0.090mm and 0.106mm) to culture, capture and sort 

copepods; 

 Glass Pasteur pipettes silanized with Enviro-Coat, OmniPur*, Non-hazardous silane 

replacement to prevent sticking of copepods onto the glass surface. Enviro-Coat (EM4190) was purchased through VWR; 

 Adjustable single channel volumetric pipettes (5-50μL, 100-1000μL) for inoculating 

algae cultures used as food for cultures and tests 

 Adjustable multi-channel volumetric pipette (1-10 μL) for adding food and for adding 

primary stock solution during the preparation of test solutions; 

 10, 50, and 250 μL Hamilton glass volumetric syringes with beveled stainless-steel 

needles for microplate water changes and test solution preparation; 

 Haemacytometer cell counting chamber (Neubauer depth of chamber 0.1mm) to count 

algae during food preparation; 

 47mm Glass Filter Holder (VWR USA, Port Washington, USA); 

 0.2 μm sterile polyethersulfone membrane, 47 mm (Pall, Port Washington, USA) 

2.3.4 Collection of copepod nauplii in preparation for the test 

Approximately 300 gravid female copepods were collected from laboratory stock mud cultures 

by sieving across 106 μm steel mesh. Gravids retained on the 106 μm sieve were rapidly rinsed 

with 30 ‰ dilution water into polystyrene Petri dishes. The organisms were transferred using 

enviro-coat treated glass transfer pipettes into wells of 12-well Netwell® microplates filled with 

0.2μm filtered seawater. Each well of the Netwell® microplate is equipped with a 74 μm 

polycarbonate mesh filter (Corning Costar 3477), suspended 4 mm above the bottom of the 

microplate. The 74 μm polyester mesh retains gravid female copepods but allows hatched 

nauplii to fall through to the bottom of the well. Each well contained approximately 50-60 

gravid females. 

Newly hatched nauplii (≤24-h) that had migrated through the mesh filter were collected, pooled, 

and haphazardly allocated to individual microwells of ultra-low attachment Hydrogel® coated 

(i.e., hydrophilic) polystyrene 96-well microplates (Corning Costar 3474) containing 250 μL of 

each test solution. For each Vinclozolin test concentration and control, a total of 90 individuals 

were allocated across three 96-well plates. Each 96-well plate had 30 individuals placed into 

columns 2 through 11 of rows B, D and F (column 1 and 12 were purposely left empty). . 
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The 96-well plates were randomly assigned a concentration using a random number table. All 

subsequent handling, loading, observations and feeding were done without respecting any order 

to ensure that handling bias would not be construed as being a chemical-dependent effect. For 

example, if experimental units are handled in treatment or concentration order, then some timerelated effect, such as operator fatigue or other error, could lead to greater or lesser effects at the 

higher or lower concentrations. Only one test chemical concentration was tested per microplate 

to reduce chances for microwell cross contamination. 

2.3.5 Diet during testing 

During the chemical exposure period, copepods were fed every six days. Individual copepods in 

test chambers were fed 2μL of a mixed-algal cell suspension (centrifuged, pelletised and reconstituted in fresh 30 ‰ seawater to 107 cells/mL) at a ratio of 1:1:1 Dunaliella tertiolecta, 

Isochrysis galbana and Phaeodactylum tricornutum. The algal cell density added to each test 

microwell at each feeding was 2 x 104 cells/microwell. 

2.3.6 Daily observations 

Nauplii were monitored daily via inverted stereomicroscope for development into the copepodite 

juvenile stage. Once becoming copepodites, exposed A. tenuiremis were then monitored for the 

development of secondary sexual characteristics and time, in days, to reproductive adult. Upon 

reaching adulthood, individual virgin female and male copepods were randomly mated (pairwise 

within each treatment) in fresh microwells (wells C and E) using the exposure parameters and 

conditions described above. Mating pairs were monitored daily for reproductive success (egg 

extrusion and fertility) and numbers of offspring successfully hatching for each pair. 

2.3.7 Test conditions 

Test conditions are summarized in Table 3. Essential physico-chemical parameters of the 

dilution water used to prepare the test solutions were measured on each day a water change took 

place. Aliquots of the dilution water were analyzed for conductivity/salinity, pH, and dissolved 

oxygen on YSI 30, Orion 230A, and YSI 550A hand held meters, respectively. Test vessels were 

stored in a temperature and light controlled environmental chamber set at 25°C. Due to small 

volumes within the microwells, direct temperature was not measured in the test vessel solutions. 

Temperature was recorded twice daily from a calibrated alcohol thermometer immersed in a 

bottle containing dilution water placed near test vessels. The thermometer is calibrated quarterly 

against an NIST traceable thermometer. Physico-chemical parameters were not measured in 

freshly made test solutions (referred to here as ‘Ups’) and test solutions drawn out of test vessels 

(i.e. ‘Downs’) in order to prevent Vinclozolin cross contamination to other ongoing toxicity 

studies. 

2.3.8 Vinclozolin concentrations in test solutions 

Samples for determining Vinclozolin concentration were collected from freshly prepared test 

solutions in the case of Up samples and from pooled solutions from test vessels in the case of 

Down samples. Samples were collected and sealed into 125 ml amber glass bottles stored in 
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Sample Locker 1 at 4°C. Samples were sent throughout the study to North Coast Laboratories, 

LTD. (Arcata, California, USA; CA-ELAP #1247) in an ice chest cooled with packs of Blue 

Ice® (Rubbermaid). Chemical analysis of the test solutions were performed by Gas 

Chromatography – Mass Spectrometry (GC-MS). Quantities were determined by correlating 

signal responses against reference calibration standards of known Vinclozolin concentrations 

prepared in hexane. Results were submitted in audited reports 1110574 and 1111154 (Appendix 

III). 

2.3.9 Statistical analyses 

Mean and standard deviations (SD) were calculated using MicrosoftTM Excel 2000. Survival, 

development, and reproduction data were analyzed using SigmaStat version 2.03 for Windows 

(IBM, Inc. (formerly SPSS, Inc.), Armonk, New York, USA). All statistical conclusions were 

made at the 95% level of certainty except in the cases of Levene’s Test and Shapiro-Wilk's Test, 

in which the 99% level of certainty was applied. The following procedures were used: 

 Data for each endpoint were analyzed using Levene's Test (USEPA, 2002) as a 

check on the assumption of homogeneity of variance implicit in parametric 

statistics. 

 Shapiro-Wilk's Test for normality (USEPA, 2002) was conducted in order to 

compare the observed sample distribution with a normal distribution. The 

assumption that observations are normally distributed must be validated before 

subsequent analyses following parametric procedures, can be performed. If the 

data were not normally distributed, then a non-parametric procedure was used for 

subsequent analyses. 

 Prior to analysis for statistically significant effects, an analysis of variance 

(ANOVA) was conducted on each data set (except survival and sex ratio data) to 

determine if a treatment group in the data set was different from the other groups 

in that data set. 

 Post-hoc pair wise comparisons between the solvent control and each treatment 

level, including the dilution water control, were performed using Dunnett's Test 

(Dunnett, 1955, 1964), which is a parametric procedure. If necessary, mean values 

were transformed by using square root, arcsine square root, or log conversion 

procedures. The multiple comparison adjustment was utilized to determine the 

NOEC and LOEC for each test endpoint. The Bonferroni pairwise mutlitple 

comparison procedure was utilized to obtain P values between the carrier control 

and treatment groups since the SigmaStat program does not calculate individual 

pairwise P values when performing the Dunnett’s test. 

 All proportional data points such as survival rates and sex ratios were analyzed 

using Pearson’s chi- square goodness-of-fit test (R X C contingency tables) 

(Piegorsch and Bailer,1997). 
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For clarity, the definition for NOEC and LOEC are described below: 

 No-Observed-Effect Concentration (NOEC) is the concentration of Vinclozolin tested which has 

no effect on the organism in the test, according to the statistical test used for analysis. 

 Lowest-Observed-Effect Concentration (LOEC) is the lowest tested concentration of Vinclozolin 

which has an effect on the organism in the test that is different from the control (or solvent 

control), according to the statistical test used for analysis. 

3 RESULTS 

3.1 PHYSICO-CHEMICAL PROPERTIES OF TEST SOLUTIONS 

The baseline physico-chemical measurements for pH, salinity and dissolved oxygen are 

presented in Table 3. Daily temperature measurements from the environmental chamber dilution 

water vessel are presented in Table 4. Table 5 provides an overall summary of these 

measurements and shows that temperature was 24.7 ± 0.7°C, pH was 7.9 ± 0.2, salinity was 30.3 

± 0.8‰, and dissolved oxygen was 97.4 ± 5.3 of air saturated value. In terms of physicochemical properties, recommendations listed in the QAPP for this study (Appendix I Table 3) 

were met. 

3.2 MEASURED VINCLOZOLIN CONCENTRATIONS IN TEST SOLUTIONS 

Test concentrations of Vinclozolin were reported by North Coast Laboratories LTD, Arcata, 

California, USA. A copy of the audited reports numbered 1110574 and 1111154 is presented in 

Appendix III. Concentration data are reported in Table 6. These tables indicate concentrations of 

the test substance were all less than the laboratory reporting limit. The primary stock solutions 

used to prepare the test concentrations were measured to be within 10 percent of the nominal 

concentration. Therefore the reported concentrations of Vinclozolin in the test treatments are 

likely a result of degradation within the test vessel or sample collection bottle. Vinclozolin loss 

may have occurred during sample extraction prior to analysis. Accordingly, in this report, all 

results are expressed in term of nominal values. 

3.3 BIOLOGICAL DATA 

A summary of the statistical analyses performed on the biological observations is presented in 

Section 2.3.9. Reports from all statistical analysis are also provided in Appendix IV. 

3.3.1 Survival on day 16 

Copepod survival data on Day 16 of the study are presented in Table 7. Treatment means, 

including the dilution water control, were statistically compared to the carrier control using 

Pearson’s chi- square goodness-of-fit test. In summary, mean survival in the control and the 

carrier control group was 80% and 90%, respectively. A statistical difference between the 

control means was not evident (P=0.095). Survival in the test treatments ranged from 82 to 91%. 
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No significant differences relative to the carrier control group were observed in the 12.96 

(P=0.273), 21.6 (P=0.196), 36 (P=0.372), 60 (P=0.372) and 100μg/L (P=1.0) test treatments. 

3.3.2 Survival on day 36 

Recording survival observations of individual copepods was concluded upon test termination on 

day 36. Table 8 lists the percentage survival for all groups. Treatment means, including the 

dilution water control, were statistically compared to the carrier control using Pearson’s chisquare goodness-of-fit test. On Day 36, the control and carrier control group had a 75% and 

84% survival rate, respectively. A statistical difference between the control means was not 

evident (P=0.140). Survival in the test treatments ranged from 75 to 81%.relative to the carrier 

control group were observed in the 12.96 (P=0.192), 21.6 (P=0.693), 36 (P=0.559), 60 (P=0.192) 

and 100μg/L (P=0.693) test treatments. 

3.3.3 Day 16 Versus Day 36 Survival 

Day 16 and Day 36 mean survival rates were statistically compared within treatment using 

Pearson’s chi- square goodness-of-fit test. differences (p>0.05) in survival were observed 

within control (P=0.477), carrier control (P=0.372), 12.96 (P=0.268), 21.6 (P=1.0), 36 

(P=0.559), 60 (P=0.192) and 100μg/L (P=0.085) Vinclozolin treatment groups between the test 

midpoint and test termination. 

3.3.4 Development – Maturation time between naupliar and copepodite stages 

Copepod development from the nauplius to copepodite stages is presented in 1Table 9. Treatment 

means, including the dilution water control, were statistically compared to the carrier control 

using a One Way ANOVA test. In summary, mean development time in the control and the 

carrier control group was 11.2 and 11.9 days, respectively. A statistical difference between the 

control means was not evident (P=0.299). relative to the carrier control group were observed for 

any Vinclozolin test treatment (P=0.140). Based on these statistical results, NOEC and LOEC 

values were determined to be 100 and >100μg/L, respectively. 

3.3.5 Development – Maturation time between copepodite and adult stages 

Copepod development from the copepodite stage into virgin adults is presented in 1Table 10. 

Treatment means, including the dilution water control, were statistically compared to the carrier 

control using a One Way ANOVA test. In summary, mean development time in the control and 

the carrier control group was 7.5 and 7.9 days, respectively. A statistical difference between the 

control means was not evident (P=0.163).). No statistically significant effects (P=0.130) relative 

to the carrier control group were observed for any Vinclozolin test treatment (P=0.130). Based 

on these statistical observations, NOEC and LOEC values were determined to be 100 and 

>100μg/L, respectively. 
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3.3.6 Development – Maturation time between naupliar and adult stages 

Copepod development from the nauplius to adult stages is presented in 1Table 11. Treatment 

means, including the dilution water control, were statistically compared to the carrier control 

using a One Way ANOVA test. In summary, mean development time in the control and the 

carrier control group was 18.4 and 19.0 days, respectively. A statistical difference between the 

control means was not evident (P=0.225). No statistically significant effects relative to the carrier 

control group were observed for any Vinclozolin test treatment (P=0.081). Based on these 

statistical observations, NOEC and LOEC values were determined to be 100 and >100μg/L, 

respectively. 

3.3.7 Sex ratio 

The proportion of females to males is presented in 1Table 12. Treatment means, including the 

dilution water control, were statistically compared to the carrier control using Pearson’s chisquare goodness-of-fit test. The average gender ratio (female to male) in the control and carrier 

control groups was 1.0 and 1.1, respectively. There was not a statistically significant difference 

(p>0.05) between the control means (P=0.865). No significant differences relative to the carrier 

control group were observed in the 12.96 (P=0.461), 21.6 (P=0.935), 36 (P=0.245), 60 (P=0.264) 

and 100μg/L (P=0.802) test treatments. The sex ratios observed in the control and carrier control 

treatments were within the Quality Criteria specified in Appendix I, Table 3, although the 

standard deviation (± 0.3) for the carrier control group exceeded ± 0.2. 

3.3.8 Reproduction – time to first egg extrusion 

The average time to first egg extrusion in females following the mating of virgin male and 

female copepods is presented in 1Table 13. Treatment means, including the dilution water control, 

were statistically compared to the carrier control using a One Way ANOVA test. The average 

time to first egg extrusion in the control and carrier control groups was 3.1 and 2.7 days, 

respectively. There was not a statistically significant difference (p>0.05) between the control 

means (P=0.800). There was a 1statistically significant (p<0.05) increase in the time to first egg 

extrusion in the 21.6 (P=0.038) and 36μg/L (P=0.023) test treatments relative to the carrier 

control. No statistically significant differences in the time to egg extrusion were observed among 

the 12.96 (P=1.0), 60 (P=1.0), and 100μg/L (P=1.0) test treatments when compared to the carrier 

control group. Organisms were observed for a minimum of six days and a maximum of twelve 

days following mating. The time to first clutch observed in the control and carrier control 

treatments were within the Quality Criteria specified in Appendix I, Table 3. 

3.3.9 Reproduction – number of hatched nauplii from first brood 

The average number of hatched nauplii from the first egg extrusion following the mating of 

virgin male and female copepods is presented in 1Table 14. Treatment means, including the 

dilution water control, were statistically compared to the carrier control using a One Way 

ANOVA test. The average number of first brood nauplii in the control and carrier control groups 
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Page 19 

was 6.4 and 8.1 nauplii per female, respectively. There was not a statistically significant 

difference (p>0.05) between the control means (P=0.296). No statistically significant effects 

relative to the carrier control group were observed for any Vinclozolin test treatment (P=0.163). 

3.3.10 Reproduction – time to second egg extrusion 

The average time to second egg extrusion in female copepods is presented in 1Table 15. 

Treatment means, including the dilution water control, were statistically compared to the carrier 

control using a One Way ANOVA test. The average time to second egg extrusion in the control 

and carrier control groups was 2.4 and 2.9 days, respectively. There was not a statistically 

significant difference (p>0.05) between the control means (P=0.570). No statistically significant

differences in the time to egg extrusion were observed among the test treatments when compared 

to the carrier control group (P=0.428). 

3.3.11 Reproduction – number of hatched nauplii from second brood 

The average number of hatched nauplii from the second egg extrusion following the mating of 

male and female copepods is presented in 1Table 16. Treatment means, including the dilution 

water control, were statistically compared to the carrier control using a One Way ANOVA test. 

The average number of second brood nauplii in the control and carrier control groups was 8.6 

and 6.6 nauplii per female, respectively. There was not a statistically significant difference 

(p>0.05) between the control means (P=0.251). No statistically significant differences in the 

number of second brood offspring were observed among the test treatments when compared to 

the carrier control group (P=0.912). 

3.3.12 Reproduction – number of nauplii hatched from two successive broods 

The average number of nauplii per female from mating pairs with two broods is presented in 

1Table 17. Treatment means, including the dilution water control, were statistically compared to 

the carrier control using a One Way ANOVA test. The total number of offspring/female in the 

control and carrier control groups was 14.9 and 14.7, respectively. There was not a statistically 

significant difference (p>0.05) between the control means (P=0.932). No statistically significant

differences were observed among the test treatments when compared to the carrier control group 

(P=0.524). The total number of viable offspring produced through two clutches in the control and 

carrier control treatments were within the Quality Criteria specified in Appendix I, Table 3. 

Based on these statistical results, NOEC and LOEC values were determined to be 100 and 

>100μg/L, respectively. 

3.3.13 Reproduction - fertilization success 

Fertilization success is defined as when mated gravid females extrude two successive clutches 

that produce viable offspring. Treatment means, including the dilution water control, were 

statistically compared to the carrier control using a One Way ANOVA test. The average 

fertilization success rates in the control and carrier control treatments were 65 and 53%, 

respectively (1Table 18). There was not a statistically significant difference (p>0.05) between the 
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control means (P=0.409). The average fertilization success rate in the vinclozolin treatments 

ranged from 24% to 52%. No statistically significant differences were observed among the test 

treatments when compared to the carrier control group (P=0.524). Gravid female copepods that 

did not hatch their second brood hatch by Day 36 were excluded from statistical analysis. Based 

on these statistical results, NOEC and LOEC values were determined to be 100 and >100μg/L, 

respectively. 

3.4 SUMMARY OF NOEC AND LOEC FOR ALL ENDPOINTS 

A summary of NOEC and LOEC for all endpoints is presented Table 19. 

4 DISCUSSION 

4.1 QUALITY OBJECTIVES AND CRITERIA 

This study achieved the minimum quality criteria specified in Appendix I, Table 3 for the 

following performance objectives: 

• Survival of A. tenuiremis in the control group was ≥ 75% up to day 27 

• The water quality parameters (dissolved oxygen, temperature, pH, and salinity) were all 

within requirements 

• The sex ratio in the control group was within the recommended 0.6 to 1.8 (±0.2). 

• The average number of viable offspring hatched from two successive broods in the 

control group was greater than 8 offspring/female. 

• The average number of days to the first brood was < 25 days. 

4.2 DISCUSSION OF RESULTS 

The chemical analysis of Vinclozolin test concentrations showed that concentrations of the test 

substance were all less than the laboratory reporting limit. A review of the procedure for 

preparing the test solutions indicated the solutions were prepared from the primary stock 

solutions properly. Moreover, the primary stock solutions used to prepare the test concentrations 

were measured to be within 10 percent of the expected concentration of 200mg/L. A review of 

the sample handling follow collection indicated the samples were submitted to the laboratory 

outside the recommended holding time. These data suggest that test chemical was likely lost 

through adsorption onto test chambers, sample storage bottles, as well as through naturally 

degradative processes prior to analysis. It is possible that some Vinclozolin was lost during the 

sample extraction process, however, this does not appear likely considering the matrix spike 

recovery data were within 90% of the expected value. A study by the Dutch Health Council 

(Greve, 1996) indicates the half-life of vinclozolin in water is 0.08 to 35 days, while the half-life for 

hydrolysis is 0.25 to 2 days from pH 6 to pH 9. 

The results from the biological data are discussed for survival, developmental and reproductive 

endpoints. Vinclozolin did not affect copepod survival at any exposure level by day 16 of the 
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test, with the survival rate not significantly reduced further by test termination on day 36. 

Similarly, the developmental endpoints tested were also not significantly affected, as Vinclozolin 

did not significantly affect the time for A. tenuiremis nauplii to mature into the copepodite stage 

and subsequent transformation to the adult stage at test concentrations up to 100μg/L. 

Vinclozolin had no effect on gender assignment for all concentrations tested. vinclozolin did, 

however, significantly delay the time for mated females to extrude their first brood at the 21.6 

and 36μg/L exposure levels, although the time for females to extrude their second brood was not 

delayed at any exposure level tested. Vinclozolin did not significantly reduce or increase the 

number of hatched offspring at any test concentration, although an appreciable decrease in 

hatched offspring in the 100μg/L treatment was noted. Lastly, with regards to reproductive 

endpoints, fertilization success was not significantly affected for any test treatment when 

compared to the carrier control. An appreciable decrease in the number of successful mating 

pairs was noted in organisms exposed vinclozolin concentrations of 21.6, 36, and 60μg/L. 

5 ACKNOWLEDGEMENTS 

Our thanks to Professor G.T. Chandler of the University of South Carolina for generously 

providing BES A. tenuiremis stock mud cultures as well as growth media for algae culturing. 

Acknowledgement also needs to be made to Kate Washburn with the Chandler laboratory for 

assisting with the training of BES technical staff and for providing technical advice. Thanks to 

the staff of North Coast Laboratories LTD for analyzing the analytical chemistry data. Many 

thanks go to BES staff scientists Terry Clohesy, Nanette Bradbury, Sebastian Castro, and Amy 

Ayala for performing the studies. 

6 REFERENCES 

Un-published material:

OECD Draft Guidelines for the Testing of Chemicals. Harpacticoid Copepod Development and 

Reproduction Test with Amphiascus tenuiremis. Post June 2009. 

Published material:

ASTM E 2317-04. (2004). Standard guide for conducting renewal microplate-based life-cycle 

toxicity tests with a marine meiobenthic copepod. American Society for Testing and Materials, 

West Conshohocken, PA, 22 pp. 

Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a 

control. Journal of the American Statistical Association, 50, 1,096-1,121 

Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics, 20, 482-

491 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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Greve, (1996). (Dutch Health Council). Hormoon-verstorende stoffen in Nederland. Gebruik, 

emissie, milieuconcentraties en fysisch/chemische karakteristieken. Included by reference from 

“Summary profiles of chemicals with information on use, production, emission, monitoring and 

legal status”. Annex 14, European Commission. 

Piegorsch WW, Bailer AJ (1997) Statistics for environmental biology and toxicology. Chapman 

and Hall, London, UK 

USEPA. 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and 

Receiving Water to Marine Organisms. EPA-800-R-02-014. 4th Edition. 

USEPA. Good Laboratory Practice Standards. 2007a. 40CFR160. 

USEPA. Good Laboratory Practice Standards. 2007b. 40CFR792. 
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Page 23 

7 FIGURES 

 Figure 1: Layout of Microplate Test Chamber 

Test organisms are reared from nauplius to sexually mature adult in the grey shaded wells. 

Virgin male and female adults are mated in the red shaded wells. 

8 TABLES 

Table 1: Method for preparation of Vinclozolin 200 mg/L primary stock solutions 

Preparation 

Date 

Amount of 

Vinclozolin 

(mg) 

Solvent Carrier 

Volume of 

Solvent 

(mL) 

Final Measured 

Concentration 

(mg/L) 

9/26/2011 10.0 Acetone 50.0 200 

10/3/2011 10.2 Acetone 51.0 190 

10/11/2011 10.3 Acetone 51.5 210 

10/18/2011 10.0 Acetone 50.0 200 

10/24/2011 10.2 Acetone 51.0 220 

Average Primary Stock 

Concentration Throughout Test 204 
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Table 2: Method for preparation of Vinclozolin test solutions 

Solution ID 

Volume of Primary Stock 

Solution used 

(in Acetone) 

Volume of 

dilution water 

used 

Total Test 

Solution 

Volume 

Percent of 

Acetone 

Carrier Used 

Control 500 mL 500 mL 

Carrier 499.75 mL 500 mL 0.05%

100 μg/L 250μL primary stock solution 499.75 mL 500 mL 0.05%

60 μg/L 150μL primary stock solution 499.85 mL 500 mL 0.03%

36 μg/L 90μL primary stock solution 499.91 mL 500 mL 0.018%

21.6 μg/L 54μL primary stock solution 499.95 mL 500 mL 0.011%

12.96 μg/L 32.4μL primary stock solution 499.97 mL 500 mL 0.006%

Table 3: Water quality parameters: pH, salinity, dissolved oxygen 

Date Dissolved Oxygen 

(%ACV) pH Salinity (‰) 

9/27/2011 95.5 8.0 31.4 

9/30/2011 92.0 8.1 30.2 

10/3/2011 94.6 8.1 29.7 

10/6/2011 NM1 NM1 NM1 

10/9/2011 99.6 8.1 30.9 

10/12/2011 95.1 8.1 30.8 

10/152011 NM1 NM1 NM1

10/18/2011 99.9 8.0 31.4 

10/21/2011 103.2 7.7 29.7 

10/24/2011 94.3 7.5 29.3 

10/27/2011 88.8 7.7 29.7 

10/30/2011 96 7.9 29.7 

Note: measurements were made on dilution water prior to preparation of fresh test solutions. 

1

No measurements were taken on 10/6 and 10/15 Up Dilution Water 

Table 4: Water quality parameter: daily temperature 

Date Temperature 

(°C) 1st read

Temperature 

(°C) 2nd read Date Temperature (°C) 1st read

Temperature 

(°C) 2nd read

9/27/11 25.0 24.1 10/16/11 25.0 25.0 

9/28/11 25.0 24.0 10/17/11 25.0 25.0 

9/29/11 25.0 25.0 10/18/11 25.0 24.0 

9/30/11 25.9 25.1 10/19/11 24.0 24.1 

10/1/11 25.0 25.0 10/20/11 24.1 24.5 

10/2/11 NM 25.0 10/21/11 24.0 25.0 
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Date Temperature 

(°C) 1st read

Temperature 

(°C) 2nd read Date Temperature (°C) 1st read

Temperature 

(°C) 2nd read

10/3/11 24.5 24.5 10/22/11 24.0 24.0 

10/4/11 25.0 25.0 10/23/11 24.5 24.0 

10/5/11 23.0 24.0 10/24/11 25.0 26.0 

10/6/11 24.0 24.0 10/25/11 25.0 25.0 

10/7/11 23.0 24.0 10/26/11 25.2 25.0 

10/8/11 24.0 24.0 10/27/11 24.5 24.1 

10/9/11 24.5 24.5 10/28/11 24.0 24.0 

10/10/11 27.0 26.0 10/29/11 24.8 24.5 

10/11/11 26.0 25.0 10/30/11 25.0 25.0 

10/12/11 24.9 24.5 10/31/11 25.0 25.0 

10/13/11 24.5 24.8 11/1/11 25.0 25.0 

10/14/11 24.1 25.5 11/2/11 24.0 24.0 

10/15/11 24.0 25.0 

Note: Temperature was measured in dilution water from a glass bottle in environmental chamber used for study. 

NM = Not Measured 

Table 5: Summary of water quality parameters 

 Temperature (°C) Dissolved 

Oxygen (%) pH Salinity (‰) 

Min 23.0 88.8 7.5 29.3 

Max 27.0 103.2 8.1 31.4 

Mean ± S.D. 24.7 ± 0.7 96.0 ± 3.9 7.92 ± 0.2 30.3 ± 0.8 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Table 6: Measured Vinclozolin test concentrations 

Nominal 

Concentration 

Day 0 

Up 

Day 0 

Dwn 

Day 6 

Up 

Day 6 

Dwn 

Day 12 

Up 

Day 12 

Dwn 

Day 18 

Up 

Day 18 

Dwn 

Day 24 

Up 

Day 2

Dwn 

Dilution Water 

Control <3.6 <12 <4.2 <10 <2.5 <10 <2.5 <14 <2.6 NM 

Carrier Control <2.6 <12 <2.5 <10 <2.5 <10 <2.5 <14 <5.0 NM 

12.96 μg/L <2.8 <12 <2.5 <10 <2.5 <10 <2.5 <12 <5.0 NM 

21.6μg/L <2.6 <12 <2.5 <10 <2.5 <12 <2.5 <14 <2.5 NM 

36μg/L <2.5 <11 <2.5 <10 <2.5 <12 <2.5 <13 <2.5 NM 60μg/L <5 <5 <5.0 <11 <5.0 <11 <2.5 <16 <5.0 NM 

100μg/L <10 <11 <10 <11 <10 <10 <10 <14 <10 <13 

25 μg/L (Spike 

1) 101% Recovery 

25μg/L (Spike 

2) 89.7% Recovery

25μg/L (Spike 

3) 92.5% Recovery

25μg/L (Spike 

4) 98.3% Recovery

25μg/L (Spike 

5) 92.3% Recovery

25μg/L (Spike 

6) 74.0% Recovery 

Mean Spike 

recovery* 91.3% ± 9.5% 

50μg/L (Matrix 

Spike 1) 92.2% Recovery (Solvent Control Up) 

50μg/L (Matrix 

Spike 1) 92.1% Recovery (12.96μg/L Up) 

Mean Matrix 

Spike recovery* 92.2%± 0.1% 

Note: Analysis performed by North Coast Laboratories, LTD (Arcata, California, USA) using GC-MS. Table compiled from NCL final report number

NM = Not Measured * Spike recoveries were not used to correct sample results. 
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Table 7: Survival on day 16 

Percent survival at day 16 

Treatment group Replicate Percent survival Mean Standard deviation 

plate 1 77 

Control plate 2 83 

plate 3 80 

80 3 

plate 1 90 

Carrier Control plate 2 93 

plate 3 87 

90 3 

plate 1 93 

plate 2 80 12.96μg/L 

Vinclozolin plate 3 77 

83 9 

plate 1 83 

plate 2 83 21.6μg/L 

Vinclozolin plate 3 80 

82 2 

plate 1 77 

plate 2 93 36μg/L 

Vinclozolin plate 3 83 

84 8 

plate 1 83 

plate 2 87 60μg/L 

 Vinclozolin plate 3 83 

84 2 

plate 1 93 

plate 2 93 100 μg/L 

Vinclozolin plate 3 87 

91 4 
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Table 8: Survival on day 36 

Percent survival at day 36 

Treatment group Replicate Percent survival Mean Standard deviation 

plate 1 77 

Control plate 2 70 

plate 3 77 

75 4 

plate 1 83 

Carrier Control plate 2 87 

plate 3 83 

84 2 

plate 1 90 

plate 2 70 12.96μg/L 

Vinclozolin plate 3 67 

76 13 

plate 1 83 

plate 2 63 21.6μg/L 

Vinclozolin plate 3 80 

75 11 

plate 1 73 

plate 2 90 36μg/L 

Vinclozolin plate 3 77 

80 9 

plate 1 73 

plate 2 83 60μg/L 

Vinclozolin plate 3 70 

75 7 

plate 1 80 

plate 2 87 100 μg/L 

Vinclozolin plate 3 77 

81 5 
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Table 9: Time to transformation to copepodite stage 

Number of days to transformation to copepodite stage 

Treatment group Replicate Mean number of 

days 

Total 

Mean Standard deviation 

plate 1 11.6 

Control plate 2 11.2 

plate 3 10.7 

11.2 0.5 

plate 1 11.5 

Carrier Control plate 2 13.0 

plate 3 11.2 

11.9 1.0 

plate 1 10.7 

plate 2 10.2 12.96μg/L 

Vinclozolin plate 3 6.6 

9.2 2.2 

plate 1 8.4 

plate 2 8.0 21.6μg/L 

Vinclozolin plate 3 8.2 

8.2 0.2 

plate 1 10.8 

plate 2 7.4 36μg/L 

Vinclozolin plate 3 12.4 

10.2 2.5 

plate 1 11.2 

plate 2 10.7 60μg/L 

Vinclozolin plate 3 13.2 

11.7 1.3 

plate 1 8.7 

plate 2 11.6 100 μg/L 

Vinclozolin plate 3 12.8 

11.0 2.1 
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Table 10: Time in copepodite stage 

Number of days in copepodite stage 

Treatment group Replicate Mean number of 

days 

Total 

Mean Standard deviation 

plate 1 7.3 

Control plate 2 8.0 

plate 3 7.2 

7.5 0.4 

plate 1 7.9 

Carrier Control plate 2 7.9 

plate 3 8.0 

7.9 0.1 

plate 1 7.6 

plate 2 7.7 12.96μg/L 

Vinclozolin plate 3 8.1 

7.8 0.3 

plate 1 8.3 

plate 2 8.2 21.6μg/L 

Vinclozolin plate 3 7.8 

8.1 0.3 

plate 1 8.1 

plate 2 8.3 36μg/L 

Vinclozolin plate 3 7.9 

8.1 0.2 

plate 1 8.2 

plate 2 8.3 60μg/L 

Vinclozolin plate 3 8.3 

8.3 0.1 

plate 1 7.9 

plate 2 8.2 100 μg/L 

Vinclozolin plate 3 7.9 

8.0 0.2 
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Table 11: Time to transformation to adult stage 

Number of days to transformation to adult stage 

Treatment Group Replicate Mean number of 

days 

Total 

Mean Standard deviation 

plate 1 18.2 

Control plate 2 18.9 

plate 3 18.0 

18.4 0.5 

plate 1 18.6 

Carrier Control plate 2 19.6 

plate 3 18.7 

19.0 0.6 

plate 1 18.0 

plate 2 17.9 12.96μg/L 

Vinclozolin plate 3 15.0 

17.0 1.7 

plate 1 16.7 

plate 2 15.9 21.6μg/L 

Vinclozolin plate 3 16.0 

16.2 0.4 

plate 1 18.9 

plate 2 15.7 36μg/L 

Vinclozolin plate 3 20.2 

18.3 2.3 

plate 1 20.2 

plate 2 19.0 60μg/L 

Vinclozolin plate 3 20.9 

20.0 1.0 

plate 1 16.7 

plate 2 20.0 100 μg/L 

Vinclozolin plate 3 20.7 

19.1 2.1 
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Table 12: Sex ratio 

Sex ratio 

Treatment group Replicate 

Number 

of males 

(% or #) 

Number 

of 

females 

(% or #) 

Ratio 

(female:male) 

Total 

Mean 

Standard 

deviation 

plate 1 47% 53% 1.11 

plate 2 52% 48% 0.91 

plate 3 48% 52% 1.09 Control 

Plate 1, 2, 

and 3 31 32 

1.0 0.1 

plate 1 52% 48% 0.92 

plate 2 41% 59% 1.44 

Carrier Control plate 3 55% 45% 0.83 

Plate 1, 2, 

and 3 33 34 

1.1 0.3 

plate 1 42% 58% 1.40 

plate 2 45% 55% 1.22 

12.96 μg/L plate 3 37% 63% 1.71 

Vinclozolin Plate 1, 2, 

and 3 26 37 

1.4 0.2 

plate 1 43% 57% 1.30 

plate 2 44% 56% 1.25 

21.6 μg/L plate 3 57% 43% 0.77 

Vinclozolin Plate 1, 2, 

and 3 31 33 

1.1 0.3 

plate 1 73% 27% 0.38 

plate 2 52% 48% 0.93 

plate 3 59% 41% 0.69 

36 μg/L 

Vinclozolin 

Plate 1, 2, 

and 3 43 28 

0.7 0.3 

plate 1 53% 47% 0.90 

plate 2 61% 39% 0.64 

plate 3 68% 32% 0.46 

60 μg/L 

Vinclozolin 

Plate 1, 2, 

and 3 37 24 

0.7 0.2 

plate 1 52% 48% 0.91 

plate 2 50% 50% 1.00 

plate 3 57% 43% 0.77 100 μg/L 

Vinclozolin Plate 1, 2, 

and 3 37 33 

0.9 0.1 
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Table 13: Time to first egg extrusion since date of pairing 

Time to first brood (days) since date of pairing 

Treatment group Replicate Mean number of 

days 

Total 

Mean Standard deviation 

plate 1 2.6 

plate 2 3.8 Control 

plate 3 2.8 

3.1 0.6 

plate 1 3.0 

Carrier Control plate 2 3.0 

plate 3 2.1 

2.7 0.5 

plate 1 3.6 

plate 2 1.7 12.96 μg/L 

Vinclozolin plate 3 3.4 

2.9 1.0 

plate 1 5.7 

plate 2 4.2 21.6 μg/L 

Vinclozolin plate 3 4.7 

4.9 0.8 

plate 1 4.5 

plate 2 5.8 36 μg/L 

Vinclozolin plate 3 5.0 

5.1 0.7 

plate 1 3.0 

plate 2 2.5 60 μg/L 

Vinclozolin plate 3 2.3 

2.6 0.4 

plate 1 3.0 

plate 2 2.1 100 μg/L 

Vinclozolin plate 3 2.3 

2.5 0.5 
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Table 14: Number of nauplii hatched from first brood 

Number of nauplii hatched from first brood 

Treatment group Replicate Mean number of 

nauplii 

Total 

Mean Standard deviation 

plate 1 6.4 

plate 2 4.9 Control 

plate 3 7.8 

6.4 1.5 

plate 1 6.3 

Carrier Control plate 2 7.7 

plate 3 10.2 

8.1 2.0 

plate 1 5.5 

plate 2 5.7 12.96 μg/L 

Vinclozolin plate 3 7.0 

6.1 0.8 

plate 1 8.0 

plate 2 6.7 21.6 μg/L 

Vinclozolin plate 3 15.0 

9.9 4.5 

plate 1 6.5 

plate 2 5.0 36 μg/L 

 Vinclozolin plate 3 4.0 

5.2 1.3 

plate 1 2.5 

plate 2 7.0 60 μg/L 

 Vinclozolin plate 3 6.5 

5.3 2.5 

plate 1 5.0 

plate 2 5.5 100 μg/L 

Vinclozolin plate 3 6.7 

5.7 0.9 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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Table 15: Time to second egg extrusion following hatching of first brood offspring 

Time to second brood (days) since hatching of first brood offspring 

Treatment group Replicate Mean number of 

nauplii 

Total 

Mean Standard deviation 

plate 1 2.8 

plate 2 2.7 Control 

plate 3 1.6 

2.4 0.7 

plate 1 3.0 

Carrier Control plate 2 1.8 

plate 3 3.8 

2.9 1.0 

plate 1 2.3 

plate 2 2.7 12.96 μg/L 

Vinclozolin plate 3 1.0 

2.0 0.9 

plate 1 2.0 

plate 2 3.0 21.6 μg/L 

Vinclozolin plate 3 2.0 

2.3 0.6 

plate 1 3.5 

plate 2 2.0 36 μg/L 

 Vinclozolin plate 3 2.0 

2.5 0.9 

plate 1 1.5 

plate 2 3.3 60 μg/L 

 Vinclozolin plate 3 2.0 

2.3 0.9 

plate 1 1.5 

plate 2 2.0 100 μg/L 

Vinclozolin plate 3 1.0 

1.5 0.5 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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Table 16: Number of nauplii hatched from second brood 

Number of nauplii hatched from second brood 

Treatment group Replicate Mean number of 

nauplii 

Total 

Mean Standard deviation 

plate 1 8.6 

plate 2 7.8 Control 

plate 3 9.3 

8.6 0.8 

plate 1 7.0 

Carrier Control plate 2 4.0 

plate 3 8.8 

6.6 2.4 

plate 1 8.5 

plate 2 6.0 12.96 μg/L 

Vinclozolin plate 3 12.5 

9.0 3.3 

plate 1 4.0 

plate 2 9.3 21.6 μg/L 

Vinclozolin plate 3 16.0 

9.8 6.0 

plate 1 14.0 

plate 2 7.7 36 μg/L 

 Vinclozolin plate 3 4.0 

8.6 5.1 

plate 1 5.0 

plate 2 10.0 60 μg/L 

 Vinclozolin plate 3 7.0 

7.3 2.5 

plate 1 5.0 

plate 2 10.0 100 μg/L 

Vinclozolin plate 3 7.0 

7.3 2.5 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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Table 17: Total number of nauplii that hatched from two successive broods 

Total number of nauplii hatched from two successive broods 

Treatment group Replicate Mean number of 

nauplii 

Total 

Mean Standard deviation 

plate 1 15.0 

Control plate 2 11.7 

plate 3 15.8 

14.2 2.2 

plate 1 13.3 

Carrier Control plate 2 11.7 

plate 3 18.4 

14.5 3.5 

plate 1 14.0 

plate 2 11.7 12.96 μg/L 

Vinclozolin plate 3 19.5 

15.1 4.0 

plate 1 12.0 

plate 2 16.0 21.6 μg/L 

Vinclozolin plate 3 31.0 

19.7 10.0 

plate 1 13.5 

plate 2 12.7 36 μg/L 

 Vinclozolin plate 3 8.0 

11.4 3.0 

plate 1 5.0 

plate 2 17.0 60 μg/L 

 Vinclozolin plate 3 13.5 

11.8 6.2 

plate 1 6.8 

plate 2 10.1 100 μg/L 

Vinclozolin plate 3 10.0 

9.0 1.9 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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Table 18: Fertilization success 

Fertilization success: mating pairs able to produce two successive broods that hatched 

Treatment Replicate 

Number of 

successful 

pair 

Total number 

of pairs 

% Successful 

mating pair Mean Standard deviation 

Control PLATE 1 5 6 83% 

Control PLATE 2 6 9 67% 

Control PLATE 3 5 11 45% 

65% 19% 

Carrier control PLATE 1 4 7 57% 

Carrier control PLATE 2 3 8 38% 

Carrier control PLATE 3 7 11 64% 

53% 14% 

12.96 μg/L 

Vi l li

PLATE 1 4 7 57% 

12.96 μg/L PLATE 2 3 9 33% 

12.96 μg/L PLATE 3 2 6 33% 

41% 14% 

21.6 μg/L 

Vi l li

PLATE 1 1 6 17% 

21.6 μg/L PLATE 2 3 7 43% 

21.6 μg/L PLATE 3 1 6 17% 

26% 15% 

36μg /L 

Vi l li

PLATE 1 1 6 17% 

36μg /L PLATE 2 3 9 33% 

36μg /L PLATE 3 1 7 14% 

21% 10% 

60μg/L 

Vi l li

PLATE 1 1 6 17% 

60μg/L PLATE 2 2 9 22% 

60μg/L PLATE 3 2 6 33% 

24% 8% 

100 μg/L 

Vi l li

PLATE 1 2 7 29% 

100 μg/L PLATE 2 7 9 78% 

100 μg/L PLATE 3 3 6 50% 

52% 14% 

Note: The total number of pairs listed in this table is derived from mating pairs that could be 

observed until the end of the test or until they produced two broods that hatched. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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Table 19: Summary table of NOEC and LOEC for all endpoints 

Endpoint 

NOEC in 

μg Vinclozolin /L 

LOEC in 

μg Vinclozolin /L 

Day 16 survival (%) 100 >100 

Day 27 survival (%) 100 >100 

Time to copepodite stage 100 >100 

Time in copepodite stage 100 >100 

Time to adult 100 >100 

Sex ratio 100 >100 

Time to 1st Brood 12.96 21.6 

Time to 2nd Brood 100 >100 

Number offspring 1st Brood 100 >100 

Number offspring 2nd Brood 100 >100 

Number of mating pair with successful 

reproduction 100 >100 

Number nauplii hatched over two clutches 100 >100 

Note: 12.96 μg Vinclozolin /L was the lowest concentration tested and 100 μg Vinclozolin /L 

was the highest concentration tested. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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9 APPENDIX I: QUALITY ASSURANCE PROJECT PLAN (QAPP) 
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10 APPENDIX II: QUALITY OF LABORATORY WATER AT BES 

Baseline chemical analyses of laboratory reagent water used for preparation of 

synthetic seawater and Vinclozolin test solutions at the BES laboratory in 2011 

Samples collected 05/10/2011 by BES staff and analyzed on 5/12/2011 by Basic Laboratory 

(Redding, California, USA) 

Chemical/Constituent 

Laboratory 

Processed 

Reagent 

Water 

Chemical/Constituent 

Laboratory 

Processed 

Reagent 

Water 

Cobalt (μg/L) <0.1 Chlordane (μg/L) <0.05 

Copper (μg/L) 2.30 Dieldrin (μg/L) <0.010 

Iron (μg/L) <100 Endosulfan I(μg/L) <0.010 

Zinc (μg/L) 5.0 Endosulfan II(μg/L) <0.020 

Aluminum (μg/L) 5.3 Endosulfan Sulfate(μg/L) <0.020 

Arsenic (μg/L) <0.5 Endrin(μg/L) <0.010 

Cadmium (μg/L) 0.05 Endrin aldehyde(μg/L) <0.010 

Chromium (μg/L) 0.2 Heptachlor(μg/L) <0.010 

Lead (μg/L) 0.3 Heptachlor epoxide(μg/L) <0.010 

Mercury (μg/L) <0.2 Methoxychlor(μg/L) <0.010 

Nickel (μg/L) <1.0 4,4-DDT(μg/L) <0.010 

Silver (μg/L) <0.5 4,4-DDE(μg/L) <0.010 

TOC (mg/L) 0.2 4,4-DDD(μg/L) <0.020 

alpha-BHC(μg/L) <0.010 2,4-DDT(μg/L) <0.010 

beta-BHC(μg/L) <0.005 2,4-DDE(μg/L) <0.010 

gamma-BHC(μg/L) <0.010 2,4-DDD(μg/L) <0.020 

delta-BHC(μg/L) <0.005 Toxaphene(μg/L) <0.50 

Aldrin (μg/L) <0.005 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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11 APPENDIX III: VINCLOZOLIN CHEMISTRY DATA REPORT 
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12 APPENDIX IV: STATISTICAL DATA OUTPUTS 

Descriptive Statistics: Tuesday, April 17, 2012, 17:24:52 

Data source: Water Quality in USEPA TO30 Vinclozolin 

Column Size Missing Mean Std Dev Std. Error C.I. of Mean 

DO 13 2 95.973 3.927 1.184 2.638 

pH 13 2 7.918 0.204 0.0615 0.137 

Salinity 13 2 30.336 0.763 0.230 0.513 

Column Range Max Min Median 5% 95% 

DO 14.400 103.200 88.800 95.500 88.960 103.035 

pH 0.600 8.100 7.500 8.000 7.510 8.100 

Salinity 2.100 31.400 29.300 30.200 29.320 31.400 

Column Skewness Kurtosis K-S Dist. K-S Prob. Sum Sum of Squares 

DO 0.0937 0.494 0.154 0.586 1055.700 101472.650 

pH -0.989 0.0185 0.201 0.237 87.100 690.090 

Salinity 0.197 -1.672 0.252 0.049 333.700 10129.070 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

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Descriptive Statistics: Tuesday, April 17, 2012, 17:28:36 

Data source: Temperature in USEPA TO30 Vinclozolin 

Column Size Missing Mean Std Dev Std. Error C.I. of Mean 

Temperature 74 1 24.653 0.666 0.0780 0.156 

Column Range Max Min Median 5% 95% 

Temperature 4.000 27.000 23.000 24.800 24.000 25.985 

Column Skewness Kurtosis K-S Dist. K-S Prob. Sum Sum of Squares 

Temperature 0.446 1.641 0.192 <0.001 1799.700 44400.750 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

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One Way Analysis of Variance Tuesday, April 17, 2012, 17:44:45 

Data source: Day 16 Survival Data in USEPA TO30 Vinclozolin 

Normality Test: Passed (P = 0.075) 

Equal Variance Test: Passed (P = 0.431) 

Group Name N Missing Mean Std Dev SEM 

DWC 3 0 80.000 3.000 1.732 

SC 3 0 90.000 3.000 1.732 

12.960 3 0 83.333 8.505 4.910 

21.600 3 0 82.000 1.732 1.000 

36.000 3 0 84.333 8.083 4.667 

60.000 3 0 84.333 2.309 1.333 

100.000 3 0 91.000 3.464 2.000 

Source of Variation DF SS MS F P 

Between Groups 6 296.000 49.333 1.962 0.140 

Residual 14 352.000 25.143 

Total 20 648.000 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.140). 

Power of performed test with alpha = 0.050: 0.259 

The power of the performed test (0.259) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

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Chi-square Tuesday, April 17, 2012, 18:07:21 

Data source: Day 16 Survival Data in USEPA TO30 Vinclozolin 

Subjects DWC SC 

Row 1 72.000 81.000 Counts 

 76.500 76.500 Expected Counts 

 47.059 52.941 Row % 

 80.000 90.000 Column % 

 40.000 45.000 Total % 

 

Row 2 18.000 9.000 Counts 

 13.500 13.500 Expected Counts 

 66.667 33.333 Row % 

 20.000 10.000 Column % 

 10.000 5.000 Total % 

 Power of performed test with alpha = 0.050: 0.369 

The power of the performed test (0.369) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 2.789 with 1 degrees of freedom. (P = 0.095) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.095) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 120 

Fisher Exact Test Monday, September 24, 2012, 23:31:15 

Data source: Day 16 Survival Data in USEPA TO30 Vinclozolin 

Test execution ended by user request, Chi-square begun 

Chi-square Monday, September 24, 2012, 23:31:15 

Data source: Day 16 Survival Data in USEPA TO30 Vinclozolin 

Subjects SC 12.96 ug/L 

Row 1 81.000 75.000 Counts 

 78.000 78.000 Expected Counts 

 51.923 48.077 Row % 

 90.000 83.333 Column % 

 45.000 41.667 Total % 

 

Row 2 9.000 15.000 Counts 

 12.000 12.000 Expected Counts 

 37.500 62.500 Row % 

 10.000 16.667 Column % 

 5.000 8.333 Total % 

 Power of performed test with alpha = 0.050: 0.181 

The power of the performed test (0.181) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 1.202 with 1 degrees of freedom. (P = 0.273) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.273) 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Page 121 

Fisher Exact Test Monday, September 24, 2012, 23:32:08 

Data source: Day 16 Survival Data in USEPA TO30 Vinclozolin 

Test execution ended by user request, Chi-square begun 

Chi-square Monday, September 24, 2012, 23:32:08 

Data source: Day 16 Data in USEPA TO30 Vinclozolin 

Subjects SC 21.6 ug/L 

Row 1 81.000 74.000 Counts 

 77.500 77.500 Expected Counts 

 52.258 47.742 Row % 

 90.000 82.222 Column % 

 45.000 41.111 Total % 

 

Row 2 9.000 16.000 Counts 

 12.500 12.500 Expected Counts 

 36.000 64.000 Row % 

 10.000 17.778 Column % 

 5.000 8.889 Total % 

 Power of performed test with alpha = 0.050: 0.236 

The power of the performed test (0.236) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 1.672 with 1 degrees of freedom. (P = 0.196) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.196) 
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Fisher Exact Test Monday, September 24, 2012, 23:32:28 

Data source: Day 16 Survival Data in USEPA TO30 Vinclozolin 

Test execution ended by user request, Chi-square begun 

Chi-square Monday, September 24, 2012, 23:32:28 

Data source: Day 16 Data in USEPA TO30 Vinclozolin 

Subjects SC 36 ug/L 

Row 1 81.000 76.000 Counts 

 78.500 78.500 Expected Counts 

 51.592 48.408 Row % 

 90.000 84.444 Column % 

 45.000 42.222 Total % 

 

Row 2 9.000 14.000 Counts 

 11.500 11.500 Expected Counts 

 39.130 60.870 Row % 

 10.000 15.556 Column % 

 5.000 7.778 Total % 

 Power of performed test with alpha = 0.050: 0.135 

The power of the performed test (0.135) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.798 with 1 degrees of freedom. (P = 0.372) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.372) 
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Fisher Exact Test Monday, September 24, 2012, 23:32:41 

Data source: Day 16 Survival Data in USEPA TO30 Vinclozolin 

Test execution ended by user request, Chi-square begun 

Chi-square Monday, September 24, 2012, 23:32:41 

Data source: Day 16 Data in USEPA TO30 Vinclozolin 

Subjects SC 60ug/L 

Row 1 81.000 76.000 Counts 

 78.500 78.500 Expected Counts 

 51.592 48.408 Row % 

 90.000 84.444 Column % 

 45.000 42.222 Total % 

 

Row 2 9.000 14.000 Counts 

 11.500 11.500 Expected Counts 

 39.130 60.870 Row % 

 10.000 15.556 Column % 

 5.000 7.778 Total % 

 Power of performed test with alpha = 0.050: 0.135 

The power of the performed test (0.135) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.798 with 1 degrees of freedom. (P = 0.372) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.372) 
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Fisher Exact Test Monday, September 24, 2012, 23:32:53 

Data source: Day 16 Survival Data in USEPA TO30 Vinclozolin 

Test execution ended by user request, Chi-square begun 

Chi-square Monday, September 24, 2012, 23:32:53 

Data source: Day 16 Data in USEPA TO30 Vinclozolin 

Subjects SC 100 ug/L 

Row 1 81.000 82.000 Counts 

 81.500 81.500 Expected Counts 

 49.693 50.307 Row % 

 90.000 91.111 Column % 

 45.000 45.556 Total % 

 

Row 2 9.000 8.000 Counts 

 8.500 8.500 Expected Counts 

 52.941 47.059 Row % 

 10.000 8.889 Column % 

 5.000 4.444 Total % 

 Power of performed test with alpha = 0.050: 0.047 

The power of the performed test (0.047) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 1.002E-014 with 1 degrees of freedom. (P = 1.000) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 1.000) 
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One Way Analysis of Variance Tuesday, April 17, 2012, 18:15:11 

Data source: Day 36 Survival Data in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.918) 

Group Name N Missing Mean Std Dev SEM 

DWC 3 0 74.667 4.041 2.333 

SC 3 0 84.333 2.309 1.333 

12.960 3 0 75.667 12.503 7.219 

21.600 3 0 75.333 10.786 6.227 

36.000 3 0 80.000 8.888 5.132 

60.000 3 0 75.333 6.807 3.930 

100.000 3 0 81.333 5.132 2.963 

Source of Variation DF SS MS F P 

Between Groups 6 257.810 42.968 0.674 0.673 

Residual 14 892.000 63.714 

Total 20 1149.810 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.673). 

Power of performed test with alpha = 0.050: 0.050 

The power of the performed test (0.050) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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Chi-square Tuesday, April 17, 2012, 18:16:35 

Data source: Day 36 Survival Data in USEPA TO30 Vinclozolin 

Subjects DWC-36 SC-36 

Row 1 67.000 76.000 Counts 

 71.500 71.500 Expected Counts 

 46.853 53.147 Row % 

 74.444 84.444 Column % 

 37.222 42.222 Total % 

 

Row 2 23.000 14.000 Counts 

 18.500 18.500 Expected Counts 

 62.162 37.838 Row % 

 25.556 15.556 Column % 

 12.778 7.778 Total % 

 Power of performed test with alpha = 0.050: 0.296 

The power of the performed test (0.296) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 2.177 with 1 degrees of freedom. (P = 0.140) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.140) 
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Fisher Exact Test Monday, September 24, 2012, 23:38:25 

Data source: Day 36 Survival Data in USEPA TO30 Vinclozolin 

Test execution ended by user request, Chi-square begun 

Chi-square Monday, September 24, 2012, 23:38:25 

Data source: Day 36 Data in USEPA TO30 Vinclozolin 

Subjects SC-36 12.96 

Row 1 76.000 68.000 Counts 

 72.000 72.000 Expected Counts 

 52.778 47.222 Row % 

 84.444 75.556 Column % 

 42.222 37.778 Total % 

 

Row 2 14.000 22.000 Counts 

 18.000 18.000 Expected Counts 

 38.889 61.111 Row % 

 15.556 24.444 Column % 

 7.778 12.222 Total % 

 Power of performed test with alpha = 0.050: 0.240 

The power of the performed test (0.240) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 1.701 with 1 degrees of freedom. (P = 0.192) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.192) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 128 

Chi-square Monday, September 24, 2012, 23:38:44 

Data source: Day 36 Survival Data in USEPA TO30 Vinclozolin 

Subjects SC-36 21.6 

Row 1 76.000 73.000 Counts 

 74.500 74.500 Expected Counts 

 51.007 48.993 Row % 

 84.444 81.111 Column % 

 42.222 40.556 Total % 

 

Row 2 14.000 17.000 Counts 

 15.500 15.500 Expected Counts 

 45.161 54.839 Row % 

 15.556 18.889 Column % 

 7.778 9.444 Total % 

 Power of performed test with alpha = 0.050: 0.064 

The power of the performed test (0.064) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.156 with 1 degrees of freedom. (P = 0.693) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.693) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 129 

Chi-square Monday, September 24, 2012, 23:38:53 

Data source: Day 36 Survival Data in USEPA TO30 Vinclozolin 

Subjects SC-36 36.0 

Row 1 76.000 72.000 Counts 

 74.000 74.000 Expected Counts 

 51.351 48.649 Row % 

 84.444 80.000 Column % 

 42.222 40.000 Total % 

 

Row 2 14.000 18.000 Counts 

 16.000 16.000 Expected Counts 

 43.750 56.250 Row % 

 15.556 20.000 Column % 

 7.778 10.000 Total % 

 Power of performed test with alpha = 0.050: 0.084 

The power of the performed test (0.084) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.342 with 1 degrees of freedom. (P = 0.559) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.559) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 130 

Chi-square Monday, September 24, 2012, 23:39:02 

Data source: Day 36 Survival Data in USEPA TO30 Vinclozolin 

Subjects SC-36 60.0 

Row 1 76.000 68.000 Counts 

 72.000 72.000 Expected Counts 

 52.778 47.222 Row % 

 84.444 75.556 Column % 

 42.222 37.778 Total % 

 

Row 2 14.000 22.000 Counts 

 18.000 18.000 Expected Counts 

 38.889 61.111 Row % 

 15.556 24.444 Column % 

 7.778 12.222 Total % 

 Power of performed test with alpha = 0.050: 0.240 

The power of the performed test (0.240) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 1.701 with 1 degrees of freedom. (P = 0.192) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.192) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 131 

Chi-square Monday, September 24, 2012, 23:39:12 

Data source: Day 36 Survival Data in USEPA TO30 Vinclozolin 

Subjects SC-36 100.0 

Row 1 76.000 73.000 Counts 

 74.500 74.500 Expected Counts 

 51.007 48.993 Row % 

 84.444 81.111 Column % 

 42.222 40.556 Total % 

 

Row 2 14.000 17.000 Counts 

 15.500 15.500 Expected Counts 

 45.161 54.839 Row % 

 15.556 18.889 Column % 

 7.778 9.444 Total % 

 Power of performed test with alpha = 0.050: 0.064 

The power of the performed test (0.064) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.156 with 1 degrees of freedom. (P = 0.693) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.693) 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Page 132 

Chi-square Tuesday, April 17, 2012, 21:16:03 

Data source: D16 vs D36 Survival data in USEPA TO30 Vinclozolin 

Subjects DWC DWC-36 

Row 1 72.000 67.000 Counts 

 69.500 69.500 Expected Counts 

 51.799 48.201 Row % 

 80.000 74.444 Column % 

 40.000 37.222 Total % 

 

Row 2 18.000 23.000 Counts 

 20.500 20.500 Expected Counts 

 43.902 56.098 Row % 

 20.000 25.556 Column % 

 10.000 12.778 Total % 

 Power of performed test with alpha = 0.050: 0.102 

The power of the performed test (0.102) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.505 with 1 degrees of freedom. (P = 0.477) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.477) 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Page 133 

Chi-square Tuesday, April 17, 2012, 21:16:21 

Data source: D16 vs D36 Survival data in USEPA TO30 Vinclozolin 

Subjects SC SC-36 

Row 1 81.000 76.000 Counts 

 78.500 78.500 Expected Counts 

 51.592 48.408 Row % 

 90.000 84.444 Column % 

 45.000 42.222 Total % 

 

Row 2 9.000 14.000 Counts 

 11.500 11.500 Expected Counts 

 39.130 60.870 Row % 

 10.000 15.556 Column % 

 5.000 7.778 Total % 

 Power of performed test with alpha = 0.050: 0.135 

The power of the performed test (0.135) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.798 with 1 degrees of freedom. (P = 0.372) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.372) 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Page 134 

Chi-square Tuesday, April 17, 2012, 21:16:40 

Data source: D16 vs D36 Survival data in USEPA TO30 Vinclozolin 

Subjects 12.96 ug/L 12.96 

Row 1 75.000 68.000 Counts 

 71.500 71.500 Expected Counts 

 52.448 47.552 Row % 

 83.333 75.556 Column % 

 41.667 37.778 Total % 

 

Row 2 15.000 22.000 Counts 

 18.500 18.500 Expected Counts 

 40.541 59.459 Row % 

 16.667 24.444 Column % 

 8.333 12.222 Total % 

 Power of performed test with alpha = 0.050: 0.184 

The power of the performed test (0.184) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 1.225 with 1 degrees of freedom. (P = 0.268) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.268) 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Page 135 

Chi-square Tuesday, April 17, 2012, 21:19:37 

Data source: D16 vs D36 Survival data in USEPA TO30 Vinclozolin 

Subjects 21.6 ug/L 21.6 

Row 1 74.000 73.000 Counts 

 73.500 73.500 Expected Counts 

 50.340 49.660 Row % 

 82.222 81.111 Column % 

 41.111 40.556 Total % 

 

Row 2 16.000 17.000 Counts 

 16.500 16.500 Expected Counts 

 48.485 51.515 Row % 

 17.778 18.889 Column % 

 8.889 9.444 Total % 

 Power of performed test with alpha = 0.050: 0.047 

The power of the performed test (0.047) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 2.788E-014 with 1 degrees of freedom. (P = 1.000) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 1.000) 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Page 136 

Chi-square Tuesday, April 17, 2012, 21:16:51 

Data source: D16 vs D36 Survival data in USEPA TO30 Vinclozolin 

Subjects 36 ug/L 36.0 

Row 1 76.000 72.000 Counts 

 74.000 74.000 Expected Counts 

 51.351 48.649 Row % 

 84.444 80.000 Column % 

 42.222 40.000 Total % 

 

Row 2 14.000 18.000 Counts 

 16.000 16.000 Expected Counts 

 43.750 56.250 Row % 

 15.556 20.000 Column % 

 7.778 10.000 Total % 

 Power of performed test with alpha = 0.050: 0.084 

The power of the performed test (0.084) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.342 with 1 degrees of freedom. (P = 0.559) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.559) 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Page 137 

Chi-square Tuesday, April 17, 2012, 21:17:02 

Data source: D16 vs D36 Survival data in USEPA TO30 Vinclozolin 

Subjects 60ug/L 60.0 

Row 1 76.000 68.000 Counts 

 72.000 72.000 Expected Counts 

 52.778 47.222 Row % 

 84.444 75.556 Column % 

 42.222 37.778 Total % 

 

Row 2 14.000 22.000 Counts 

 18.000 18.000 Expected Counts 

 38.889 61.111 Row % 

 15.556 24.444 Column % 

 7.778 12.222 Total % 

 Power of performed test with alpha = 0.050: 0.240 

The power of the performed test (0.240) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 1.701 with 1 degrees of freedom. (P = 0.192) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.192) 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Page 138 

Chi-square Tuesday, April 17, 2012, 21:17:14 

Data source: D16 vs D36 Survival data in USEPA TO30 Vinclozolin 

Subjects 100 ug/L 100.0 

Row 1 82.000 73.000 Counts 

 77.500 77.500 Expected Counts 

 52.903 47.097 Row % 

 91.111 81.111 Column % 

 45.556 40.556 Total % 

 

Row 2 8.000 17.000 Counts 

 12.500 12.500 Expected Counts 

 32.000 68.000 Row % 

 8.889 18.889 Column % 

 4.444 9.444 Total % 

 Power of performed test with alpha = 0.050: 0.390 

The power of the performed test (0.390) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 2.973 with 1 degrees of freedom. (P = 0.085) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.085) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 139 

One Way Analysis of Variance Tuesday, September 25, 2012, 00:00:20 

Data source: Nauplius to Copepodite in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.533) 

Group Name N Missing Mean Std Dev SEM 

DWC 3 0 11.167 0.451 0.260 

SC 3 0 11.900 0.964 0.557 

Source of Variation DF SS MS F P 

Between Groups 1 0.807 0.807 1.424 0.299 

Residual 4 2.267 0.567 

Total 5 3.073 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.299). 

Power of performed test with alpha = 0.050: 0.080 

The power of the performed test (0.080) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 140 

One Way Analysis of Variance Tuesday, September 25, 2012, 00:00:45 

Data source: Nauplius to Copepodite in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.527) 

Group Name N Missing Mean Std Dev SEM 

SC 3 0 11.900 0.964 0.557 

12.960 3 0 9.167 2.237 1.291 

21.600 3 0 8.200 0.200 0.115 

36.000 3 0 10.200 2.553 1.474 

60.000 3 0 11.700 1.323 0.764 

100.000 3 0 11.033 2.108 1.217 

Source of Variation DF SS MS F P 

Between Groups 5 32.207 6.441 2.068 0.140 

Residual 12 37.373 3.114 

Total 17 69.580 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.140). 

Power of performed test with alpha = 0.050: 0.256 

The power of the performed test (0.256) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 141 

One Way Analysis of Variance Tuesday, September 25, 2012, 00:01:02 

Data source: Copepodite To Adult in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 1.000) 

Group Name N Missing Mean Std Dev SEM 

DWC 3 0 7.500 0.436 0.252 

SC 3 0 7.933 0.0577 0.0333 

Source of Variation DF SS MS F P 

Between Groups 1 0.282 0.282 2.914 0.163 

Residual 4 0.387 0.0967 

Total 5 0.668 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.163). 

Power of performed test with alpha = 0.050: 0.185 

The power of the performed test (0.185) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 142 

One Way Analysis of Variance Tuesday, September 25, 2012, 00:01:15 

Data source: Copepodite To Adult in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.947) 

Group Name N Missing Mean Std Dev SEM 

SC 3 0 7.933 0.0577 0.0333 

12.960 3 0 7.800 0.265 0.153 

21.600 3 0 8.100 0.265 0.153 

36.000 3 0 8.100 0.200 0.115 

60.000 3 0 8.267 0.0577 0.0333 

100.000 3 0 8.000 0.173 0.100 

Source of Variation DF SS MS F P 

Between Groups 5 0.387 0.0773 2.142 0.130 

Residual 12 0.433 0.0361 

Total 17 0.820 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.130). 

Power of performed test with alpha = 0.050: 0.272 

The power of the performed test (0.272) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 143 

One Way Analysis of Variance Tuesday, September 25, 2012, 00:01:30 

Data source: Nauplius to Adult in USEPA TO30 Vinclozolin 

Normality Test: Passed (P = 0.109) 

Equal Variance Test: Passed (P = 0.925) 

Group Name N Missing Mean Std Dev SEM 

DWC 3 0 18.367 0.473 0.273 

SC 3 0 18.967 0.551 0.318 

Source of Variation DF SS MS F P 

Between Groups 1 0.540 0.540 2.051 0.225 

Residual 4 1.053 0.263 

Total 5 1.593 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.225). 

Power of performed test with alpha = 0.050: 0.123 

The power of the performed test (0.123) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 144 

One Way Analysis of Variance Tuesday, September 25, 2012, 00:01:43 

Data source: Nauplius to Adult in USEPA TO30 Vinclozolin 

Normality Test: Passed (P = 0.108) 

Equal Variance Test: Passed (P = 0.621) 

Group Name N Missing Mean Std Dev SEM 

SC 3 0 18.967 0.551 0.318 

12.960 3 0 16.967 1.704 0.984 

21.600 3 0 16.200 0.436 0.252 

36.000 3 0 18.267 2.316 1.337 

60.000 3 0 20.033 0.961 0.555 

100.000 3 0 19.133 2.136 1.233 

Source of Variation DF SS MS F P 

Between Groups 5 30.969 6.194 2.609 0.081 

Residual 12 28.493 2.374 

Total 17 59.463 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.081). 

Power of performed test with alpha = 0.050: 0.379 

The power of the performed test (0.379) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 145 

Chi-square Tuesday, September 25, 2012, 00:13:05 

Data source: Sex Ratio in USEPA TO30 Vinclozolin 

Subjects 0.000 1.000 

DWC 31.000 32.000 Counts 

 31.015 31.985 Expected Counts 

 49.206 50.794 Row % 

 48.438 48.485 Column % 

 23.846 24.615 Total % 

 

SC 33.000 34.000 Counts 

 32.985 34.015 Expected Counts 

 49.254 50.746 Row % 

 51.563 51.515 Column % 

 25.385 26.154 Total % 

 Power of performed test with alpha = 0.050: 0.050 

The power of the performed test (0.050) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.0289 with 1 degrees of freedom. (P = 0.865) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.865) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 146 

Chi-square Tuesday, September 25, 2012, 00:16:49 

Data source: Sex Ratio in USEPA TO30 Vinclozolin 

Subjects 0.000 1.000 

SC 33.000 34.000 Counts 

 30.408 36.592 Expected Counts 

 49.254 50.746 Row % 

 55.932 47.887 Column % 

 25.385 26.154 Total % 

 

12.96ug/L 26.000 37.000 Counts 

 28.592 34.408 Expected Counts 

 41.270 58.730 Row % 

 44.068 52.113 Column % 

 20.000 28.462 Total % 

 Power of performed test with alpha = 0.050: 0.106 

The power of the performed test (0.106) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.544 with 1 degrees of freedom. (P = 0.461) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.461) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 147 

Chi-square Tuesday, September 25, 2012, 00:17:00 

Data source: Sex Ratio in USEPA TO30 Vinclozolin 

Subjects 0.000 1.000 

SC 33.000 34.000 Counts 

 32.733 34.267 Expected Counts 

 49.254 50.746 Row % 

 51.563 50.746 Column % 

 25.191 25.954 Total % 

 

21.6ug/L 31.000 33.000 Counts 

 31.267 32.733 Expected Counts 

 48.438 51.563 Row % 

 48.438 49.254 Column % 

 23.664 25.191 Total % 

 Power of performed test with alpha = 0.050: 0.048 

The power of the performed test (0.048) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.00663 with 1 degrees of freedom. (P = 0.935) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.935) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 148 

Chi-square Tuesday, September 25, 2012, 00:17:09 

Data source: Sex Ratio in USEPA TO30 Vinclozolin 

Subjects 0.000 1.000 

SC 33.000 34.000 Counts 

 36.899 30.101 Expected Counts 

 49.254 50.746 Row % 

 43.421 54.839 Column % 

 23.913 24.638 Total % 

 

36ug/L 43.000 28.000 Counts 

 39.101 31.899 Expected Counts 

 60.563 39.437 Row % 

 56.579 45.161 Column % 

 31.159 20.290 Total % 

 Power of performed test with alpha = 0.050: 0.199 

The power of the performed test (0.199) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 1.354 with 1 degrees of freedom. (P = 0.245) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.245) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 149 

Chi-square Tuesday, September 25, 2012, 00:17:21 

Data source: Sex Ratio in USEPA TO30 Vinclozolin 

Subjects 0.000 1.000 

SC 33.000 34.000 Counts 

 36.641 30.359 Expected Counts 

 49.254 50.746 Row % 

 47.143 58.621 Column % 

 25.781 26.563 Total % 

 

60ug/L 37.000 24.000 Counts 

 33.359 27.641 Expected Counts 

 60.656 39.344 Row % 

 52.857 41.379 Column % 

 28.906 18.750 Total % 

 Power of performed test with alpha = 0.050: 0.186 

The power of the performed test (0.186) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 1.247 with 1 degrees of freedom. (P = 0.264) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.264) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 150 

Chi-square Tuesday, September 25, 2012, 00:17:30 

Data source: Sex Ratio in USEPA TO30 Vinclozolin 

Subjects 0.000 1.000 

SC 33.000 34.000 Counts 

 34.234 32.766 Expected Counts 

 49.254 50.746 Row % 

 47.143 50.746 Column % 

 24.088 24.818 Total % 

 

100ug/L 37.000 33.000 Counts 

 35.766 34.234 Expected Counts 

 52.857 47.143 Row % 

 52.857 49.254 Column % 

 27.007 24.088 Total % 

 Power of performed test with alpha = 0.050: 0.054 

The power of the performed test (0.054) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 

Yates correction for continuity was used in calculating this test. 

Chi-square= 0.0629 with 1 degrees of freedom. (P = 0.802) 

The proportions of observations in different columns of the contingency table do not vary from 

row to row. The two characteristics that define the contingency table are not significantly 

related. (P = 0.802) 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 151 

One Way Analysis of Variance Tuesday, September 25, 2012, 00:25:56 

Data source: Time to First Brood Ln transformed in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 1.000) 

Group Name N Missing Mean Std Dev SEM 

Control 3 0 1.107 0.201 0.116 

Sol. Cntrl 3 0 0.980 0.206 0.119 

Source of Variation DF SS MS F P 

Between Groups 1 0.0242 0.0242 0.584 0.487 

Residual 4 0.166 0.0414 

Total 5 0.190 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.487). 

Power of performed test with alpha = 0.050: 0.050 

The power of the performed test (0.050) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

Page 152 

One Way Analysis of Variance Tuesday, September 25, 2012, 00:26:52 

Data source: Time to First Brood Ln Transformedin USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.755) 

Group Name N Missing Mean Std Dev SEM 

Sol. Cntrl 3 0 0.980 0.206 0.119 

12.96 ug/L 3 0 1.012 0.418 0.241 

21.6 ug//L 3 0 1.574 0.154 0.0892 

36 ug/L 3 0 1.624 0.127 0.0736 

60 ug/L 3 0 0.949 0.136 0.0785 

100 ug/L 3 0 0.891 0.185 0.107 

Source of Variation DF SS MS F P 

Between Groups 5 1.671 0.334 6.475 0.004 

Residual 12 0.620 0.0516 

Total 17 2.291 

The differences in the mean values among the treatment groups are greater than would be 

expected by chance; there is a statistically significant difference (P = 0.004). 

Power of performed test with alpha = 0.050: 0.921 

Multiple Comparisons versus Control Group (Dunnett's Method) : 

Comparisons for factor: Col 21 

Comparison Diff of Means q' P P<0.050 

Sol. Cntrl vs. 36 ug/L 0.644 3.472 -- Yes 

Sol. Cntrl vs. 21.6 ug//L 0.595 3.205 -- Yes 

Sol. Cntrl vs. 100 ug/L 0.0886 0.477 -- No 

Sol. Cntrl vs. 12.96 ug/L 0.0321 0.173 -- Do Not Test 

Sol. Cntrl vs. 60 ug/L 0.0304 0.164 -- Do Not Test 

Note: The P values for Dunnett's and Duncan's tests are currently unavailable except for 

reporting that the P's are greater or less than the critical values of .05 and .01. 

A result of "Do Not Test" occurs for a comparison when no significant difference is found 

between two means that enclose that comparison. For example, if you had four means sorted in 

order, and found no difference between means 4 vs. 2, then you would not test 4 vs. 3 and 3 vs. 

2, but still test 4 vs. 1 and 3 vs. 1 (4 vs. 3 and 3 vs. 2 are enclosed by 4 vs. 2: 4 3 2 1). Note that 

not testing the enclosed means is a procedural rule, and a result of Do Not Test should be treated 

as if there is no significant difference between the means, even though one may appear to exist. 
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One Way Analysis of Variance Tuesday, September 25, 2012, 00:27:12 

Data source: Time to First Brood, Ln Transformed in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.755) 

Group Name N Missing Mean Std Dev SEM 

Sol. Cntrl 3 0 0.980 0.206 0.119 

12.96 ug/L 3 0 1.012 0.418 0.241 

21.6 ug//L 3 0 1.574 0.154 0.0892 

36 ug/L 3 0 1.624 0.127 0.0736 

60 ug/L 3 0 0.949 0.136 0.0785 

100 ug/L 3 0 0.891 0.185 0.107 

Source of Variation DF SS MS F P 

Between Groups 5 1.671 0.334 6.475 0.004 

Residual 12 0.620 0.0516 

Total 17 2.291 

The differences in the mean values among the treatment groups are greater than would be 

expected by chance; there is a statistically significant difference (P = 0.004). 

Power of performed test with alpha = 0.050: 0.921 

Multiple Comparisons versus Control Group (Bonferroni t-test): 

Comparisons for factor: Col 21 

Comparison Diff of Means t P P<0.050 

Sol. Cntrl vs. 36 ug/L 0.644 3.472 0.023 Yes 

Sol. Cntrl vs. 21.6 ug//L 0.595 3.205 0.038 Yes 

Sol. Cntrl vs. 100 ug/L 0.0886 0.477 1.000 No 

Sol. Cntrl vs. 12.96 ug/L 0.0321 0.173 1.000 Do Not Test 

Sol. Cntrl vs. 60 ug/L 0.0304 0.164 1.000 Do Not Test 

A result of "Do Not Test" occurs for a comparison when no significant difference is found 

between two means that enclose that comparison. For example, if you had four means sorted in 

order, and found no difference between means 4 vs. 2, then you would not test 4 vs. 3 and 3 vs. 

2, but still test 4 vs. 1 and 3 vs. 1 (4 vs. 3 and 3 vs. 2 are enclosed by 4 vs. 2: 4 3 2 1). Note that 

not testing the enclosed means is a procedural rule, and a result of Do Not Test should be treated 

as if there is no significant difference between the means, even though one may appear to exist. 
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One Way Analysis of Variance Tuesday, September 25, 2012, 00:28:10 

Data source: First Brood Offspring in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.461) 

Group Name N Missing Mean Std Dev SEM 

Control 3 0 6.367 1.450 0.837 

Sol. Cntrl 3 0 8.067 1.976 1.141 

Source of Variation DF SS MS F P 

Between Groups 1 4.335 4.335 1.443 0.296 

Residual 4 12.013 3.003 

Total 5 16.348 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.296). 

Power of performed test with alpha = 0.050: 0.081 

The power of the performed test (0.081) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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One Way Analysis of Variance Tuesday, September 25, 2012, 00:28:30 

Data source: First Brood Offspring in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.585) 

Group Name N Missing Mean Std Dev SEM 

Sol. Cntrl 3 0 8.067 1.976 1.141 

12.96 ug/L 3 0 6.067 0.814 0.470 

21.6 ug//L 3 0 9.900 4.464 2.577 

36 ug/L 3 0 5.167 1.258 0.726 

60 ug/L 3 0 5.333 2.466 1.424 

100 ug/L 3 0 5.733 0.874 0.504 

Source of Variation DF SS MS F P 

Between Groups 5 52.984 10.597 1.931 0.163 

Residual 12 65.853 5.488 

Total 17 118.838 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.163). 

Power of performed test with alpha = 0.050: 0.225 

The power of the performed test (0.225) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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One Way Analysis of Variance Tuesday, September 25, 2012, 00:29:39 

Data source: Time to Second Brood, Ln Transformed in USEPA TO30 Vinclozolin 

Normality Test: Passed (P = 0.185) 

Equal Variance Test: Passed (P = 0.769) 

Group Name N Missing Mean Std Dev SEM 

Control 3 0 0.831 0.313 0.181 

Sol. Cntrl 3 0 1.007 0.382 0.220 

Source of Variation DF SS MS F P 

Between Groups 1 0.0466 0.0466 0.382 0.570 

Residual 4 0.488 0.122 

Total 5 0.534 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.570). 

Power of performed test with alpha = 0.050: 0.050 

The power of the performed test (0.050) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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One Way Analysis of Variance Tuesday, September 25, 2012, 00:29:58 

Data source: Time to Second Brood, Ln transformed in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.945) 

Group Name N Missing Mean Std Dev SEM 

Sol. Cntrl 3 0 1.007 0.382 0.220 

12.96 ug/L 3 0 0.609 0.533 0.308 

21.6 ug//L 3 0 0.828 0.234 0.135 

36 ug/L 3 0 0.880 0.323 0.187 

60 ug/L 3 0 0.764 0.399 0.230 

100 ug/L 3 0 0.366 0.348 0.201 

Source of Variation DF SS MS F P 

Between Groups 5 0.769 0.154 1.060 0.428 

Residual 12 1.740 0.145 

Total 17 2.508 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.428). 

Power of performed test with alpha = 0.050: 0.059 

The power of the performed test (0.059) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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One Way Analysis of Variance Tuesday, September 25, 2012, 00:31:16 

Data source: Second Brood Offspring in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.111) 

Group Name N Missing Mean Std Dev SEM 

Control 3 0 8.567 0.751 0.433 

Sol. Cntrl 3 0 6.600 2.425 1.400 

Source of Variation DF SS MS F P 

Between Groups 1 5.802 5.802 1.801 0.251 

Residual 4 12.887 3.222 

Total 5 18.688 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.251). 

Power of performed test with alpha = 0.050: 0.106 

The power of the performed test (0.106) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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One Way Analysis of Variance Tuesday, September 25, 2012, 00:31:30 

Data source: Second Brood Offspring in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.064) 

Group Name N Missing Mean Std Dev SEM 

Sol. Cntrl 3 0 6.600 2.425 1.400 

12.96 ug/L 3 0 9.000 3.279 1.893 

21.6 ug//L 3 0 9.767 6.014 3.472 

36 ug/L 3 0 8.567 5.056 2.919 

60 ug/L 3 0 7.333 2.517 1.453 

100 ug/L 3 0 7.333 2.517 1.453 

Source of Variation DF SS MS F P 

Between Groups 5 21.693 4.339 0.286 0.912 

Residual 12 182.047 15.171 

Total 17 203.740 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.912). 

Power of performed test with alpha = 0.050: 0.050 

The power of the performed test (0.050) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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One Way Analysis of Variance Tuesday, September 25, 2012, 00:45:34 

Data source: Two Brood Offspring in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.880) 

Group Name N Missing Mean Std Dev SEM 

Control 3 0 14.933 3.301 1.906 

Sol. Cntrl 3 0 14.667 3.837 2.215 

Source of Variation DF SS MS F P 

Between Groups 1 0.107 0.107 0.00833 0.932 

Residual 4 51.233 12.808 

Total 5 51.340 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.932). 

Power of performed test with alpha = 0.050: 0.050 

The power of the performed test (0.050) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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One Way Analysis of Variance Tuesday, September 25, 2012, 00:45:57 

Data source: Two Brood Offspring in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.467) 

Group Name N Missing Mean Std Dev SEM 

Sol. Cntrl 3 0 14.667 3.837 2.215 

12.96 ug/L 3 0 15.067 4.008 2.314 

21.6 ug//L 3 0 19.667 10.017 5.783 

36 ug/L 3 0 12.233 4.020 2.321 

60 ug/L 3 0 13.167 4.010 2.315 

100 ug/L 3 0 11.900 1.825 1.054 

Source of Variation DF SS MS F P 

Between Groups 5 122.112 24.422 0.879 0.524 

Residual 12 333.393 27.783 

Total 17 455.505 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.524). 

Power of performed test with alpha = 0.050: 0.050 

The power of the performed test (0.050) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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Descriptive Statistics: Tuesday, September 25, 2012, 00:58:14 

Data source: Fertilization Success in USEPA TO30 Vinclozolin 

Column Size Missing Mean Std Dev Std. Error C.I. of Mean 

Control-1 3 0 65.150 18.986 10.961 47.163 

Sol. Cntrl-1 3 0 52.760 13.609 7.857 33.807 

12.96 ug/L-1 3 0 41.267 13.747 7.937 34.149 

21.6 ug//L-1 3 0 25.400 15.121 8.730 37.562 

36 ug/L-1 3 0 21.430 10.374 5.990 25.771 

60ug/L-1 3 0 24.073 8.483 4.898 21.073 

100 ug/L-1 3 0 52.117 24.673 14.245 61.292 

Column Range Max Min Median 5% 95% 

Control-1 37.880 83.330 45.450 66.670 45.450 83.330 

Sol. Cntrl-1 26.140 63.640 37.500 57.140 37.500 63.640 

12.96 ug/L-1 23.810 57.140 33.330 33.330 33.330 57.140 

21.6 ug//L-1 26.190 42.860 16.670 16.670 16.670 42.860 

36 ug/L-1 19.040 33.330 14.290 16.670 14.290 33.330 

60ug/L-1 16.660 33.330 16.670 22.220 16.670 33.330 

100 ug/L-1 49.210 77.780 28.570 50.000 28.570 77.780 

Column Skewness Kurtosis K-S Dist. K-S Prob. Sum Sum of Squares 

Control-1 -0.358 -- 0.199 0.632 195.450 13454.480 

Sol. Cntrl-1 -1.298 -- 0.293 0.342 158.280 8721.279 

12.96 ug/L-1 1.732 -- 0.385 0.089 123.800 5486.757 

21.6 ug//L-1 1.732 -- 0.385 0.089 76.200 2392.757 

36 ug/L-1 1.630 -- 0.343 0.179 64.290 1592.982 

60ug/L-1 0.936 -- 0.253 0.486 72.220 1882.506 

100 ug/L-1 0.383 -- 0.201 0.629 156.350 9365.973 
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One Way Analysis of Variance Tuesday, September 25, 2012, 01:05:09 

Data source: Fertilization Success in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.360) 

Group Name N Missing Mean Std Dev SEM 

Control 3 0 0.947 0.206 0.119 

Sol. Cntrl 3 0 0.816 0.136 0.0785 

Source of Variation DF SS MS F P 

Between Groups 1 0.0257 0.0257 0.847 0.409 

Residual 4 0.121 0.0304 

Total 5 0.147 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.409). 

Power of performed test with alpha = 0.050: 0.050 

The power of the performed test (0.050) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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One Way Analysis of Variance Tuesday, September 25, 2012, 01:05:33 

Data source: Fertilization Success in USEPA TO30 Vinclozolin 

Normality Test: Passed (P > 0.200) 

Equal Variance Test: Passed (P = 0.271) 

Group Name N Missing Mean Std Dev SEM 

Sol. Cntrl 3 0 0.816 0.136 0.0785 

12.96 ug/L 3 0 0.693 0.141 0.0812 

21.6 ug//L 3 0 0.522 0.168 0.0967 

36 ug/L 3 0 0.473 0.122 0.0703 

60 ug/L 3 0 0.508 0.0951 0.0549 

100 ug/L 3 0 0.812 0.258 0.149 

Source of Variation DF SS MS F P 

Between Groups 5 0.367 0.0734 2.810 0.066 

Residual 12 0.314 0.0261 

Total 17 0.681 

The differences in the mean values among the treatment groups are not great enough to exclude 

the possibility that the difference is due to random sampling variability; there is not a statistically 

significant difference (P = 0.066). 

Power of performed test with alpha = 0.050: 0.424 

The power of the performed test (0.424) is below the desired power of 0.800. 

You should interpret the negative findings cautiously. 
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13 APPENDIX V: STUDY PROTOCOL (P27568) 

PROTOCOL 

Effect of Vinclozolin on the Survival, Development, and 

Reproduction of the Marine Copepod Amphiascus Tenuiremis

 Prepared for: RTI International 

 Protocol Number: P27568 

 BES Study Number: 27568 

 PROTOCOL WRITTEN BY: NANETTE BRADBURY, B.SC. 

 MANAGEMENT APPROVAL: DAVID S. BLOCK, PH.D. 
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PROTOCOL APPROVAL (STUDY NO: 27568) 

Pages 1 - 13 

____________________________________ ___April 25, 2011___ 

David S. Block Date 

Study Director 

Block Environmental Services, Inc. 

____________________________________ ____________________ 

Carol Sloan Date 

Sponsors Agent 

RTI International 
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SCHEDULE OF WORK 

STUDY TITLE Effect of Vinclozolin on the Survival, Development, and 

Reproduction of the Marine Copepod Amphiascus Tenuiremis

RELEVANT 

GUIDELINE 

Draft OECD Test Guideline ‘Harpacticoid copepod development 

and reproduction test with Amphiascus tenuiremis’

STUDY NUMBER 27568 

SPONSOR 

(name & address) 

Hernan Navarro, Ph.d.

RTI International

Hermann 120

3040 Cornwallis Rd

RTP, NC 27709, USA.

SPONSORS AGENT 

(name & address) 

Carol Sloan

RTI International

Hermann 120

3040 Cornwallis Rd

RTP, NC 27709, USA.

STUDY DIRECTOR David S. Block, Ph.D

REPORT DUE TO 

SPONSOR 15 December 2011

TEST SUBSTANCE 

& CAS NUMBER 

Vinclozolin CAS# 50471-44-8

PROPOSED 

EXPERIMENTAL 

DATES 

START: June 6, 2011 COMPLETION: June 24, 2011

[Amended]

START: September 27, 2011 COMPLETION: November 2, 2011

PRINCIPLE 

SCIENTIST(S) 

Terence Clohesy

Nanette Bradbury

ASSISTANT 

SCIENTIST(S) 

Sebastian Castro

Amy Ayala

MAIN TEST 

LOCATION 

2451 Estand Way, Pleasant Hill, California, USA

Testing Room #4
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SCHEDULE OF WORK 

STANDARD 

OPERATING 

PROCEDURES 

All SOPS used will be documented in the study book. 

ANALYTICAL 

CHEMISTRY METHOD 

Vinclozolin will be measured in test solutions using USEPA Method 

provided (GC/MS) 

TEST 

CONCENTRATION(S) 

TO BE EMPLOYED 

The test concentrations will be a dilution water control, solvent control 

and 12.96, 21.6, 36, 60, and 100 μg/L. 

CIRCULATION LIST Sponsor or Sponsor’s Agent 

Client Manager 

QA 

Principal Scientists 

Assistant Scientists 

INTRODUCTION 

The objectives of this study are to determine the effect of Vinclozolin on the survival rate, 

development, mating success, and reproductive output of the copepod Amphiascus tenuiremis. 

This method is based on a draft OECD Test Guideline ‘Harpacticoid copepod development and 

reproduction test with Amphiascus tenuiremis’ (OECD, 2009) and the published ASTM 

Guideline entitled “Standard guide for conducting renewal microplate-based life-cycle 

toxicity tests with a marine meiobenthic copepod.” (ASTM, 2004). 

Principles of the test

Newly hatched nauplii (< 24 hours old) will be exposed in individual microwell (250 μL total 

volume) test chambers to a range of Vinclozolin concentrations in seawater at 25°C for a period 

of 36 days. Once maturing into sexually dimorphic virgin adults (typically between 15-18 days 

at 25 ̊C), male and female copepods will be paired within treatment in order to determine the 

effect of Vinclozolin on mating success and offspring production. The test will be terminated 

when all mating pairs have successfully produced two viable broods or on Day 36 of the 

exposure period, whichever comes first. All phases of the test will be performed under semistatic test conditions with water renewal occurring every three days. The test duration should 

allow sufficient time for the control animals to reach adulthood, mate and produce at least two 

clutches of offspring. 

Test endpoints, such as the survival rate, time to developmental milestone, mating success, and 

offspring production will be statistically compared between Vinclozolin exposed copepods and 
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laboratory controls in order to calculate no observed effect (NOEC) and lowest observed effect 

(LOEC) concentrations. 

MATERIALS 

Test Chemical

A Vinclozolin certificate of analysis and purity information will be supplied by the Sponsor. 

The Sponsor will outline all precautions required in the handling, storage and disposal of 

Vinclozolin.Unused Vinclozolin will be resubmitted back to the Sponsor upon completion of the 

study. 

TEST ORGANISM 

The test organism will be the marine crustacean Amphiascus tenuiremis. The copepods will 

originate from laboratory cultures established by Dr Tom Chandler, Professor of Environmental 

Health and Marine Sciences, University of South Carolina, Columbia, SC 29208 who will 

confirm the taxonomic identity of this species. 

Test animals should be derived from a healthy stock (i.e., showing no signs of stress such as high 

mortality, poor fecundity etc). The stock animals must be maintained in culture conditions 

(light, temperature, seawater medium and feeding regimen) similar to those to be used in the test. 

Justification for the selection of the test species

Amphiascus tenuiremis’c.f. Mielke is an easily-cultured, diosaccid harpacticoid copepod that is 

amphi-Atlantic in distribution ranging from the North Sea/Baltic intertidal to the southern Gulf 

of Mexico (Ref 2). It is an epifaunal to deep burrowing (0 to 15 mm) species that ingests 

sediments for diatoms and bacteria, and produces burrowing larvae with no swimming ability. 

Sexes are dimorphic with females reaching 0.4 mm in length and males 0.25 to 0.30 mm. Males 

are also streamlined in shape and have a swollen geniculate segment on their first antenna to 

clasp the female. 

Amphiascus tenuiremis’ has a generation time (egg to egg) of 17 - 18 days at 25°C in microplate 

culture, or 14 - 15 days in 25°C sediment culture. At 21°C in sediments, the generation time is 

20 - 21 days egg to egg. Females are sexually mature after the fifth copepodite stage (that is, in 

the twelfth life stage). They then mate and produce their first clutch in two to three days. Sperm 

transfer is via a membranous spermatophore sac attached externally by the male to the female 

genital pore. Isolated virgin females can be fertilized successfully up to 14 days post adulthood, 

and females will not extrude viable eggs unless paired with a reproductively-functional mature 

male. Females will produce eight to nine clutches over their median lifetime of 49 ± 2 days. 

Nauplii hatch within two to three days of egg extrusion and reach the copepodite stage in six to 

seven days. 
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CULTURE MEDIUM 

Natural or synthetic seawater may be used as the test medium. If natural seawater is used, it 

should be collected from a location distant from any known sources of pollution and filtered to 

remove indigenous organisms. If synthetic seawater is used, it should be prepared by dissolving 

reagents of recognized analytical grade, or a commercially available formulation, in distilled or 

deionized water. Any water in which the copepods show suitable long-term survival with 

normal development and fecundity may be used as the test medium. 

The salinity of the dilution water will be 30 ± 3‰. The dilution water must have a dissolved 

oxygen concentration above 70% of the air saturation value, and a pH of 8.0 ± 0.4 before being 

used to prepare the test solutions. The dilution water should be aerated for 24 hours before use. 

The dilution water will be filtered to 0.2 μm prior to test solution preparation to minimize 

bacterial contamination. 

The dilution water will be of constant quality and measurement of heavy metals (e.g., Cu, Pb, 

Zn, Hg, Cd, Ni), major anions and cations (e.g., Ca, Mg, Na, K, Cl, SO4), pesticides (e.g., total 

organophosphorus and total organochlorine pesticides), total organic carbon and suspended 

solids should be made. 

TEST APPARATUS 

Disposable polystyrene microwell plates will be used as test vessels. Polystyrene plates are not 

ideal for testing organic compounds because of non-specific binding which may reduce the 

chemical availability of the test compound. The polystyrene microplates will therefore be precoated with glass or polyacrylamide gel unless it can be demonstrated that there is no significant 

loss of the test compound between water renewals using ‘uncoated’ polystyrene microwell 

plates. The microplates will be 96 well plates each well having a 300 μL total volume and a 250 

μL working volume. 

EXPERIMENTAL DESIGN 

The test solutions will be maintained at 25 ± 2°C by housing the test vessels in a temperaturecontrolled room or in climate controlled chambers. Glass or plastic ‘humidity chambers’ will be 

used to reduce evaporative losses. Potential for evaporative loss will be measured before 

beginning any tests by placing seawater filled microplates in the test room or chamber for 72 

hours and measuring salinity change over this period. Photoperiod will be controlled to provide 

12 h light: 12 h dark. 

In addition, some or all of the following equipment will be required: 

• oxygen meter (with microelectrode or other suitable equipment for measuring 

dissolved oxygen in low volume (e.g., 10-mL) samples); 

• pH meter; 

• conductivity meter, osmometer or salinity refractometer; 

• adequate apparatus for the control of the lighting regime; 
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• stereo compound microscope, preferably inverted with dark-field or Hoffman DIC 

illumination; 

• dissection stereomicroscope, preferably with dark-field illumination and fiber 

optic episcopic illumination. 

• climate-controlled environmental chambers or a temperature regulated room; 

• 0.5 m3

 glass or plastic rectangular containers with sealable lids to create humidity 

chambers for the microplates (to minimize evaporative loss). 

• various plastic or stainless-steel mesh sieves (0.05 mm to 0.25 mm) to culture, 

capture and sort copepods; 

• Pasteur pipettes, preferably silanized or equivalent treatment to prevent copepods 

from adhering to pipette walls; 

• adjustable single and multi-channel volumetric pipettors (1-10 μL, 100-500 μL) 

for food additions; 

• 250 μL Hamilton glass volumetric syringes with 23 gauge beveled stainless-steel 

needles; 

• haemacytometer cell counting chamber or electronic particle counter/analyser 

(e.g., a Coulter principle impedance-based system) 

Development to adult

Nauplii (< 24 hours old) will be obtained by isolating ovigerous females from a mass culture at 

least 3 weeks old. Nauplii will be exposed in individual microwells of a 96 microwell plate (30 

animals per microplate) with three microplates per treatment (total of 90 animals) for the control, 

solvent control and each test concentration in the definitive test. The outer microwells of the 

plate will be left empty. 

The positions of the microplates in the test area will be randomized. Addition of nauplii, 

subsequent handling, loading, observation and feeding will be performed in an irregular plate 

order. If experimental units are handled in treatment or concentration order, then some timerelated effect, such as operator fatigue or other error, could lead to greater or lesser effects at the 

higher or lower concentrations. 

The number of days it takes for development of each individually isolated copepod, from a first 

stage nauplius to a juvenile copepodite to a reproductively mature adult, will be checked daily. 

Survival of larvae (nauplii), juveniles (copepodites) and all adults will also be recorded daily up 

to and including the last day of the test. The sex of each adult will be recorded after sexual 

dimorphism is reached. 

Creation of sexed pairs

On the first test solution renewal day upon reaching adulthood, when the individual copepods 

can be sexed visually (after 15 - 18 days), mature virgin males and females will be removed from 

their respective treatment or control microplates and paired impartially with an opposite sex 

individual (i.e., from the same microplate within the same treatment) in a new or unused 

microwell filled with test medium for each treatment or control replicate. If necessary the males 

and females may be transferred to a separate vessel containing the appropriate test solution (or 
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Page 172 

control) media to aid identification of sex. If not all the animals have achieved sexual maturity at 

this renewal day then unpaired animals will be left until the next renewal day and paired with 

copepods that have become adult during the preceding three days. 

If survival was 100% and the sex ratios are balanced then this would result in 15 pairs. If sex 

ratios are skewed, or mortality rates have produced insufficient numbers of males and/or females 

to produce multiple mating pairs within a given microplate within a given treatment, then virgin 

adults may be mated across microplates within treatments. However, this must be noted in any 

report of results, and an a priori assumption of no significant microplate to microplate variability 

within the given treatment(s) must be made by the investigator. 

Once individual mating pairs are established for each treatment and control adults, 90-95% of 

transfer seawater will be aspirated and replaced with the appropriate control or test substance 

concentration. This replacement of media after matings are in place will help to ensure that 

transfer seawater is diluted to negligible levels at the beginning of the mating time window. In 

the case of uneven sex ratios, spare unmated males or females may be retained and monitored for 

survival as individuals to the end of the test exposure period. 

Offspring production

After pairing of male: female mating pairs, observations will be made daily for the presence of 

females carrying their first and subsequent egg sacs. In controls, two fertile egg sacs per female 

(> 4 eggs/sac normally) are usually extruded simultaneously from the abdomen in 1 - 2 days. 

Total egg number per mating pair will be counted by direct observation of all eggs in each egg 

sac if desired. Numbers of successfully hatching nauplii (i.e., total viable clutch, or fecundity) 

will be recorded for the first and second successive clutches of eggs. Hatching number can be 

obtained by subtracting any non-hatched or necrotic eggs from the total clutch size at the clutchone and clutch-two time points. Alternatively, numbers of hatched nauplii crawling on the 

bottom of the microwell may be counted by direct microscopic observation. 

Once the second clutch is hatched, then they will be counted and the bioassay is complete for a 

given mating pair. All such mating pairs should be recorded as a “successful mating”. Percent 

mating success will be calculated for each group of mating pairs in each microplate within each 

treatment and control as the ratio of successful mating pairs versus unsuccessful pairs (i.e., those 

pairs unable to produce 2 viable clutches of eggs). An average percent mating success and 

standard deviation will be calculated from these data for each treatment and control. The mean 

number of viable offspring produced through two broods should be > 8 offspring per mating pair 

in the controls. At the end of the exposure period, all remaining copepods (i.e., including 

unfertilized females and males) will be discarded. 

Observations

Copepod lifecycle endpoints will be monitored daily in each microplate. Test observations 

include mean development rates from nauplius to the copepodite and adult stages, respectively, 

fertilization success (i.e., number of mating pairs able to produce two successive clutches), total 

mean viable offspring production per mating pair (i.e. through two clutches averaged over the 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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number of mating pairs created and tested), time to production of first clutch, time interval 

between successive clutches, and stage-specific mortalities. Observations may also be made for 

any abnormal behaviour (relative to the controls), including uncoordinated swimming, loss of 

equilibrium, change in pigmentation and atypical quiescence. Sex ratio may be determined by 

observations of male and female secondary sexual characteristics which in harpacticoid 

copepods normally can be distinguished in the 5th copepodite stage at the earliest. Numbers of 

aborted egg sacs and necrotic/infertile eggs may also be counted. 

Feeding

The test animals will be fed on Day 0 and at every other microwell renewal day (every 6th day of 

the test) with cultured algae. Each microwell will receive the equivalent of 2 μl of a 1:1:1 

volume mixed-algal cell suspension (centrifuged, pelletized and re-constituted in fresh seawater 

to 107

 cells/mL) of a chlorophyte (e.g., Dunaliella tertiolecta), a chrysophyte (e.g., Isochrysis 

galbana) and the diatom Phaeodactylum tricornutum. Therefore the cell density added to each 

test microwell at each feeding should target ~ 2 x 104 cells/microwell. This density will support 

copepod maturation and reproduction without fouling the microwell environment. 

Test medium renewal

The test will be run as a semi-static renewal with the medium in each test microwell being 

renewed every third day of the test. Potential evaporation losses of media from the test chambers 

will be monitored daily and, should not exceed 10% on a daily basis. To prevent such a 

scenario, microplates will be placed over a platform over a water bath inside a humidity chamber 

in the temperature regulated incubator. If necessary excess evaporative losses can be corrected 

by more frequent medium renewal. 

When the medium is renewed, a fixed volume (preferably 90-95% of the total test medium 

volume) will be aspirated from each microwell chamber under dissection stereomicroscopic 

observation and cool fiber optic or tangential lighting using a Hamilton gas syringe of suitable 

needle diameter (e.g., 23 gauge bevelled). Care should be taken that copepods are not aspirated 

into the syringe. If copepods are aspirated they may experience stress or egg sac damage; thus 

such aspirated copepods should be disqualified from the test. 

PREPARATION OF TEST SOLUTIONS 

Stock solutions of Vinclozolin will be prepared in an organic solvent (acetone) on a weekly 

basis. The test solutions will be prepared by addition of the current primary stock solution to 

dilution water. Each test concentration and the solvent control will contain the same 

concentration of solvent, which will not exceed 1 ml/L. The volume of each test solution 

prepared will be in excess of that required for the replicate test vessels to allow for initial 

physical and chemical analysis. Further details of the test solution preparation will be 

documented in the study book. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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PHYSICAL AND CHEMICAL ANALYSIS 

During the test, dissolved oxygen, pH and temperature should be measured in the controls and all 

test concentrations each time test medium is renewed. As a minimum, these measurements 

should be made in the control(s) and in the highest test concentration. Temperature should 

preferably be monitored continuously in at least one blank test microplate or in a 100-mL Griffin 

beaker filled with deionized water and placed inside the microplate humidity chamber.

The analytical chemistry method used will be provided by USEPA. Chemical analysis will be 

conducted on all newly prepared primary stock solutions, solutions of controls, all renewal test 

concentrations (Ups), and on the solutions removed from the microwells (Downs). Chemical 

analysis will be conducted at the start of the test and then on at least one water renewal per week 

for the duration of the test. If timings between test renewals varies then a mixture of different 

length renewals will be included in the analysis (e.g. 2 or 3 days between renewals). 

BIOLOGICAL DATA ANALYSIS 

The mortality data for selected time periods will be analyzed to calculate the median effect 

concentrations (EC50) and 95% confidence intervals. 

Fisher’s Exact Test or Pearson’ Chi-square goodness of fit tests (Ref) can be used to identify 

significant differences in survival between the control(s) and each treatment after selected time 

periods (e.g. 10 and 21 days). 

Larval development rates, stage specific mortality, percent mating success and sex ratios may all 

be analyzed for significant differences relative to the controls. Reproduction data, expressed as 

the mean number of F1 offspring produced from fertilized females (F0) in each replicate will be 

calculated. Generally, the reproduction data are not adjusted for mortality but are presented on a 

per female or per mating pair basis. If an animal dies after producing young, the total number of 

offspring produced up to the time of death will be used. On occasion, it may be necessary to 

make an adjustment for mortality and those animals that died during the exposure will not be 

included in the statistical analysis. One-way nested analysis of variance (ANOVA) will be used 

to evaluate effects of the test substance on the reproduction (and other chronic endpoints) of the 

copepods, and a multiple comparison procedure (e.g., Dunnett's test) will be used to define the 

treatment level resulting in e.g., significantly different reproduction from the control(s) (p = 

0.05). For ANOVA, individual microwell mating pairs will be nested within their particular 

microplate, which will be replicated within a given treatment. A nested model will be ideal if 

data are balanced across microplates and treatments because microwells within a microplate are 

not strictly independent of one another. 

Analysis of variance is a parametric procedure and is based on the assumptions that the 

observations are independent and normally distributed, and that the variance of the observations 

is homogeneous across all test substance treatments and the control(s). Data will be tested for 

normality and homogeneity of variance prior to analysis of variance techniques. Data that fail to 

meet these assumptions for ANOVA will be analyzed using non-parametric (distribution-free) 

procedures. 
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The biological data may also be analyzed using a regression model in order to estimate the 

concentration that would cause an x% reduction in reproductive output, i.e., ECx (e.g., EC50, 

EC20 or EC10). Depressed intrinsic or instantaneous rates of population increase (rm or λ), may 

also be calculated. Details of these calculations will be recorded in the study book. 

If a statistically significant difference in survival, development rate, or reproduction is detected 

between the control and solvent control, only the solvent control will be used as the basis for the 

calculation of results. If no significant differences exist between control and solvent control data, 

these may be pooled for greater statistical power in comparisons of control response with test 

substance treatments. 

For the test to be valid, the following performance criteria will be met: 

Parameter Units Expected Results

Survival: Daily assessment of survival will 

be made to provide a basis for expression 

and interpretation of reproductive output. 

Not 

Applicable 

75% or greater survival in controls. No individual control 

microplates survival should be lower than 70%. At the end of 

the exposure period. 

Dissolved Oxygen Concentration: 

Expressed as % of the air saturation value. Percentage 

The dissolved oxygen concentration must have been at least 

70% of the air saturation value (ASV) throughout the exposure 

period. 

Temperature: Low variation in exposure 

temperature. needed o C The temperature should not vary more than 4 o C during the entire test with a 36 day mean temperature of 25 ± 2 o C. 

pH: Low variation in exposure pH needed. pH units pH must not vary more than 0.5 units from the average control 

pH with a 36 day mean pH of 8.0 ± 0.4. 

Conductivity/salinity: Low variation in 

conductivity/salinity needed. % Conductivity/salinity must not vary more than 10% from the control start value of 30‰ 

Sex Ratio: Number The sex ratio in F0 controls (females/males) should on the 

average range between 0.4 to 2.0. 

Viable Offspring: Number The average number of viable offspring produced through two 

clutches in the control(s) should not be less than 8 individuals. 

Average Number of Days to the First 

Clutch Number 

The average number of days to the first clutch in the control(s) 

should at a room temperature of 25oC not exceed the number of 

days representing 70% of the total exposure period or 25 days, 

whichever is less. 

REPORTING 

Following completion of the experimental phase and internal scientific review, a draft report will 

be submitted to the Sponsor for comment. The content and format of the draft report will 

comply with the requirements of the relevant regulatory authorities. Any amendments to the 

content and format of the draft report requested by the Sponsor that are in addition to the 

requirements of relevant regulatory authorities will incur an extra charge. Unless the Sponsor 

requests any amendments within three months of receipt of the draft report, the project shall be 

deemed completed and final reports issued. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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COMPLIANCE 

GLP compliance

The study will be conducted in accordance with USEPA Good Laboratory Practice Regulations. 

Quality assurance evaluation

The study described in this protocol will be subject to Quality Assurance Audit. The form of this 

inspection will be described in the QA Standard Operating Procedures.

ARCHIVING PROCEDURES 

 

All raw data and other relevant documents from this study will be archived, identified by the 

study number. 

REFERENCES 

ASTM E 2317-04. (2004). Standard guide for conducting renewal microplate-based life-cycle 

toxicity tests with a marine meiobenthic copepod. American Society for Testing and Materials, 

West Conshohocken, PA, 22 pp. 

OECD Draft Guidelines for the Testing of Chemicals. Harpacticoid Copepod Development and 

Reproduction Test with Amphiascus tenuiremis. Post June 2009. 

Lang, K (1948) Monographie der Hapacticiden. Hanken Ohlsson, Lund, Sweden 

Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a 

control. Journal of the American Statistical Association, 50, 1,096-1,121 

Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics, 20, 482-

491 

Piegorsch WW, Bailer AJ (1997) Statistics for environmental biology and toxicology. Chapman 

and Hall, London, UK 

USEPA. 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and 

Receiving Water to Marine Organisms. EPA-800-R-02-014. 4th Edition. 

USEPA. Good Laboratory Practice Standards. 2007a. 40CFR160. 

USEPA. Good Laboratory Practice Standards. 2007b. 40CFR792. 
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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14 APPENDIX VI: DEVIATIONS FROM STUDY PROTOCOL 

14.1 LIST OF DEVIATIONS

Below is a list of the deviations encountered during the study.

Deviation

number

Deviation title Deviation description

1 Protocol not distributed or

signed.

The completed test protocol was not submitted to Sponsor or

signed by Sponsor prior to initiation of study.

2 Starting number of 

nauplii/Empty test wells 

On D1, several wells across all test treatments were observed

to contain >1 nauplius per well.  These extra nauplii were

transferred to test wells observed to be empty on the same

replicate within each treatment at the first water change on

day 3.  Extra organisms were haphazardly selected to be

discarded if there were not a sufficient number of empty test

wells available.

3 Water quality measurements

not recorded

Dissolved Oxygen, pH, and Salinity readings were not recorded

during water renewal on D9 and D18

4 Temperature not recorded Temperature in the humidity chamber was not measured on

D5‐reading #1  

5 Water chemistry values not

collected in test solutions

Dissolved Oxygen and pH were not collected in the Up and

Down test solutions.  Operators instead collected data from

dilution water prior to water change.  The basis for this

deviation is that operators interpreted the language of the

OECD 2009 guideline to indicate this was an optional

component (“should”, as opposed to “must” or “will”)  

6 Potential for evaporation data

not recorded.

A 14‐day dry run study was performed in June/July for the

operators to practice performing the test.  Salinity results on

water change days were reported to have been performed,

but the data was not recorded.

7 Lost copepods during pairing

process

Approximately 6 animals were lost in various plates spread

across the concentrations.

8 Mature male/female copepods

not immediately mated.  

Sexually mature copepods were scheduled to be paired for

mating on Day 18, a water change day.  D18 fell on a Saturday

and the laboratory workload coupled with reduced staff was

such that a water change and mating could not occur on the

same day.  BES elected to prioritize the water change and

delay the creation of mating pairs until D21.

9 Mating pairs not from same

microplate

In order to maximize the number of mating pairs, males and

females were paired across replicates but within treatment.  

However, we attempted to create male/female pairs within

replicate when possible.

10 Survival not consistently

recorded after mating

Survival was not consistently recorded after D27 due to the

difficulty tracking individual copepods after mating.
BES Project No. 27568 EPA Contract No. EP-W-06-026 

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Deviation

number

Deviation title Deviation description

11 Vinclozolin chemistry samples

not stored under recommended

conditions

Up and Dwn chemistry samples were stored at 4 degrees C

until submission to analyzing laboratory. BES was instructed

by the analyzing laboratory to store samples at –20 degrees C

12 Vinclozolin chemistry samples

not analyzed in a timely manner

Analyzing laboratory did not have method specific standard

curve until mid to late October 2011. Up and Dwn chemistry

samples were stored until test was completed, and then

submitted for analysis.