BES Project No. 27569 EPA Contract No. EP-W-06-026

Study Title

Effect of Lindane on the Survi val, Development, and Reproduc tion of the Marine Copepod Amphiascus tenuiremis

Block Environmental Services Authors

David S. Block, Ph.D. Nanette Bradbury

Study Completion Date

March 12, 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. 27569 RTI Study No. 0210114.030 EPA Contract No. EP-W-06-026BES Project No. 27569 EPA Contract No. EP-W-06-026

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### QUALITY ASSURANCE STATEMENT

The study conduct, raw data and report for “ Effect of Lindane 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

27569 ( Lindane ) 8/16/2011 in-test

, setu p 8/16/2011 9/9/2011 in-test

, matin g 9/9/2011 9/26/2011 data

, anal y tical methods verification 9/26/2011 10/5/2011 data

, chemistr y 10/10/2011 12/21/2011 data

, biolo gy s p readsheets 12/23/2011

2/24/2012 draft re p ort 2/27/2012 3/5/2012 revised draft re p ort 3/6/2012

BLOCK ENVIRONMENTAL SERVICES, INC.

________________________________________________ ____March, 9, 2012____ Ronald M. Block, Ph.D. Date Quality Systems DirectorBES Project No. 27569 EPA Contract No. EP-W-06-026



### 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. 3/12/2012 Program Manger/Technical Report Writer Date

Ronald M. Block, Ph.D Quality Systems Director

Nanette Bradbury Senior Biologist/Technical Report Writer

Terence Clohesy Biologist

Sebastian Castro Biologist

Amy Ayala Biologist

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### 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.

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### TABLE OF CONTENTS

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

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

DATA RETENTION

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............................................................................................................ ABSTRACT

............................................................................................................................... ... 1 INTRODUCTION

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

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

2.2 Culturing test organisms

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

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

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

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

..................................................................................................... 2.2.5 Photoperiod

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

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

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

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

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

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

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

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

......................................................................................................... 2.3.8 Lindane concentrations in test solutions..................................................................

2.3.9 Statistical analyses

................................................................................................... 3 RESULTS

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

.............................................................. 3.2 Measured lindane concentr ations in test solutions

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

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

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

................................................................................................... 3.3.3 Day 16 Versus Day 36 Survival

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3.3.4 Development – Maturation time between naupliar and copepodite stages

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. 34 3.3.5 Development – Maturation time betw een copepodite and adult stages

................... 3.3.6 Development – Maturation time be tween naupliar and adult stages

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

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

.............................................................. 3.3.9 Reproduction – number of hatc hed nauplii from first brood

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

...................................................... 3.3.11 Reproduction – number of hatc hed nauplii from second brood

.......................... 3.3.12 Reproduction – Total number of nauplii per two brood females

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

..................................................................... 3.4 Summary of NOEC and LO EC for all endpoints

........................................................ 4 DISCUSSION

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

.................................................................................... 4.2 discussion of results

..................................................................................................... 5 ACKNOWLEDGEMENTS

................................................................................................ 6 REFERENCES

..................................................................................................................... 7 FIGURES

.............................................................................................................................. Figure 1: Layout of Mi croplate Test Chamber

................................................................ 8 TABLES

............................................................................................................................... .. Table 1: Method for preparation of lindane primary stock solution

................................ Table 2: Method for preparati on of lindane test solutions

............................................... Table 3: Water quality paramete rs: pH, salinity, dissolved oxygen

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

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

............................................................... Table 6: Measured lindane test concentrations

................................................................ Table 7: Summary of measured lindane test concentration

............................................. Table 8: Survival on day 16............................................................................................. Table 9: Survival on day 36............................................................................................. Table 10: Time to transformation to copepodite stage

.................................................... Table 11: Time in copepodite stage

................................................................................. Table 12: Time to transformation to adult stage

.............................................................. Table 13: Sex ratio

........................................................................................................... Table 14: Time to first egg extrusion since date of pairing

............................................. Table 15: Number of nauplii hatched from first brood

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Table 16: Time to second brood since date of pairing

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............................................... Table 18: Total number of nauplii that hatched from two successive broods

................. Table 19: Fertilization success

......................................................................................... Table 20: Summary table of NO EC and LOEC for all endpoints

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

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

.............................. 11 APPENDIX III: LINDANE CH EMISTRY DATA REPORT

......................................... 12 APPENDIX IV: STUDY PROTOCOL (P27569)

............................................................ 13 APPENDIX V: DEVIATIONS FROM STUDY PROTOCOL

...................................... 13.1 List of deviations

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Page 8 ABSTRACT

The current study assessed the chronic sensitivity of the meiobenthic copepod Amphiascus tenuiremis to lindane concentrations of 2.5, 5, 10, 15, and 20- μ g/L using a micropl ate-based lifecycle screening assay. Copepod surv ival in the laboratory control and carrier control treatments on Day 16 was 82% and 79%, respectively with survival in each control treatment decreasing to 77% by Day 36. The 15 and 20 μg/L lindane con centrations significantly affected copepod survival on both Day 16 and 36 time points relative to the control response . None of the lower lindane concentrations signifi cantly affected copepod survival . Lindane had a significant inhibitory effect on A. tenuiremis development into both cope podite and adult life-stages. Specifically, lindane significantly increased th e time to develop from the naupliar to the copepodite life-stages and also significantly in creased the time necessary for exposed copepods to mature into sexually mature adults. Based on the development data obtained during the study, NOEC and LOEC values were calculated to be 2.5μg/L and 5μg/L respectively. None of the lindane concentrations significan tly affected the ratio of male:female copepod development. Lindane was also observed to ha ve a significant effect on cope pod reproduction, with a reduction in the percentage of successful mating pairs and a reduction in the total nu mber of viable eggs and hatched nauplii. Based on the mating success da ta, NOEC and LOEC values were calculated to be 5μg/L and 10μg/L, respectively. Based on the total offspring production data, the NOEC and LOEC were calculated to be 10 μg/L and 15 μg/L, respectively. Time-weighted measured lindane concentrations were with in 20% of the nominal values. All statistical analyses were performed using measured values.BES Project No. 27569 EPA Contract No. EP-W-06-026

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, ascert ain 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 Prevention, Pesticides and Toxic Substa nces 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” and the June 2009 proposed OECD Test Guidelines for the Harpacticoid Copepod Development and Reproduction Test with Amphiascus tenuiremis ( OECD 2009 ). As indicated above, the test is intended to serve as a high er tier test with an aquatic arthropod for collecting definitive concentration-re sponse information on adverse effects suitable for use in ecological risk assessment. Specificall y, the design enables the collection of biological data from this copepod species in order to facilitate the measurement of development, reproductive and life-table parameters, which pr ovide sensitive and eco logically relevant endpoints that can be used to determine the po tential chronic toxicity of many water-borne contaminants.

Block Environmental Services (BES) conducted one copepod development and reproduction test with lindane using the proposed OECD ( OECD, 2009 ) and published ASTM test guidelines ( ASTM, 2004 ) Endpoints ranged from survival, devel opment, and specific reproductive output measures, including time to first and second broo d, number of offspring, reproductive success.

The experimental phase of the study was c onducted from August 16, 2011 to September 21, 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.

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2 MATERIALS AND METHODS

2.1 QAPP/PROTOCOL

This study was conducted according 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 lindane on the survival rate, development, mating succe ss 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

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2.2 CULTURING TEST ORGANISMS

2.2.1 Source of copepods

A gift of A. tenuiremis was received from Professor G.T. Chandler (University of South Carolina, School of Public Health, 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 lit ers 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 th e instructions presented by the Chandler Laboratory (Columbia, South Caro lina). Synthetic Instant Ocean ® seawater was used to prepare the copepod culture medium. The synthetic seaw ater was prepared by dissolving Instant Ocean ® sea salt into laboratory grade deionized water in accordance with the manuf acturer’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 proces sing carbon-scrubbed municipal 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

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2.2.4 Culture Sediment

Concentrated sediment previously filtered through a 63μm wire mesh sieve was received as a gift from Professor G.T. Chandler and stored in the dark at 4 ° C prior to use. According to the Chandler Laboratory, the 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 (min-max: 4,090-4,520 Lux) and 12-h dark photoperiod was maintained throughout the culturing of A. tenuiremis

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2.3 TOXICITY TEST PROCEDURE

2.3.1 Test chemical

Three samples of the test chemical were received from the EDSP Chemical Repository, housed at Battelle's Pacific Northwest Division, Sequim, Washington. Upon receipt at BES, the test substances were stored at room te mperature in the original container in a dark, ventilated cabinet. Concentrations were adjusted for the purity of the test substance and are presented as active ingredient (a.i.). The follow ing information was provided:

Name: Lindane CAS registry number: 58-89-9

Source: Battelle Marine Scie nce Laboratory, Sequim WA 98382, U.S.A. Lot / Batch number: Lot: 11419EB Purity: >99.6% Form White powder Date of receipt at BES March 11, 2011 Location of storage Fume hood in chemical storage room

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2.3.2 Preparation of test solutions

The primary stock solution of lindane was prep ared only once on 7/16/2011 and stored in an amber glass bottle at 4°C in Sample Locker 1. This primary stock solution was used throughout the remainder of the 36-day test. Lindane was we ighed using an analytical balance (Sartorius Series A 200S) and dissolved into pesticide gr ade acetone to achieve a stock concentration of 80μg/ml as detailed in Table 1 . The primary stock concentration was selected so that the total acetone carrier volume would not exceed 0.05% in any test treatment. Acetone was stored at room temperature in a self-closing organic solven t safety cabinet. Acetone was measured using a glass graduated cylinder.

Nominal lindane test concentr ations of 2.5, 5, 10, 15, and 20 μg/L were prepared from the lindane primary stock as detailed in Table 2 . Test solutions were prep ared every 3 days, on the day of water change. Calculated amounts of th e 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 st ainless-steel needles. Test solutions were not prepared by serial dilution, but instead prepared independen tly by pipetting the calculated amount of primary stock solution directly into the designated volumetric flask. To prevent bias, the acetone solvent carrier was a dded to an aliquot of the contro l solution in a volume equivalent to the highest amount added into the lindane treatments. Once prepared, test solutions were sealed, mixed for 1-hr using Teflon ® coated stir-bars, and stored in the chemical storage room until used.

2.3.3 Test equipment

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

 96-well polystyrene microplates (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);

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 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

Approximately 300 gravid female copepods were collected from laboratory stock mud cultures by sieving across 106 μ m steel mesh. Gravid females 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 pipettes 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 lindane test concentrat ion 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 (colum n 1 and 12 were purposely left empty). See Figure 1

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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 chemical 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 monitored daily via inverted stereomicroscope for development into the copepodite juvenile stage. Once becoming copepodites, exposed A. tenuiremis were then monitored for the development of secondary sexual characteristics and time, in 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 summarized 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 handheld meters, respectively. Test vessels were stored in a temperature and light controlled environmental chamber set at 25°C with a light intensity ranging from 4,090 to 4,520 Lux. Due to small volumes within the microwells, direct temperature was not measured in the test vessel solutions. Temperature was recorded twice daily from a calibrated alcohol thermometer immersed in a bottle containing di lution water placed near test vessels. The thermometer is calibrated quarte rly against an NIST traceable thermometer. Physico-chemical parameters were not measured in freshly made test solutions (referred to here as ‘Ups’) and test solutions draw n out of test vessels (i.e. ‘Dow ns’) in order to prevent lindane cross contamination to other ongoing toxicity studies.

2.3.8 Lindane concentrations in test solutions

Samples for determining lindane concentration 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 in

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Sample Locker 1 at 4°C. Samples were sent throughout the study to North Coast Laboratories, LTD. (Arcata, California, USA; CA-ELAP #1247) in an ice chest cooled with packs of Blue Ice ® (Rubbermaid). Chemical analysis of the test solutions were performed by gas chromatography – electron capture detection (GC-ECD) using EPA method 505. Quantities were determined by correlating signal responses against reference calibra tion standards of known lindane concentrations prepared in hexane. Results were subm itted in audited reports 1109247 and 1109490 ( Appendix III ).

2.3.9 Statistical analyses

Mean and standard deviations (SD) were calculated using Microsoft TM Excel 2000. Survival, 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 distribution with a nor mal distribution. The assumption that observations are normally distributed must be validated before subsequent analyses following parametric procedures, can be perf ormed. If the data were not normally distributed, then a non-parametric proce dure was used for subsequent analyses.

 Prior to analysis for statistically significant effects, an analysis of variance (ANOVA) was conducted on each data set (except survival and sex ratio data) to determine if a treatment group in the data set was different from the other groups in that data set.

 Post-hoc pair wise comparisons between the solvent control and each treatment level, including the dilution wate r control, were performe d using Dunnett's Test ( Dunnett, 1955, 1964 ), which is a parametric procedure. If necessary, mean values were transformed by using square root, arcsine squa re root, or log conversion procedures. The multiple comparison adjustment was utilized to determine the NOEC and LOEC for each test endpoint. The Bonferroni pairwise mutlitp le comparison procedure was utilized to obtain P values between the carrier control and treatemen t groups since the SigmaStat program does not calculate individual pair wise P values in the Dunnett’s test.

 All proportional data points such as survival rates and sex ratios were analyzed using Pearson’s chi- square go odness-of-fit test (R X C contingency tables) ( Piegorsch and Bailer,1997 ).

For clarity, the definition for NOE C and LOEC are described below:

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 No-Observed-Effect Concentra tion (NOEC) is the concentration of lindane tested which has no effect on the organism in the test, accord ing to the statistical test used for analysis.

 Lowest-Observed-Effect Concentration (LOEC) is the lowest tested concentration of lindane which has an effect on the organism in the test that is different from the control (or solvent control), according to the statistical test used for analysis.

3 RESULTS

3.1 PHYSICO-CHEMICAL PROPERTIES OF TEST SOLUTIONS

The baseline physico-chemical measurements for pH, salinity and dissolved oxygen are presented in Table 3 . Daily temperature measurements from the environmental chamber dilution water vessel are presented in Table 4

. Table 5 provides an overall summary of these measurements and shows that temperature wa s 25.1 ± 0.6°C, pH was within 8.2 ± 0.1, salinity was within 29.7 ± 0.6‰, and dissolved oxygen was 97.4 ± 5.3 of air saturated value. In terms of physico-chemical properties, recommendati ons listed in the QAPP for this study ( Appendix I Table 3 ) were met.

3.2 MEASURED LINDANE CONCENTRAT IONS IN TEST SOLUTIONS

Test concentrations of lindane were repor ted by North Coast Laboratories LTD, Arcata, California, USA. A copy of the audited re ports numbered 1109247 and 1109490 is presented in Appendix III. Concentration data, including Quality Assurance/Quality Control spikes, are reported in Table 6 . Time weighted values of the measur ed lindane concentr ations and percent of nominal calculations are summarized in Table 7 . Time weighted values were generated by calculating the arithmetic mean between all Up and Down samples throughout the 36-day test period for each treatment level. These tables indi cate concentrations of the test substance ranged from 75 to 87 percent of the nominal concentrat ion, which is within the criteria specified in Appendix I . In this report, all results are expr essed in term of nominal values.

3.3 BIOLOGICAL DATA

A summary 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 8 . 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 82% a nd 79%, respectively. A statistical difference between the control means was not evident (P=0 .706). Survival in the test tr eatments ranged from 63 to 79%. Copepod survival between the ca rrier control and 20 ug/L lindane treatment was statistically

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reduced (P=0.033). No significant effects relative to th e carrier control group were observed in the 2.5 (P=0.855), 5 (P=0.175), 10 (P=0.597), and 15μg/L (P=0.067) test treatments.

3.3.2 Survival on day 36

Recording survival observations of individual copepods was concluded upon test termination on day 36. Treatment means, including the dilution water control, were statistically compared to the carrier control using Pearson’s chi- square goodness-of-fit test . On Day 36, the control and carrier control group each had a me an survival rate of 77%(P=0.860). Table 9 lists the percentage survival for all groups. There was a statistically significant difference for survival (p<0.05) for the 15 (P=0.017) and 20μg/L (P=0.025) c oncentrations relative to the carrier control group. No significant effects relative to the carrier control group were observed in the 2.5 (P=0.862), 5 (P=1.0), and 10μg/L(P=0.497) test treatments. The surviv al result in the 5μg/L test concentration reflects data from only two repli cates; replicate 1 was accidentally dropped by an operating technician on day 25.

3.3.3 Day 16 Versus Day 36 Survival

Day 16 and Day 36 mean survival rates were st atistically compared within treatment using Pearson’s chi- square go odness-of-fit test. No statistically significant differences (p>0.05) in survival was observed within control (P= 0.461), carrier control (P=0.858), 2.5 (P=0.597), 5 (P=0.405), 10 (P=0.738), 15 (P=0.442), and 20 μg /L (P=0.759) lindane treatment groups between the test midpoint and test termination.

3.3.4 Development – Maturation time between naupliar and copepodite stages

Copepod development from the nauplius to copepodite stages is presented in 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 conjunction with Dunnett’s post-hoc multiple comparison procedure. In summary, mean deve lopment time in the control and the carrier control group was 9.5 and 9.3 days, respectively. A statistical differen ce between the control means was not evident (P=0.593). There was a statistically significant (p<0.05) increase in the number of days for nauplii to maturate into the copepodite stage for lindane concentrations of 5 (P=0.008), 10 (P=0.001), 15 (P=0.001), and 20 μg/L (P =0.001). No significant effect relative to the carrier control group was observed in the 2.5 μg/L (P=1.0) test treatment. Based on these statistical results, NOEC and LOEC values were determined to be 2.5 and 5μg/L, respectively.

3.3.5 Development – Maturation time between copepodite and adult stages

Copepod development from the copepodite stage into virgin adults is presented in Table 11

. Treatment means, including the dilu tion water control, were statistically 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 8.1 and 8.2 days, resp ectively. A statistical difference between the

Page 17BES Project No. 27569 EPA Contract No. EP-W-06-026

control means was not evident (P=0 .725).). No statistica lly significant effects (P=0.1) relative to the carrier control group were observed in the 2.5, 5, 10, 15and 20μg/L test treatments. Based on these statistical observations, NOEC and LOEC valu es were determined to be 20 and >20μg/L, respectively.

3.3.6 Development – Maturation time between naupliar and adult stages

Copepod development from the nauplius to adult stages is presented in Table 12 . Treatment means, including the dilution water control, were statistically compared to the carrier control using a One Way ANOVA test in conjunction with Dunnett’s post-hoc multiple comparison procedure. In summary, mean development time in the control and the carrier control group was 17.4 and 17.5 days, respectively. A statistical difference between the control means was not evident (P=0.856). There was a statistically significant (p<0.05) increase in the number of days from nauplius to virgin adult for lindane concentrations of 5 (P=0.032), 10 (P=0.001), 15 (P=0.001), and 20 μg/L (P=0.001). No significant eff ect relative to the car rier control group was observed in the 2.5μg/L (P=1.0) te st treatment. Based on thes e statistical results, NOEC and LOEC values were determined to be 2.5 and 5μg/L, respectively.

3.3.7 Sex ratio

The proportion of females to males is presented in Table 13 . 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 1.4 and 1.2, respectively. There was not a statistically significant difference (p>0.05) between the control means (P=0.763). No significant effect re lative to the carrier control group was observed in the 2.5 (P=0.337), 5 (P=0.567), 10 (P=0.900), 15 (P=0.909) and 20μg/L (P=0.209) test treatments. The sex ratio s observed in the contro l and carrier control treatments were within the Quality Criteria specified in Appendix I, Table 3 , although the standard deviations exceeded ± 0.2 (± 0.4 and ± 0.5, respectively).

3.3.8 Reproduction – time to first egg extrusion

The average time to first egg extrusion in fe males following the mating of virgin male and female copepods is presented in Table 14 . 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 2.4 and 2.3 days, respectively). There was not a statistically significant differen ce (p>0.05) between the control means (P=0.656). No statistica lly significant differences in th e time to egg extrusion were observed among the test treatments when compar ed to the carrier control group (P=0.396). It should be noted that no mated females became gr avid in replicate 2 of both the 15 and 20μg/L test treatments. Organisms were observed for a minimum of six days and a maximum of twelve days following mating. The time to first clutch observed in the contro l and carrier control treatments were within the Quality Criteria specified in Appendix I, Table 3

.

Page 18BES Project No. 27569 EPA Contract No. EP-W-06-026

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 Table 15 . Treatment means, including the dilution water control, were statistically compared to the carrier control using a One Way ANOVA test in conjunction with Dunnett’s pos t-hoc multiple comparison procedure. The average number of first brood nauplii in the co ntrol and carrier control groups was 5.9 and 5.8 nauplii per female, respectively). There was not a statistically signifi cant difference (p>0.05) between the control means (P=0.970). There was a statistically significant (P=0.031) decrease in first brood offspring in the 20μg/L (P=0.022) test treatment when compared to the carrier control. No statistically signi ficant effects rela tive to the carrier control group were observed in the 2.5 (P=1.0), 5 (P=1.0), 10 (P=1.0), and 15μg/ L (P=0.459) lindane concentrations. However, a large standard deviation in the 15μg/L treatm ent may have precluded a calculated statistical difference since the offspring produced were 39% of the offspring measured in the carrier control
group.

3.3.10 Reproduction – time to second egg extrusion

The average time to second egg extrusion in female copepods is presented in Table 16

. Treatment means, including the dilu tion water control, were statistically compared to the carrier control using a One Way ANOVA test. The average time to second egg extrusion in the control and carrier control groups was 2.2 and 2.3 days, respectively. There was not a statistically significant difference (p>0.05) between the control means (P=0.709). No statistically significant differences in the time to egg extrusion were observed among the test treatments when compared to the carrier control group (P=0.517). The 20μg/ L test treatment data were excluded from statistical analysis since mate d females did not produce a second brood in any of the treatment replicates.

3.3.11 Reproduction – number of hatched nauplii from second brood

The average number of hatched nauplii from th e second egg extrusion following the mating of male and female copepods is presented in Table 17 . Treatment means, including the dilution water control, were statistically compared to th e carrier control using a One Way ANOVA test in conjunction with Dunnett’s post-hoc multiple co mparison procedure. The average number of second brood nauplii in the control and carrier control groups was 6.0 and 6.7 nauplii per female, respectively. There was not a st atistically significant differen ce (p>0.05) betwee n the control means (P=0.420). There was a statistically significant (p<0.05) decrease in second brood offspring in the 15 (P=0.002) and 20μg/L (P=0.002) test treatments when compared to the carrier control. No statistically signi ficant effects rela tive to the carrier control group were observed in the 2.5 (P=1.0), 5 (P=1.0), and 10μg/L (P=1.0) lindane concentrations

3.3.12 Reproduction – Total number of nauplii per two brood females

Page 19BES Project No. 27569 EPA Contract No. EP-W-06-026

The average number of nauplii per female from mating pairs with two broods is presented in Table 18 . Treatment means, including the dilution water control, were statistically compared to the carrier control us ing a One Way ANOVA test in conj unction with Dunnett’s post-hoc multiple comparison procedure. The total number of offspring/female in the control and carrier control groups was 11.7 and 12.5, respectively. There was not a statistically significant difference (p>0.05) between the control means (P=0.625). There was a statistically significant (p<0.05) decrease in nauplii per female in the 15 (P=0.033) and 20μg/L (P=0.007) test treatment when compared to the carrier control. No statis tically significant effects relative to the carrier control group were observed in the 2.5 (P =0.468), 5 (P=1.0), and 10μg/L (P=1.0), lindane concentrations. The total numbe r of viable offspring produced through two clutches in the control and carrier control tr eatments were within the Quality Criteria specified in Appendix I, Table 3 . Based on these statistical results, NOEC and LOEC values were determined to be 10 and 15μg/L, respectively.

3.3.13 Reproduction - fertilization success

Fertilization success is defined as when mated gravid females extrude two successive clutches that produce viable offspring. Treatment means, including the dilution water control, were statistically compared to the carri er control using t-tests. The aver age fertilization success rates in the control and carrier control treatm ents were 85 and 59%, respectively ( Table 19 ). While the fertilization rate in the carri er control group was reduced by 26% relative to the control group, the difference was not statistica lly significant (P=0.102). Neverthele ss, the reduced rate may be indicative of a solvent effect in this particular study. There was a statistically significant (p<0.05) decrease in the fertilization success rate in the 10 (P<0.001), 15 (P<0.001) and 20μg/L (P<0.001) test treatments relative to the carrier control. No sign ificant effects relative to the control group were observed in the 2.5 (P=0.8) and 5μg/L (P=0.583) lindane concentrations. Gravid female copepods that did not hatch their second brood hatch by Day 36 were excluded from statistical analysis. Based on these stat istical results, NOEC and LOEC values were determined to be 5 and 10μg/L, respectively.

3.4 SUMMARY OF NOEC AND LOEC FOR ALL ENDPOINTS

A summary of NOEC an d LOEC for all endpoints is presented Table 20

.

4 DISCUSSION

4.1 QUALITY OBJECTIVES AND CRITERIA

This study achieved the minimum quality criteria specified in Appendix I, Table 3 for the following performance objectives:

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

• The water quality parameters (dissolved oxyge n, temperature, pH, and salinity) were all within requirements

Page 20BES Project No. 27569 EPA Contract No. EP-W-06-026

• 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

Based on a time weighted average over the 36 day exposure period, A. tenuiremis were exposed lindane concentrations ranging from 75 to 87% of the nominal exposure level. This range indicates that, on average, linda ne exposure concentrations th roughout the testing period were within 25% of the predicted level. These meas ured levels were calcul ated as time-weighted mean concentrations accounting for the concentr ation of lindane in freshly prepared test solutions on water change days (referred to as “U P” samples) and the aged test solutions being replaced (referred to as “DOWN” samples). Meas ured lindane concentrations in the UP samples ranged from 95 to 106% of the nominal levels, wh ile test chemical in the DOWN samples ranged from 56 to 74% of the nominal concentrations. Possible explanations for the loss of test chemical are adsorption onto test chambers and sample storage bot tles, as well as through natural degradation.

Lindane did not affect copepod su rvival in the 2.5, 5, and 10μg/L test treatments throughout the 36 day exposure period. Lindane, in contrast, significantly reduced survival of copepods exposed to 20μg/L by day 16 of the test, and significantly reduced copepod survival in the 15 and 20μg/L treatments by day 36 of the test. This was unexpect ed as the concentration series was specifically selected to allow meaningful observations of chronic developmental a nd reproductive endpoints without affecting organism survival. Copepod su rvival was not significantly different between sea water controls and the 0.05% acetone control, indicating effe cts observed in copepods are most likely attributed to the test chemical and not the solvent. The reduced survival rate might be attributed to increased organism stress during water changes as operators aspirated test solution too quickly; however, as the contro ls are subjected to the same st ressors, this is most likely accounted for.

Lindane was shown to significantl y delay development time for A. tenuiremis nauplii to mature into the copepodite stage and subs equent transformation to the adu lt stage at concentrations of 5μg/L and above. Lindane had no significant effect on sex ratio for all co ncentrations tested. Lastly, lindane did not significan tly delay the time for mated females to extrude their first or second broods at any level tested. The most st riking reproductive effect of lindane was the reduced number of successful mating pairs and th e number of offspring that hatched. Lindane concentrations of 10μg/L, 15μg/L, and 20μg/L significantly reduced or completely prevented exposed mating pairs from successfully produci ng two successive viable broods. Moreover, lindane reduced the number of viable nauplii hatc hed from the first brood at the 20μg/L test level as well as the second brood at the 15μg/L concentration.

Page 21BES Project No. 27569 EPA Contract No. EP-W-06-026

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- 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 Practi ce Standards. 2007b. 40CFR792.

Page 22BES Project No. 27569 EPA Contract No. EP-W-06-026



### 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 lindane primary stock solution Test chemical weight: 4.0mg
Type of solvent: Acetone

Volume of solvent used: 50mL
Test chemical primary stock concentration: 80 mg/L

Page 23BES Project No. 27569 EPA Contract No. EP-W-06-026

Table 2: Method for preparatio n of lindane test solutions

Solution

ID Volume of Primary Stock

Solution used Volume of

acetone used Percent of

Acetone

Carrier Used Volume of

dilution

water used

Control 500 mL Carrier 125 μ L 0.025% 500 mL 20 μg /L 125 μ L of p rimar y stock solution 125 μ L 0.025% 500 mL 15 μg /L 94 μ L of p rimar y stock solution 94 μ L 0.019% 500 mL 10 μg /L 62.5 μ L of p rimar y stock solution 62.5 μ L 0.013% 500 mL 5 μg /L 31.25 μ L of p rimar y stock solutio n 31.25 μ L 0.006% 250 mL 2.5 μg /L 15.6 μ L of p rimar y stock solution 15.6 μ L 0.003% 250 mL

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

Date Dissolved Oxygen pH Salinity

8/16/2011 96.3 8.5 30.2 8/19/2011 93.4 8.1 30 8/22/2011 105.0 8.1 30 8/25/2011 89.5 8.1 28.2 8/28/2011 107.8 8.1 30.1 8/31/2011 98.0 8.0 30.6 9/3/2011 94.6 8.3 29.8 9/6/2011 100.1 8.2 29.5 9/9/2011 93.1 8.1 29.8 9/12/2011 97.1 8.1 29.3 9/15/2011 97.4 8.1 29.7 Note: measurements were made on dilution water prior to preparation of fresh test solutions.

Page 24BES Project No. 27569 EPA Contract No. EP-W-06-026

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
8/16/2011 25.0 24.1 9/4/2011 25.0 25.0 8/17/2011 25.0 ND 9/5/2011 25.0 25.0 8/18/2011 ND ND 9/6/2011 25.0 25.0 8/19/2011 25.0 24.9 9/7/2011 25.0 25.0 8/20/2011 25.0 25.7 9/8/2011 25.0 25.0 8/21/2011 25.0 25.0 9/9/2011 25.0 24.0 8/22/2011 25.0 25.0 9/10/2011 25.0 25.0 8/23/2011 25.0 25.0 9/11/2011 25.0 25.0 8/24/2011 25.0 24.7 9/12/2011 25.0 25.0 8/25/2011 24.5 25.0 9/13/2011 25.0 25.1 8/26/2011 24.8 25.0 9/14/2011 25.8 25.9 8/27/2011 24.4 24.9 9/15/2011 25.0 24.1 8/28/2011 24.8 25.0 9/16/2011 28.0 24.0 8/29/2011 25.0 25.0 9/17/2011 24.1 24.1 8/30/2011 25.0 25.0 9/18/2011 27.5 26.0 8/31/2011 25.0 25.0 9/19/2011 24.9 24.9 9/1/2011 25.0 25.0 9/20/2011 25.0 26.0 9/2/2011 25.0 24.9 9/21/2011 25.5 ND 9/3/2011 25.0 25.0 Note: Temperature was measured in dilution water from a glass bottle in environmental chamber used for study. ND = Not Determined

Table 5: Summary of wa ter quality parameters

Temperature (°C) Dissolved Oxygen (%) pH Salinity (‰)

Min 24.0 89.5 8.0 28.2
Max 28.0 107.8 8.5 30.6
Mean ± S.D. 25.0 ± 0.6 97.5 ± 5.3 8.16 ± 0.14 29.7 ± 0.6

Page 25BES Project No. 27569 EPA Contract No. EP-W-06-026Page 26Table 6: Measured lindane test concentrationsNominalConcentrationDay 0UpDay 0DwnDay 6UpDay 6DwnDay 12UpDay 12DwnDay 21UpDay 21DwnDay 27UpDay 27DwnDay 33UpDay 33DwnDilution WaterControl<0.010<0.010<0.0100.021<0.0100.04<0.010<0.010<0.0100.021<0.010<0.010Carrier Control<0.010<0.010<0.0100.026<0.010<0.010<0.010<0.010<0.010<0.010<0.010<0.0102.5 μg/L2.51.12.51.42.51.62.61.62.71.62.91.65.0μg/L52.24.62.73.82.54.72.85.13.55.3310μg/L1258.65.6116.69.95.67.414117.815μg/L156.9147.8179.815**1610171020μg/L208.22012236.72013221422130.5 μg/L (Spike1)101% Recovery103% Recovery0.5μg/L (Spike2)98.4% Recovery103% Recovery0.5μg/L (Spike3)101% Recovery105% Recovery0.5μg/L (Spike4)99.9% Recovery105% Recovery0.5μg/L (Spike5)95.1% RecoveryMean Spikerecovery***99.1% ± 2.5%104% ± 1.2%Note: Analysis performed by North Coast Laboratories, LTD (Arcata, California, USA) using GC-ECD.. Table compiled from NCLfinal report numbers 1109247 and 1109490.* Analysis could not be performed assample was spilled during handling.** Spike recoveries were not usedto correct sample results.BES Project No. 27569 EPA Contract No. EP-W-06-026

Page 27 Table 7: Summary of measured lindane test concentration

Nominal Concentration (μg lindane/L) Time Weighted Mean Measured Concentration (μg lindane/L) Percentage of Nominal (%)

Dilution Water Control <0.01 Carrier Control <0.01 2.5 2.1 82.0 5.0 3.8 75.3 10 8.7 87.1 15 12.6 83.9 20 16.2 80.8

Table 8: Survival on day 16 Percent survival at day 16

Treatment group Replicate Percent survival Mean Standard deviation plate 1 83 plate 2 80 Control plate 3 83 82 2

plate 1 83 plate 2 90 Carrier Control plate 3 63 79 14

plate 1 63 plate 2 87 2.5 μg/L lindane plate 3 87 79 14

plate 1 70 plate 2 50 5 μg/L lindane plate 3 87 69 19

plate 1 83 plate 2 67 10 μg/L lindane plate 3 73 74 8

plate 1 77 plate 2 63 15 μg/L lindane plate 3 57 66 10

plate 1 60 plate 2 77 20 μg/L lindane plate 3 53 63* 12

*Statistically significant difference in surv ival relative to the carrier controlBES Project No. 27569 EPA Contract No. EP-W-06-026

Table 9: Survival on day 36 Percent survival at day 36 Treatment group Replicate Percent survival Mean Standard deviation plate 1 77 plate 2 73 Control plate 3 80 77 4

plate 1 77 plate 2 90 Carrier Control plate 3 63 77 14

plate 1 53 plate 2 83 2.5 μg/L lindane plate 3 87 74 19

plate 1 70* plate 2 43 5 μg/L lindane plate 3 83 65 20

plate 1 73 plate 2 67 10 μg/L lindane plate 3 73 71 3.5

plate 1 70 plate 2 63 15 μg/L lindane plate 3 43 59** 14

plate 1 60 plate 2 67 20 μg/L lindane plate 3 53 60** 9

*denotes the survival rate on day 24; the plat e was accidentally dropped on day 25 during test maintenance.

**Statistically significant difference in surv ival relative to the carrier control

Page 28BES Project No. 27569 EPA Contract No. EP-W-06-026

Table 10: 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 9.6 plate 2 8.9 Control plate 3 9.9 9.5 0.5

plate 1 9.2 plate 2 9.0 Carrier Control plate 3 9.6 9.3 0.3

plate 1 10.0 plate 2 10.0 2.5 μg/L lindane plate 3 10.0 10.0 0.0

plate 1 12.5 plate 2 11.5 5 μg/L lindane plate 3 11.1 11.7* 0.7

plate 1 14.3 plate 2 13.3 10 μg/L lindane plate 3 14.1 13.9* 0.5

plate 1 17.0 plate 2 14.3 15 μg/L lindane plate 3 15.4 15.6* 1.4

plate 1 14.4 plate 2 16.1 20 μg/L lindane plate 3 14.5 15.0* 1.0

*Statistically significant difference when compared to the Carrier Control

Page 29BES Project No. 27569 EPA Contract No. EP-W-06-026

Table 11: Time in copepodite stage Number of days in copepodite stage
Treatment group Replicate Mean number of days Total Mean Standard deviation
plate 1 7.9 plate 2 8.3 Control plate 3 8.1 8.1 0.2

plate 1 8.9 plate 2 7.9 Carrier Control plate 3 7.9 8.2 0.6

plate 1 8.7 plate 2 8.3 2.5 μg/L lindane plate 3 8.6 8.5 0.2

plate 1 8.5 plate 2 7.9 5 μg/L lindane plate 3 7.9 8.1 0.3

plate 1 8.5 plate 2 9.2 10 μg/L lindane plate 3 8.6 8.8 0.4

plate 1 10.0 plate 2 8.4 15 μg/L lindane plate 3 9.9 9.4 0.9

plate 1 9.4 plate 2 8.9 20 μg/L lindane plate 3 8.3 8.9 0.6

Page 30BES Project No. 27569 EPA Contract No. EP-W-06-026

Table 12: 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 16.9 plate 2 17.3 Control plate 3 18.0 17.4 0.6

plate 1 18.3 plate 2 17.0 Carrier Control plate 3 17.2 17.5 0.7

plate 1 18.1 plate 2 18.1 2.5 μg/L lindane plate 3 18.6 18.3 0.3

plate 1 20.7 plate 2 20.0 5 μg/L lindane plate 3 19.0 19.9* 0.9

plate 1 23.2 plate 2 22.5 10 μg/L lindane plate 3 22.4 22.7* 0.4

plate 1 26.4 plate 2 22.9 15 μg/L lindane plate 3 24.6 24.6* 1.8

plate 1 23.8 plate 2 24.5 20 μg/L lindane plate 3 22.8 23.7* 0.9

*Statistically significant when comp ared to the Carrier Control

Page 31BES Project No. 27569 EPA Contract No. EP-W-06-026

Table 13: Sex ratio
Sex ratio

Treatment group Replicate Number of males Number of females Ratio (female:male) Total Mean Standard deviation
plate 1 35 65 1.88 plate 2 44 56 1.25 plate 3 48 52 1.09 Control
Plate 1, 2, and 3 27 37 1.4 0.4

plate 1 53 47 0.90 plate 2 36 64 1.75 plate 3 53 47 0.89 Carrier Control Plate 1, 2, and 3 27 21 1.2 0.5

plate 1 79 21 0.27 plate 2 64 36 0.57 plate 3 33 67 2.00 2.5 μg/L lindane Plate 1, 2, and 3 31 23 0.9 0.9

plate 1 80 20 0.25 plate 2 64 36 0.57 plate 3 32 68 2.13 5 μg/L lindane Plate 1, 2, and 3 28 24 1.0 1.0

plate 1 35 65 1.86 plate 2 62 38 0.63 plate 3 47 53 1.11 10 μg/L lindane
Plate 1, 2, and 3 25 28 1.2 0.6

plate 1 45 55 1.22 plate 2 31 69 2.25 plate 3 50 50 1.00 15 μg/L lindane
Plate 1, 2, and 3 20 26 1.5 0.7

plate 1 67 33 0.50 plate 2 63 37 0.58 plate 3 45 55 1.20 20 μg/L lindane Plate 1, 2, and 3 28 18 0.8 0.4

Page 32BES Project No. 27569 EPA Contract No. EP-W-06-026

Table 14: 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 2.0 plate 2 3.0 Control
plate 3 2.2 2.4 0.5

plate 1 4.0 plate 2 1.0 Carrier Control plate 3 1.8 2.3 1.5

plate 1 3.7 plate 2 2.3 2.5 μg/L lindane plate 3 1.0 2.3 1.4

plate 1 ** plate 2 4.6 5 μg/L lindane plate 3 2.8 3.7 1.3

plate 1 2.3 plate 2 1.0 10 μg/L lindane plate 3 1.0 1.4 0.8

plate 1 2.7 plate 2 NC 15 μg/L lindane plate 3 4.0 3.3 0.9

plate 1 2.0 plate 2 NC 20 μg/L lindane plate 3 4.0 3.0 1.4

** Plate dropped by technici an the day after mating NC=Could not be calculated since no females became gravid

Page 33BES Project No. 27569 EPA Contract No. EP-W-06-026

Table 15: 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 6.3 plate 2 4.9 Control
plate 3 6.4 5.9 0.8

plate 1 6.7 plate 2 4.5 Carrier Control plate 3 6.3 5.8 1.2

plate 1 4.3 plate 2 6.3 2.5 μg/L lindane plate 3 4.0 4.9 1.3

plate 1 ** plate 2 3.0 5 μg/L lindane plate 3 4.8 3.9 1.3

plate 1 3.5 plate 2 1.0 10 μg/L lindane plate 3 4.0 2.8 1.6

plate 1 7.0 plate 2 0.0 15 μg/L lindane plate 3 0.0 2.3 4.1

plate 1 1.0 plate 2 0.0 20 μg/L lindane plate 3 0.0 0.3* 0.6

*Statistically significant difference when compared to the Carrier Control

** Plate dropped by technici an the day after mating

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Table 16: Time to second brood since date of pairing Time to second brood (days) since date of pairing
Treatment group Replicate Mean number of nauplii Total Mean Standard deviation
plate 1 2.5 plate 2 1.8 Control
plate 3 2.2 2.2 0.4

plate 1 2.3 plate 2 2.0 Carrier Control plate 3 2.5 2.3 0.3

plate 1 1.5 plate 2 2.0 2.5 μg/L lindane plate 3 5.0 2.8 1.9

plate 1 *** plate 2 2.2 5 μg/L lindane plate 3 1.0 1.6 0.8

plate 1 3.0 plate 2 3.0 10 μg/L lindane plate 3 2.0 2.7 0.6

plate 1 3.5 plate 2 NC 15 μg/L lindane plate 3 NC 3.5 0

plate 1 NC plate 2 NC 20 μg/L lindane plate 3 NC NC NC

*** Plate dropped by technici an the day after mating NC=Could not be calculated since no females became gravid

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Table 17: 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 5.1 plate 2 5.5 Control
plate 3 7.3 6.0 0.8

plate 1 7.3 plate 2 7.0 Carrier Control plate 3 5.8 6.7 1.2

plate 1 4.5 plate 2 5.7 2.5 μg/L lindane plate 3 ** 5.1 1.3

plate 1 *** plate 2 5.3 5 μg/L lindane plate 3 7.4 6.4 1.3

plate 1 4.0 plate 2 5.0 10 μg/L lindane plate 3 10.0 6.3 1.6

plate 1 0.0 plate 2 0.0 15 μg/L lindane plate 3 0.0 0* 0

plate 1 0.0 plate 2 0.0 20 μg/L lindane plate 3 0.0 0* 0

*Statistically significant difference when compared to the Carrier Control ** Lone female on plate extruded second brood sac but did not hatch eggs prior to accidentally being removed from the test well by a technician during a water change. *** Plate dropped by technici an the day after mating

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Table 18: 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 11.4 plate 2 9.6 Control plate 3 14.1 11.7 2.3

plate 1 14.0 plate 2 11.5 Carrier Control plate 3 12.0 12.5 1.3

plate 1 9.0 plate 2 11.7 2.5 μg/L lindane plate 3 0** 6.9 6.1

plate 1 *** plate 2 8.3 5 μg/L lindane plate 3 12.2 10.3 2.8

plate 1 8.0 plate 2 6.0 10 μg/L lindane plate 3 14.0 9.3 4.2

plate 1 7.0 plate 2 0** 15 μg/L lindane plate 3 0** 4.1 2.3

plate 1 0** plate 2 0** 20 μg/L lindane plate 3 0** 0 0

* Statistically different when co mpared to the Carrier Control

** Replicates with no females having two broods

*** Plate dropped by technici an the day after mating

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Table 19: 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 11 11 100%
Control PLATE 2 6 8 75%
Control PLATE 3 8 10 80% 85% 13%

Carrier control PLATE 1 3 5 60%
Carrier control PLATE 2 2 4 50%
Carrier control PLATE 3 4 6 67% 59% 8%

2.5 μg/L lindane PLATE 1 2 2 100% 2.5 μg/L lindane PLATE 2 3 6 50% 2.5 μg/L lindane PLATE 3   **  75% 35%

5μg/L lindane PLATE 1 *** *** *** 5μg/L lindane PLATE 2 4 7 57% 5μg/L lindane PLATE 3 5 7 71% 64% 10%

10μg /L lindane PLATE 1 1 9 11% 10μg /L lindane PLATE 2 1 8 13% 10 μg /L lindane PLATE 3 1 10 10% 11%* 1%

15μg/L lindane PLATE 1 0 10 0% 15μg/L lindane PLATE 2 0 7 0% 15μg/L lindane PLATE 3 0 4 0% 0%* 0%

20 μg/L lindane PLATE 1 0 7 0% 20μg/L lindane PLATE 2 0 7 0% 20μg/L lindane PLATE 3 0 7 0% 0%* 0%

* Denotes statistically significant differen ce when compared to the solvent control ** Lone female on plate extruded second brood sac but did not hatch eggs prior to accidentally being removed from the test well by a technician during a water change. *** Plate dropped by technici an the day after mating

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, whichever came first. A single mating pair in both c ontrol replicate #2 and 2.5μg/L replicate #2 was excluded because the second brood ha d extruded but did not hatch prior to test termination on Day 36.

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Table 20: Summary table of NOEC and LOEC for all endpoints
Endpoint NOEC in μg lindane /L LOEC in μg lindane /L Day 16 survival (%) 15 20 Day 27 survival (%) 10 15 Time to copepodite stage 2.5 5 Time in copepodite stage >20 >20

Time to adult 2.5 5

Sex ratio >20 >20

Time to 1st Brood 20 >20

Time to 2nd Brood >15 >15 Number offspring 1st Brood 15 20 Number offspring 2nd Brood 10 15
Number of mating pair with successful reproduction 5 10

Number nauplii hatched over two clutches 10 15 Note: 2.5 μg lindane /L was the lowest concentration tested and 20 μg lindane /L was the highest concentration tested.

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### 9 APPENDIX I: QUALITY ASSUR ANCE PROJECT PLAN (QAPP)

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### 10 APPENDIX II: QUALITY OF LABORATORY WATER AT BES

Baseline chemical analyses of laboratory reagent water used for preparation of synthetic seawater and linda ne 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) <0.005

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### 11 APPENDIX III: LINDANE CHEMISTRY DATA REPORT

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### 12 APPENDIX IV: STUDY PROTOCOL (P27569)



## PROTOCOL

Effect of Lindane on the Survival , Development, and Reproduction of the Marine Copepod Amphiascus Tenuiremis

Prepared for: RTI International

Protocol Number: P27569

BES Study Number: 27569

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

MANAGEMENT APPROVAL: DAVID S. BLOCK, PH.D.

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### PROTOCOL APPROVAL (STUDY NO: 27569)

Pages 1 - 13

____________________________________ ___ April 21, 2011

___ David S. Block Date Study Director Block Environmental Services, Inc.

____________________________________ ____________________ Carol Sloan Date Sponsors Agent RTI International

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### SCHEDULE OF WORK

STUDY TITLE Effect of Lindane 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 27569

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 Lindane CAS# 58-89-9

PROPOSED EXPERIMENTAL DATES START: April 18, 2011 COMPLETION: May 6, 2011 [Amended] START: August 16, 2011 COMPLETION: September 21, 2011

PRINCIPLE SCIENTIST(S) Terence Clohesy Nanette Bradbury

ASSISTANT SCIENTIST(S) Sebastian Castro Amy Ayala

MAIN TEST LOCATION 2451 Estand Way, Pleasant Hill, California, USA Testing Room #4

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### SCHEDULE OF WORK

STANDARD OPERATING PROCEDURES All SOPS used will be documented in the study book.

ANALYTICAL CHEMISTRY METHOD Lindane will be measured in test solutions using USEPA Method 505

TEST CONCENTRATION(S) TO BE EMPLOYED The test concentrations will be a dilution water control, solvent control and 2.5, 5, 10, 15 and 20 μg/L.

CIRCULATION LIST Sponsor or Sponsor’s Agent
Client Manager
QA
Principal Scientists

Assistant Scientists

INTRODUCTION

The objectives of this study are to determine the effect of lindane 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 lindane conc entrations in seawater at 25°C for a period of 36 days. Once maturing into sexually dimorphic vi rgin adults (typically between 15-18 days at 25  ̊ C), male and female copepods will be paired with in treatment in order to determine the effect of lindane on mating success and offspring produ ction. The test will be terminated when all mating pairs have successfully produced two viab le broods or on Day 36 of the exposure period, whichever comes first. All phases of the test will be performed under semi-static test conditions with water renewal occurring every three days. The test duration should allow sufficient time for the control animals to reach a dulthood, mate and produce at leas t two clutches of offspring.

Test endpoints, such as the survival rate, time to developmental milestone, mating success, and offspring production will be statistically co mpared between lindane exposed copepods and laboratory controls in order to calculate no observed effect (NOE C) and lowest observed effect (LOEC) concentrations.

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MATERIALS

Test Chemical A lindane certificate of analys is and purity information will be supplied by the Sponsor. The Sponsor will outline all precautions required in the handling, storage and disposal of lindane. Unused lindane will be resubmitted back to the Sponsor upon completion of the study.

TEST ORGANISM

The test organism will be the marine crustacean Amphiascus tenuiremis . The copepods will originate from laboratory 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.

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

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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;

• 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 chambe rs or a temperature regulated room;

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• 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 (or 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.

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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 the 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

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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 lindane will be prepared in an organic solvent (acetone). The test solutions will be prepared by addition of a stock solution to dilution water and each test concentration and the solvent control will contain the same concentra tion of solvent, which will not exceed 1 ml/L. The volume of each test solution prepared will be in excess of that required for the replicate test vessels to allow for initial physical and chemical analysis. Further details of the test solution preparation will be documented in the study book.

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

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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 USEPA Method 505 “Analysis of Organohalide pesticides and Aroclors in Dri nking Water”. Chemical analysis will be conducted on all newly prepared solutions of controls and all test c oncentrations (Ups) and on the solutions removed from the microwells at test rene wal (Downs). Chemical analysis will be conducted at the start of the test and then on at least one water renewal pe r week for the duration of the test. If timings between test renewals varies then a mixture of di fferent length renewals will be included in the analysis (e.g. 2 or 3 days between renewals).

BIOLOGICAL DATA ANALYSIS

The mortality data for selected time periods will be analyzed to calculate the median effect concentrations (EC50) and 95% confidence intervals.

Fisher’s Exact Test or Pearson’ Chi-square 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.

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,

Page 124BES Project No. 27569 EPA Contract No. EP-W-06-026

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.

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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.

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Page 127 

### 13 APPENDIX V: DEVIATIONS FROM STUDY PROTOCOL

13.1 LIST OF DEVIATIONS Below is a list of the deviations encountered during the study.

Deviation number Deviation title Deviation description

1 Protocol not distributed or signed. The completed test protocol was not submitted to Sponsor or signed by Sponsor prior to initiation of study.
2 Starting number of nauplii/Empty test wells On D1, several wells across all test treatments were observed

to contain >1 nauplius per well. These extra nauplii were transferred to test wells observed to be empty on the same replicate within each treatment at the first water change on day 3. Extra organisms were haphazardly selected to be discarded if there were not a sufficient number of empty test wells available.
3 D21 Down sample spilled The 15ug/L (nominal) down sample reflecting day 21 test solution was accidentally knocked over during the water change process. 4 Temperature not recorded Temperature in the humidity chamber was not measured on D1

‐ reading #2 and both readings on D2
5 Water chemistry values not collected in test solutions Dissolved Oxygen and pH were not collected in the Up and
Down test solutions. Operators instead collected data from dilution water prior to water change. The basis for this deviation is that operators interpreted the language of the
OECD 2009 guideline to indicate this was an optional component (“should”, as opposed to “must” or “will”)
6 Potential for evaporation data

not recorded. A 14

‐ day dry run study was performed in June/July for the operators to practice performing the test. Salinity results on water change days were reported to have been performed, but the data was not recorded.
7 Lost copepods during pairing
process Approximately 15 animals were lost in various plates spread

across the concentrations.
8 Mature male/female copepods

not immediately mated. Sexually mature copepods were scheduled to be paired for mating on Day 18, a water change day. D18 fell on a Saturday and the laboratory workload coupled with reduced staff was such that a water change and mating could not occur on the

same day. BES elected to prioritize the water change and delay the creation of mating pairs until D21.
9 Mating pairs not from same microplate In order to maximize the number of mating pairs, males and females were paired across replicates but within treatment. However, we attempted to create male/female pairs within replicate when possible.