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

Study Title

Effect of Vinclozolin on the Survival, Deve lopment, 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-06-026BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 2 

### 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 Envi ronmental Services, Inc (BES) to determine adherence with the Quality Assurance Project Plan (QAPP) and labora tory standard operating procedures. Dates of study inspections, inspection types, and dates repo rted to the Program Manager are given below.

Inspection Date Inspection Type Reported to Program Manager

27568 ( Vinclozolin ) 9/26/2011 in-test

, setu p 9/26/2011 10/24/2011 in-test

, matin g 10/24/2011 11/15/2011 anal y tical methods verification 11/15/2011

1/6/2011 chemistr y 1/12/2012 3/30/2011 data

, biolo gy s p readsheets 4/4/2012

5/18/2012 draft re p ort 5/29/2012

BLOCK ENVIRONMENTAL SERVICES, INC.

________________________________________________ ____May 29, 2012____ Ronald M. Block, Ph.D. Date Quality Systems DirectorBES Project No. 27568 EPA Contract No. EP-W-06-026

Page 3 

### 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 BiologistBES Project No. 27568 EPA Contract No. EP-W-06-026

Page 4 

### DATA RETENTION

All records that would be required to reconstruc t the study will be maintained. The original raw
data and the original fina l report will be maintained indefinite ly 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

Page 5 

### TABLE OF CONTENTS

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

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

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

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

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

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

2 MATERI ALS 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 concen trations 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................................................................................ 17BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 6 3.3.4 Development – Maturation time between naupliar and copepodite stages............... 17 3.3.5 Development – Maturation time betw een copepodite and adult stages

.................... 17 3.3.6 Development – Maturation time be tween 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 hatc hed nauplii from first brood .................................... 18 3.3.10 Reproduction – time to second egg extrusion....................................................... 19 3.3.11 Reproduction – number of hatc hed 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 LO EC for all endpoints

......................................................... 20

4 DISCUSSI ON ........................................................................................................................ 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 Micr oplate Test Cha mber ................................................................. 23

8 TABLES

......................................................................................................................... ........ 23 Table 1: Method for preparation of Vinc lozolin 200 m g/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 Vinclozo lin 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 fo llowing hatching of first brood offspring

...... 35BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 7 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 a nd LOEC for all endpoints .................................... 39

9 APPENDI X I: QUALIT Y ASSURANCE PROJECT PLAN (QAPP)............................. 40

10 APPENDI X II: QUALITY OF LABORATORY WATER AT BES ............................... 75

11 APPENDI X III: VINCLOZOLIN CHEMISTRY DATA REPORT ............................... 76

12 APPENDI X IV: STATISTICAL DATA OUTPUTS....................................................... 116

13 APPENDI X V: STUDY PROTOCOL (P27568) .............................................................. 165

14 APPENDI X VI: DEVIATIONS FROM STUDY PROTOCOL ..................................... 177

14.1 List of deviations......................................................................................................... 177BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 8 

### 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- μ g/L using a microplatebased life-cycle screening assay. Copepod survival in the laborat ory control and carrier control on Day 16 was 80% and 90%, respectively, with resp ective survival rates d ecreasing to 75% and 84% by Day 36. No Vinclozolin test concentrat ion significantly affect ed copepod survival on Day 16 and Day 36 time points relative to the carri er 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 deve lopment data obtained during the study, NOEC and LOEC values were calculated to be 100μg/L and >100μg/L respectively. None of the Vinclozolin concentrations signifi cantly affected the ratio of female:male copepod development. Vinclozolin was observed to have a significant e ffect on copepod reproduction, with an increase in the time for virgin females to extrude th eir 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 th e total number of viable eggs and hatched nauplii, nor the percentage of su ccessful mating pairs comp ared 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 anal yses were performed using nominal values.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 9 

### 1 INTRODUCTION

The U.S. Environmental Protection Agency (E PA) is currently implementing the Endocrine Disruptor Screening Program (EDSP). To support this program, EPA has contracted with RTI International (RTI) to provide co mprehensive toxicological testing services. These services were designed to assist the EPA in validat ing a suite of longer term Tier 2 in vivo , arthropod ecotoxicological tests for identif ying and characterizing endocrin e effects through exposure to pesticides, industrial chemicals, and environmenta l contaminants. The obj ective 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 mysi d 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 standa rdize technical guidan ce and test guidelines in support of the Office of Chemical Safety and Pollution Preventi on regulatory programs. These studies were conducted under the study protocol, applicable Quality Assurance Project Plans (QAPPs), relevant program and facility standard opera ting procedures (SOPs), and published guidance documents.

The purpose of work to be conducted by Block E nvironmental Services, Inc. (BES) under this assignment is to demonstrate a copepod develo pment and reproduction test based on ASTM E 2317-04 “Standard guide for conducting renewal microplate-based life-c ycle 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 in tended to serve as a higher tier test with an aquatic arthropod for collecting defin itive concentration-response information on adverse effects suitable for use in ecological risk assessm ent. Specifically, collection of development, reproductive and life-table parameter data from this copepod species provides sensitive and ecologically relevant measures th at can be used to determine th e potential chronic toxicity of many water-borne contaminants. Endpoints incl uded 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 c onducted from September 27, 2011 to November 2, 2011. The original raw data, protocol, and technica l report produced durin g this study are stored at the BES laboratory, located in Pleasant Hill, California.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 10 

### 2 MATERIALS AND METHODS

2.1 QAPP/PROTOCOL

This study was conducted accord ing to the procedur es outlined in the Quality Assurance Project Plan (QAPP) for Task Order 30 Mysid/Copepod In ter 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 su ccess and reproductive out put 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 w as received from Professor G.T. Chandler (University of South Carolina, School of Public Health, Columb ia, 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 Laborator y, copepod cultures were maintain ed 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 Labo rato ry. Synthetic Instant Ocean ® seawater was used to prepare the copepod culture medium. The synthetic seawat er was prepared by dissolving Instant Ocean ® sea salt into laboratory grade deionized water in acc ordance with the manufacturer’s instructions at appropriate amounts and volumes necessary to achieve 30 ‰ salinity. Copepod culture media was prepared by filtering the synthetic seaw ater through a 0.2μm hydrophilic polyethersulfone membrane (VWR ® 47mm glass filter holder; Pall Cor poration, Port Washington, USA). The quality of the culture media wa s checked weekly using measurem ents of dissolved oxygen, pH, salinity, and temperature. The culture media wa s 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 2.8x10 8 algal cells/L.

2.2.3 Laboratory Reagent Water for Culture Media Preparation

Laboratory reagent water is prepared by processing carbon scrubbed m unicipal water in tandem through a reverse osmosis membrane and deionizati on resin. The quality of the laboratory water is checked once annually, at a minimum. The an alytical report for the most recent samples, which were collected and analy zed in May 2011, is provided in Appendix II

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

Page 11 2.2.4 Culture Sediment

Concentrated sedim ent 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 sedime nt was obtained from Bread a nd 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 us ing 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 (m in-max: 380-420 ft-c) and 12-h da rk photoperiod was maintained throughout the culturing of A. tenuiremis

.

2.3 TOXICITY TEST PROCEDURE

2.3.1 Test chemical

Three sam ples 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 contai ner in a dark ventilated cabinet. Concentrations were adjusted for the purity of the test substance and are presented as active ingredient (a.i.). The follow ing information was provided:

Name: Vinclozolin CAS registry number: 50471-44-8

Source: Battelle Marine Scie nce 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 roomBES Project No. 27568 EPA Contract No. EP-W-06-026

Page 12 2.3.2 Preparation of test solutions

The prim ary stock solution of Vinclozolin was pr epared 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, Vinclozoli n 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 con centration 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-clo sing organic solvent safe ty 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. Calcul ated 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 se rial 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 solv ent carrier was added to an aliquot of the control solution in a volume e quivalent 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 m icroplate test design followed the draft OECD (2009) guideline. The following equipment was used:

 96-well polystyrene microplates (Cor ning 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 retain s 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 w ithin environmental chamber (Dixon DT8 Recorder #39585);

 Alcohol thermometer immersed in a glass beak er containing dilution water located in the humidity chamber. Thermometer is calibrate d to an NIST certified thermometer every three months;

 Two inverted stereo compound microscopes with dark-field (T wo AmScope IN200B with No equipment ID);BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 13  Two dissection stereomicroscope, with dark-field illumination and LED episcopic illumination (Two AmScope SM-1TZ-PL);

 Temperature and light regulated environm ental chamber (Lab-Line Environ-Shaker #3597 designated as environmental cham ber #3, located in Testing Room #4);

 One 0.047 m 3 plastic container with sealable lid to create humidity chambers for the microplates (to minimize evaporative loss of test solutions). A plexiglass platform (perforated with 0.3cm 3 holes) was supported above standi ng 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 Envi ro-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 (5-50 μ L, 100-1000 μ L) for inoculating algae cultures used as food for cultures and tests

 Adjustable multi-channel volumetric pipette (1-10 μ L) for adding food and for adding primary stock solution during the preparation of test solutions;

 10, 50, and 250 μ L Hamilton glass volumetric syringe s 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 μ m sterile polyethersulfone membrane, 47 mm (Pall, Port Washington, USA)

2.3.4 Collection of copepod nauplii in preparation for the test

Approxim ately 300 gravid female copepods were collected from laboratory stock mud cultures by sieving across 106 μ m steel mesh. Gravids retained on the 106 μ m sieve were rapidly rinsed with 30 ‰ dilution water into polystyrene Petri dishes. The organisms were transferred using enviro-coat treated glass transfer pi pettes into wells of 12-well Netwell ® microplates filled with 0.2 μ m filtered seawater. Each well of the Netwell ® microplate is equipped with a 74 μ m polycarbonate mesh filter (Corning Costar 3477), suspended 4 mm above the bottom of the microplate. The 74 μm polyester mesh retain s gravid female copepods but allows hatched nauplii to fall through to the bottom of the well. Each well contai ned 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 u ltra-low attachment Hydrogel ® coated (i.e., hydrophilic) polystyrene 96-well micr oplates (Corning Cost ar 3474) containing 250 μ L of each test solution. For each Vinclozolin test concen tration 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 (col umn 1 and 12 were purposely left empty). .BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 14 The 96-well plates were randomly assigned a conc entration using a random number table. All subsequent handling, loading, obs ervations and feeding were done without respecting any order to ensure that handling bias would not be cons trued as being a chemical-dependent effect. For example, if experimental units are handled in tr eatment or concentration order, then some timerelated effect, such as operator fa tigue or other error, could lead to greater or lesser effects at the higher or lower concentrations. Only one test chemical concentr ation was tested per microplate to reduce chances for micr owell cross contamination.

2.3.5 Diet during testing

During the chem ical exposure period, copepods were fed every six days. Individual copepods in test chambers were fed 2 μ L of a mixed-algal cell suspensi on (centrifuged, pelletised and reconstituted in fresh 30 ‰ seawater to 10 7 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 10 4 cells/microwell.

2.3.6 Daily observations

Nauplii were m onitored 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 da ys, to reproductive adult. Upon reaching adulthood, individual virgin female and male copepods were randomly mated (pairwise within each treatment) in fresh microwells (we lls C and E) using the ex posure parameters and conditions described above. Mating pairs were monitored da ily for reproductive success (egg extrusion and fertility) and numbers of offs pring successfully hatching for each pair.

2.3.7 Test conditions

Test conditions are sum marized in Table 3 . Essential physico-chemi cal 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 en vironmental chamber set at 25°C. Due to small volumes within the microwells, direct temperatur e 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 plac ed near test vessels. The thermometer is calibrated quarterly against an NIST traceable thermometer. Physico-chemical parameters were not measured in freshly made test solutions (referr ed to here as ‘Ups’) and test so lutions drawn out of test vessels (i.e. ‘Downs’) in order to prevent Vinclozoli n cross contamination to other ongoing toxicity studies.

2.3.8 Vinclozolin concentrations in test solutions

Sa mples for determining Vinclozolin concentratio n were collected from freshly prepared test solutions in the case of Up samples and from pool ed solutions from test vessels in the case of Down samples. Samples were collected and sealed into 125 ml amber glass bottles stored inBES Project No. 27568 EPA Contract No. EP-W-06-026

Page 15 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 st andards of known Vinclo zolin concentrations prepared in hexane. Results were su bmitted in audited reports 1110574 and 1111154 ( Appendix III ).

2.3.9 Statistical analyses

Mean and standard deviations (SD) were calculated using Microsoft TM Excel 2000. S urvival, development, and reproduction data were anal yzed using SigmaStat version 2.03 for Windows (IBM, Inc. (formerly SPSS, Inc.), Armonk, New Yo rk, 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 a pplied. The following procedures were used:

 Data for each endpoint were anal yzed 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 distribu tion 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 signifi cant effects, an analysis of variance (ANOVA) was conducted on each data set (exc ept 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 diluti on 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 obtai n P values between the carrier control and treatment groups since the SigmaSta t program does not cal culate individual pairwise P values when performing the Dunnett’s test.

 All proportional data points su ch 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 ).BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 16 For clarity, the definition for NOE C 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-chem ical 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 wa s 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 CONCENT RATIONS IN TEST SOLUTIONS

Test concentrations of Vinclozolin were re ported by North Coast Laboratories LTD, Arcata, California, USA. A copy of the audited re ports num bered 1110574 and 1111154 is presented in Appendix III . Concentration data are reported in Table 6 . These tables indica te concentrations of the test substance were all less than the laborat ory reporting limit. The primary stock solutions used to prepare the test concentrations were m easured to be within 10 percent of the nominal concentration. Therefore the reported concentrati ons of Vinclozolin in the test treatments are likely a result of degradation with in 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 te rm of nominal values.

3.3 BIOLOGICAL DATA

A summ ary of the statistical anal yses 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 statis tically compared to th e carrier control using Pearson’s chi- square go odness-of-fit test. In summary, mean survival in the control and the carrier control group was 80% a nd 90%, respectively. A statistical difference between the control means was not evident (P=0 .095). Survival in the test tr eatments ranged from 82 to 91%.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 17 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 term ination on day 36. Table 8 lists the percentage survival for a ll groups. Treatment means, including thedilution water control, were statistically compared to the carrier control using Pearson’s chisquare goodness-of-fit test. On Day 36, the cont rol 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 th e 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 m ean survival rates were st atistically compared within treatment using Pearson’s chi- square go odness-of-fit test. differences (p >0.05) in survival were observed within control (P=0.477), carrier cont rol (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) Vinc lozolin treatment groups between the test midpoint and test termination.

3.3.4 Development – Maturation time between naupliar and copepodite stages

Copepod developm ent from the nauplius to copepodite stages is presented in

1 Table 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 ti me in the control and the carrier control group was 11.2 and 11.9 days, respec tively. A statistical difference between the control means was not evident (P=0.299). relativ e to the carrier contro l group were observed for any Vinclozolin test treatment (P=0.140). Base d 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 developm ent from the copepodite stage into virgin adults is presented in

1 Table 10

. Treatment means, including the dilu tion water control, were statisti cally compared to the carrier control using a One Way ANOVA test. In summar y, mean development time in the control and the carrier control group was 7.5 and 7.9 days, resp ectively. 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 an y Vinclozolin test treatment (P=0.130). Based on these statistical observations, NOEC and LOEC values were determined to be 100 and >100μg/L, respectively.BES Project No. 27568 EPA Contract No. EP-W-06-026

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

Copepod developm ent from the nauplius to adult stages is presented in

1 Table 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, respec tively. 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 Vinclozo lin 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 fe males to males is presented in

1 Table 12 . Treatment means, including thedilution water control, were statistically compared to the carrier control using Pearson’s chisquare goodness-of-fit test. The av erage gender ratio (female to ma le) 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 rati os observed in the cont rol 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 tim e to first egg extrusion in fe males following the mating of virgin male and female copepods is presented in

1 Table 13 . Treatment means, including the dilution water control, were statistically compared to the carrier co ntrol 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 differen ce (p>0.05) betwee n the control means (P=0.800). There was a

1 statistically significant (p<0.05) increas e 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 signifi cant 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 mi nimum of six days and a maximum of twelve days following mating. The time to first clutch observed in the contro l 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

1 Table 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 naupl ii in the control and carrier control groupsBES Project No. 27568 EPA Contract No. EP-W-06-026

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 significanteffects 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 tim e to second egg extrusion in female copepods is presented in

1 Table 15

. Treatment means, including the dilu tion water control, were statisti cally 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 num ber of hatched nauplii from th e second egg extrusion following the mating of male and female copepods is presented in

1 Table 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 wa s 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 offspri ng were observed among the test treatments when compared to the carrier contro l group (P=0.912).

3.3.12 Reproduction – number of nauplii hatched from two successive broods

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

1 Table 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 gr oups 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 th e test treatments when compared to the carrier control group (P=0.524). The total number of viable offspring produced through two clutch es 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 th e carrier control using a On e Way ANOVA test. The average fertilization success rates in the control and carrier control treatments were 65 and 53%, respectively (

1 Table 18 ). There was not a statistically signi ficant difference (p>0.05) between theBES Project No. 27568 EPA Contract No. EP-W-06-026

Page 20 control means (P=0.409). The average fertilizat ion 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 we re excluded from statistical analysis. Based on these statistical results, NOE C and LOEC values were determined to be 100 and >100μg/L, respectively.

3.4 SUMMARY OF NOEC AND LOEC FOR ALL ENDPOINTS

A summ ary of NOEC an d LOEC for all endpoints is presented Table 19

.



### 4 DISCUSSION

4.1 QUALITY OBJECTIVES AND CRITERIA

This study achieved the m inimum 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 oxyge n, temperature, pH, and salinity) were all within requirements

• The sex ratio in the control group was w ithin 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 chem ical analysis of Vinclozolin test concentr ations showed that con centrations of the test substance were all less than the laboratory re porting limit. A review of the procedure for preparing the test solutions indicated the solu tions were prepared from the primary stock solutions properly. Moreover, the primary stock solu tions used to prepare the test concentrations were measured to be within 10 percent of the ex pected 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 we ll as through naturally degradative processes prior to an alysis. 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 t he 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 discu ssed for survival, developmental and reproductive endpoints. Vinclozolin did not affect copepod surv ival at any exposure level by day 16 of theBES Project No. 27568 EPA Contract No. EP-W-06-026

Page 21 test, with the survival rate not significantly reduced further by test termination on day 36. Similarly, the developmental endpoints tested were also not significantly a ffected, as Vinclozolin did not significantly affect the time for A. tenuiremis nauplii to mature into the copepodite stage and subsequent transformation to the adult stag e at test concentrations up to 100μg/L. Vinclozolin had no effect on gender assignment fo r 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 te sted. Vinclozolin did not signifi cantly reduce or increase the number of hatched offspring at any test conc entration, although an appreciable decrease in hatched offspring in the 100μg/L treatment was noted. Lastly, with regards to reproductive endpoints, fertilization success wa s 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 vinclo zolin 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 gr owth media for algae culturing. Acknowledgement also needs to be made to Ka te Washburn with the Ch andler laboratory for assisting with the training of BES technical staff and for providi ng technical advice. Thanks to
the staff of North Coast Laboratories LTD for an alyzing the analytical chemistry data. Many thanks go to BES staff scientists Terry Clohe sy, Nanette Bradbury, Sebastian Castro, and Amy Ayala for performing the studies.



### 6 REFERENCES

Un-published material:

OECD Draft Guidelines for the Testing of Ch emicals. Harpacticoid Copepod Development and Reproduction Test with Amphiascu s tenuiremis. Post June 2009.

Published material:

ASTM E 2317-04. (2004). Standard guide for co nducting 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 proce dure 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 mu ltiple comparisons with a control. Biometrics , 20, 482- 491BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 22 Greve, (1996). (Dutch Health Council). Horm oon-verstorende stoffen in Nederland. Gebruik, emissie, milieuconcentraties en fysisch/chemische karakteristieken. Included by reference from “Summary profiles of chemical s with information on use, production, emission, monitoring and legal status”. Annex 14, European Commission.

Piegorsch WW, Bailer AJ (1997) Statistics for environmen tal biology an d toxicology . Chapman and Hall, London, UK

USEPA. 2002. Short-term Methods for Estimatin g the Chronic Toxicity of Effluents and Receiving Water to Marine Or ganisms. EPA-800-R-02-014. 4 th Edition.

USEPA. Good Laboratory Practi ce Standards. 2007a. 40CFR160.

USEPA. Good Laboratory Practi ce Standards. 2007b. 40CFR792.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 23 

### 7 FIGURES

Figure 1: Layout of Microplate Test Chamber

Test organisms are reared from nauplius to sexually mature adu lt 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 204BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 24 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 250 μ L p rimar y stock solution 499.75 mL 500 mL 0.05% 60 μg /L 150 μ L p rimar y stock solution 499.85 mL 500 mL 0.03% 36 μg /L 90 μ L p ri m ar y stock solution 499.91 mL 500 mL 0.018% 21.6 μg /L 54 μ L p rimar y stock solution 499.95 mL 500 mL 0.011% 12.96 μg /L 32.4 μ L p rimar y 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 NM 1 NM 1 NM 1
10/9/2011 99.6 8.1 30.9 10/12/2011 95.1 8.1 30.8 10/152011 NM 1 NM 1 NM 1
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 pa rameter: 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 25.0BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 25 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 wa ter 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 ± 0.8BES Project No. 27569 EPA Contract No. EP-W-06-026Page 26Table 6: Measured Vinclozolin test concentrationsNominalConcentrationDay 0UpDay 0DwnDay 6UpDay 6DwnDay 12UpDay 12DwnDay 18UpDay 18DwnDay 24UpDay 24DwnDay 30UpDay 30DwnDilution WaterControl<3.6 <12 <4.2 <10 <2.5<10 <2.5 <14 <2.6 NM NMNMCarrier Control <2.6 <12 <2.5 <10 <2.5 <10 <2.5 <14 <5.0 NMNMNM12.96 μg/L <2.8 <12 <2.5 <10 <2.5 <10 <2.5 <12 <5.0 NMNMNM21.6μg/L <2.6 <12 <2.5 <10 <2.5 <12 <2.5 <14 <2.5 NMNMNM36μg/L <2.5 <11 <2.5 <10 <2.5 <12 <2.5 <13 <2.5 NMNMNM60μg/L <5 <5 <5.0 <11 <5.0 <11 <2.5 <16 <5.0 NMNMNM100μg/L <10 <11 <10 <11 <10<10 <10 <14 <10 <13 <10 <1225 μg/L (Spike1)101% Recovery74% Recovery25μg/L (Spike2)89.7% Recovery25μg/L (Spike3)92.5% Recovery25μg/L (Spike4)98.3% Recovery25μg/L (Spike5)92.3% Recovery25μg/L (Spike6)74.0% RecoveryMean Spikerecovery*91.3% ± 9.5% 74% ± 0.0%50μg/L (MatrixSpike 1)92.2% Recovery (Solvent Control Up)50μg/L (MatrixSpike 1)92.1% Recovery (12.96μg/L Up)Mean MatrixSpike recovery*92.2%± 0.1%Note: Analysis performed by North Coast Laboratories, LTD (Arcata, California, USA)using GC-MS. Table compiled from NCL finalreport numbers 1110574 and 1111154.NM = Not Measured* Spike recoveries were not used to correct sample results.BES Project No. 27569 EPA Contract No. EP-W-06-026

Page 27 Table 7: Survival on day 16 Percent survival at day 16

Treatment group Replicate Percent survival Mean Standard deviation plate 1 77 plate 2 83 Control plate 3 80 80 3

plate 1 90 plate 2 93 Carrier Control 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 4BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 28 Table 8: Survival on day 36 Percent survival at day 36

Treatment group Replicate Percent survival Mean Standard deviation plate 1 77 plate 2 70 Control plate 3 77 75 4

plate 1 83 plate 2 87 Carrier Control 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 5BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 29 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 plate 2 11.2 Control plate 3 10.7 11.2 0.5

plate 1 11.5 plate 2 13.0 Carrier Control 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 2.1BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 30 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 plate 2 8.0 Control plate 3 7.2 7.5 0.4

plate 1 7.9 plate 2 7.9 Carrier Control 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 0.2BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 31 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 plate 2 18.9 Control plate 3 18.0 18.4 0.5

plate 1 18.6 plate 2 19.6 Carrier Control 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 2.1BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 32 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 plate 3 55% 45% 0.83 Carrier Control Plate 1, 2, and 3 33 34 1.1 0.3

plate 1 42% 58% 1.40 plate 2 45% 55% 1.22 plate 3 37% 63% 1.71 12.96 μg/L Vinclozolin Plate 1, 2, and 3 26 37 1.4 0.2

plate 1 43% 57% 1.30 plate 2 44% 56% 1.25 plate 3 57% 43% 0.77 21.6 μg/L 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 0.1BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 33 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 plate 2 3.0 Carrier Control 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 0.5BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 34 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 plate 2 7.7 Carrier Control 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 0.9BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 35 Table 15: Time to second egg extrusion fo llowing hatching of first brood offspring Time to second brood (days) since ha tching 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 plate 2 1.8 Carrier Control 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 0.5BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 36 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 plate 2 4.0 Carrier Control 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 2.5BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 37 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 plate 2 11.7 Control plate 3 15.8 14.2 2.2

plate 1 13.3 plate 2 11.7 Carrier Control 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 1.9BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 38 Table 18: Fertilization success

Fertilization success: mating pairs able to pr oduce 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 mati ng 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

Page 39 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 concentra tion 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 ASSUR ANCE PROJ ECT PLAN (QAPP)BES Project No. 27568 EPA Contract No. EP-W-06-026

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Page 75 

### 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 a nd 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) <0.005BES Project No. 27568 EPA Contract No. EP-W-06-026

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### 11 APPENDIX III: VINCLOZOLIN CHEMISTRY DATA REPORTBES Project No. 27569 EPA Contract No. EP-W-06-026

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Page 116 

### 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 Di st. 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 10129.070BES Project No. 27569 EPA Contract No. EP-W-06-026

Page 117 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 Di st. K-S Prob. Sum Sum of Squares Temperature 0.446 1.641 0.192 <0.001 1799.700 44400.750BES Project No. 27569 EPA Contract No. EP-W-06-026

Page 118 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 ne gative findings cautiously.BES Project No. 27569 EPA Contract No. EP-W-06-026

Page 119 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne the contingency table are not significantly related. (P = 0.196)BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 122 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne the contingency table are not significantly related. (P = 0.372)BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 123 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne the contingency table are not significantly related. (P = 0.372)BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 124 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne the contingency table are not significantly related. (P = 1.000)BES Project No. 27569 EPA Contract No. EP-W-06-026

Page 125 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 ne gative findings cautiously.BES Project No. 27569 EPA Contract No. EP-W-06-026

Page 126 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne the contingency table are not significantly related. (P = 0.140)BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 127 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 ne gative 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 ne gative 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 ne gative 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 ne gative 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 ne gative 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 colu mns of the contingency table do not vary from row to row. The two characteristics that defi ne 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 ne gative 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 Transf ormedin 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 statisti cally 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 comp arison 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 di fference between the means, even though one may appear to exist.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 153 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 statisti cally 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 comp arison 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 di fference between the means, even though one may appear to exist.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 154 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 ne gative findings cautiously.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 155 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 ne gative findings cautiously.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 156 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 ne gative findings cautiously.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 157 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 ne gative findings cautiously.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 158 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 ne gative findings cautiously.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 159 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 ne gative findings cautiously.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 160 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 ne gative findings cautiously.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 161 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 ne gative findings cautiously.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 162 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 Di st. 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 9365.973BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 163 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 ne gative findings cautiously.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 164 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 ne gative findings cautiously.BES Project No. 27569 EPA Contract No. EP-W-06-026

Page 165 

### 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 WRIT TEN BY: NANETTE BRADBURY, B.SC.

MANAGEMENT APPROVAL: DAVID S. BLOCK, PH.D.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 166 

### 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 InternationalBES Project No. 27568 EPA Contract No. EP-W-06-026

Page 167 

### 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 ‘Har pacticoid 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 #4BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 168 

### 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 th e effect of Vinclozolin on the survival rate, development, mating success, and re productive output of the copepod Amphiascus tenuiremis

. This method is based on a draft OECD Test Gu ideline ‘Harpacticoid copepod development and reproduction test with Amphiascus tenuiremis’ (OECD, 2009) and the published ASTM Guideline entitled “Standard guide for conduc ting 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 indi vidual 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 dimorphi c 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 offs pring production. The test will be terminated when all mating pairs have successfully produ ced two viable broods or on Day 36 of theexposure period, whichever comes first. All phases of the test will be performed under semistatic test conditions with water renewal occurr ing every three days. Th e 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 comp ared between Vinclozolin exposed copepods andBES Project No. 27568 EPA Contract No. EP-W-06-026

Page 169 laboratory controls in order to calculate no observed effect (NOE C) and lowest observed effect (LOEC) concentrations.

MATERIALS

Test Chemical

A Vinclozolin certificate of anal ysis 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 resubm itted 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 culture s 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 st ock (i.e., showing no signs of stress such as high mortality, poor fecundity etc). The stock anim als must be maintained in culture conditions (light, temperature, seawater medium and feeding regi men) similar to those to be used in the test.

Justification for the sel ection of the test species

Amphiascus tenuiremis’c .f. Mielke is an easily- cultured, diosaccid harpac ticoid copepod that is amphi-Atlantic in distribution ranging from the Nort h 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 swolle n 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 matu re after the fifth copepodite stage (that is, in the twelfth life stage). They then mate and produ ce their first clutch in tw o to three days. Sperm transfer is via a membranous spermatophore sac a ttached 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 clut ches 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.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 170 CULTURE MEDIUM

Natural or synthetic seawater may be used as th e test medium. If natu ral 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 seaw ater 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 cope pods show suitable l ong-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 μ m 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, SO 4 ), pesticides (e.g., total organophosphorus and total organochlorine pestic ides), total organic carbon and suspended solids should be made.

TEST APPARATUS

Disposable polystyrene microwell pl ates will be used as test vesse ls. Polystyrene plates are not ideal for testing organic compounds because of non-specific binding which may reduce the chemical availability of the test compound. Th e polystyrene microplates will therefore be precoated with glass or polyacrylam ide gel unless it can be demonstr ated that there is no significant loss of the test compound between water renewa ls using ‘uncoated’ polystyrene microwell plates. The microplates will be 96 well plates each well having a 300 μ L total volume and a 250

μ L working volume.

EXPERIMENTAL DESIGN

The test solutions will be main tained at 25 ± 2°C by housing the test vessels in a temperaturecontrolled room or in climate c ontrolled chambers. Glass or plas tic ‘humidity chambers’ will be used to reduce evaporative losses. Potential for evaporative loss will be measured before beginning any tests by placing seawater filled micr oplates in the test ro om or chamber for 72 hours and measuring salinity change over this pe riod. 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 ot her 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 c ontrol of the lighting regime;BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 171 • stereo compound microscope, preferably i nverted 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 m 3 glass or plastic rectangular containers with sealable lids to create humidity chambers for the microplates (to minimize evaporative loss).

• various plastic or stainles s-steel mesh sieves (0.05 mm to 0.25 mm) to culture, capture and sort copepods;

• Pasteur pipettes, preferably silanized or equivalent tr eatment to prevent copepods from adhering to pipette walls;

• adjustable single and multi-channe l volumetric pipettors (1-10 μ L, 100-500 μ L) for food additions;

• 250 μ L Hamilton glass volumetric syringes wi th 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 isol ating ovigerous females from a mass culture at least 3 weeks old. Nauplii will be exposed in individual microwe lls 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 th e 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 feedi ng will be performed in an irregular plate order. If experimental units are handled in tr eatment or concentration order, then some timerelated effect, such as operator fa tigue or other error, could lead to greater or lesser effects at the higher or lower concentrations.

The number of days it takes for de velopment of each individually isolated copepod, from a first stage nauplius to a juvenile copepodite to a repr oductively mature adult, will be checked daily. Survival of larvae (nauplii), juve niles (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 reach ing 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 microplat es and paired impartially with an opposite sex individual (i.e., from the same microplate with in the same treatment) in a new or unused microwell filled with test medium for each treatmen t or control replicate. If necessary the males and females may be transferred to a separate vess el containing the approp riate test solution (orBES Project No. 27568 EPA Contract No. EP-W-06-026

Page 172 control) media to aid identificati on 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 produc ed insufficient numbers of males and/or females to produce multiple mating pairs within a given micr oplate within a given treatment, then virgin adults may be mated across microplates within trea tments. However, this must be noted in any report of results, and an a priori assumption of no significant micr oplate to microplate variability within the given treatment(s) mu st be made by the investigator.

Once individual mating pairs are established for each treatment and control adults, 90-95% of transfer seawater will be aspira ted and replaced with the appropr iate control or test substance concentration. This replacement of media after ma tings 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 e nd of the test exposure period.

Offspring production

After pairing of male: female mating pairs, obser vations 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 simu ltaneously 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 succe ssfully hatching nauplii (i.e., to tal viable clutch, or fecundity) will be recorded for the first and second successi ve clutches of eggs. Hatching number can be obtained by subtracting any non-hatche d or necrotic eggs from the to tal clutch size at the clutchone and clutch-two time points. Alternativel y, numbers of hatched na uplii crawling on the bottom of the microwell may be counte d by direct microsc opic 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 mi croplate 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 aver age 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 c opepods (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 ad ult 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 tw o clutches averaged over theBES Project No. 27568 EPA Contract No. EP-W-06-026

Page 173 number of mating pairs created a nd tested), time to production of first clutch, time interval between successive clutches, and st age-specific mortalities. Obse rvations may also be made for any abnormal behaviour (relative to the contro ls), including uncoordi nated 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 5t h copepodite stage at the earliest. Numbers of aborted egg sacs and necrotic/infe rtile eggs may also be counted.

Feeding

The test animals will be fed on Day 0 and at every other microwell renewal day (every 6 th day of the test) with cultured algae. Each microw ell will receive the equivalent of 2 μ l of a 1:1:1 volume mixed-algal cell suspension (centrifuged, pelletized and re-constituted in fresh seawater to 10 7 cells/mL) of a chlorophyte (e.g., Dunaliella tertiolecta ), a chrysophyte (e.g., Isochrysis galbana ) and the diatom Phaeodactylum tricornutum . Therefore the cell de nsity added to each test microwell at each feeding should target ~ 2 x 10 4 cells/microwell. This density will support copepod maturation and reproduction without fouling the microwe ll 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 platfo rm over a water bath inside a humidity chamber in the temperature regulated incubator. If n ecessary excess evaporative losses can be corrected by more frequent medium renewal.

When the medium is renewed, a fixed volume (p referably 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 Ha milton gas syringe of suitable needle diameter (e.g., 23 gauge be velled). Care should be taken that copepods are not aspirated into the syringe. If copepods ar e 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 a nd 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 deta ils of the test soluti on preparation will be documented in the study book.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 174 PHYSICAL AND CHEMICAL ANALYSIS

During the test, dissolved oxygen, pH and temperatur e 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 concentra tion. 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 pl aced inside the microplate humidity chamber.

The analytical chemistry method used will be pr ovided by USEPA. Chemical analysis will be conducted on all newly prepared primary stock solu tions, solutions of cont rols, all renewal test concentrations (Ups), and on the solutions remo ved 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 te st renewals varies then a mixture of different length renewals will be included in the anal ysis (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 goodn ess of fit tests (Ref) can be used to identify significant differences in survival between the co ntrol(s) and each treatment after selected time periods (e.g. 10 and 21 days).

Larval development rates, stage specific morta lity, percent mating success and sex ratios may all be analyzed for significant diffe rences relative to the controls. Reproduction data, expressed as the mean number of F 1 offspring produced from fertilized females (F 0 ) in each replicate will be calculated. Generally, the reproduct ion data are not adjusted for mortality but are presented on a per female or per mating pair basis. If an animal dies afte r 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 an imals that died during the exposure will not be included in the statistical analysis. One-way ne sted analysis of variance (ANOVA) will be used to evaluate effects of the test substance on th e reproduction (and other ch ronic 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 reproduc tion from the control(s) (p = 0.05). For ANOVA, individual microwell mating pair s will be nested within their particular microplate, which will be replicat ed within a given treatment. A nested model will be ideal if data are balanced across microplates and treatmen ts because microwells within a microplate are not strictly independent of one another.

Analysis of variance is a parametric proce dure and is based on the assumptions that the observations are independent and normally distribu ted, and that the variance of the observations is homogeneous across all test subs tance 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-pa rametric (distribution-free) procedures.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 175 The biological data may also be analyzed usi ng a regression model in order to estimate the concentration that would cause an x% reducti on in reproductive outpu t, i.e., ECx (e.g., EC50, EC20 or EC10). Depressed intrinsic or instan taneous rates of population increase (rm or λ ), may also be calculated. Details of these ca lculations will be r ecorded in the study book. If a statistically significant difference in surviv al, development rate, or reproduction is detected between the control and solvent cont rol, only the solvent control will be used as the basis for the calculation of results. If no signifi cant differences exist between c ontrol 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 th e 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 char ge. Unless the Sponsor requests any amendments within three months of r eceipt 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

Page 176 COMPLIANCE

GLP compliance

The study will be conducted in accordance with USEPA Good Laboratory Pr actice Regulations.

Quality assurance evaluation

The study described in this protocol will be subject to Quality Assurance Aud it. 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 co nducting 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 Ch emicals. Harpacticoid Copepod Development and Reproduction Test with Amphiascus tenuiremis . Post June 2009.

Lang, K (1948) Monographie der Hapactic iden. Hanken Ohlsson, Lund, Sweden

Dunnett, C.W. (1955). A multiple comparison proce dure 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 mu ltiple comparisons with a control. Biometrics , 20, 482- 491

Piegorsch WW, Bailer AJ (1997) Statistics for environmen tal biology an d toxicology . Chapman and Hall, London, UK

USEPA. 2002. Short-term Methods for Estimatin g the Chronic Toxicity of Effluents and Receiving Water to Marine Or ganisms. EPA-800-R-02-014. 4 th Edition.

USEPA. Good Laboratory Practi ce Standards. 2007a. 40CFR160.

USEPA. Good Laboratory Pract ice Standards. 2007b. 40CFR792.BES Project No. 27568 EPA Contract No. EP-W-06-026

Page 177 

### 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 nu mber 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 in dicate 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. BE S 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

Page 178 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.