Study number 11-0030/B

Guideline Draft OECD Test Guideline ‘Harpacticoid copepod development and reproduction test with Amphiascus tenuiremis’

. Test Facility Brixham Environmental Laboratory, AstraZeneca UK Limited, Freshwater Quarry, Brixham, Devon, TQ5 8BA, UK Sponsor U.S. Environmental Protection Agency. EPA East Building, 1201 Constitutional Avenue, NW Room 4106-J, Mail Code 7203M, Washington, DC20004.

Authors G Le Page R J Brown
December 2011 

### Report No BR0476/B Copy number Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals) Study No. 11-0030/B



### 2 (45) CONFIDENTIAL (CATEGORY B)

Not to be photocopied or microfilmed. This doc ument contains information confidential and trade secret to U.S. Environmental Protection Ag ency. It should not be released in any form to an outside party, nor should information contai ned herein be used by a registration authority to support registration of any other product without the written permission of U.S. Environmental Protection Agency.Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals) Study No. 11-0030/B



### 5 (45) CONTRIBUTORS

Study Director Test Facility G Le Page Brixham Environmental Laboratory Freshwater Quarry AstraZeneca UK Limited Brixham Devon TQ5 8BA UK

Principal Scientist, Ecotoxicology Gareth Le Page
Principal Scientist, Analytical Chemistry Cathryn MoneyReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals) Study No. 11-0030/B



### 6 (45) CONTENTS page

TITLE PAGE.....................................................................................................................

....... 1 CONFIDENTIALITY STATEMENT ..................................................................................... 2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT .................................. 3 QUALITY ASSURANCE STATEMENT............................................................................... 4 CONTRIBUTORS...................................................................................................................

. 5 CONTENTS .......................................................................................................................

...... 6 LIST OF TABLES.................................................................................................................

... 8 LIST OF FIGURES................................................................................................................

.. 8 LIST OF APPENDICES .......................................................................................................... 8 1 SUMMARY............................................................................................................

..... 9 2 INTRODUCTION ................................................................................................. 11 3 MATERIALS......................................................................................................... 11 3.1 Test substance ........................................................................................................ 11 3.2 Test organism......................................................................................................... 11 3.3 Dilution water ........................................................................................................ 12 4 TEST METHOD AND CONDITIONS................................................................. 12 4.1 Test apparatus and conditions................................................................................ 12 4.2 Preparation of test solutions................................................................................... 13 4.3 Test procedure........................................................................................................ 13 4.4 Creation of sexed pairs........................................................................................... 14 4.5 Offspring production.............................................................................................. 14 4.6 Feeding................................................................................................................... 14 4.7 Physical and chemical parameters ......................................................................... 15 4.8 Chemical analysis .................................................................................................. 15 4.9 Biological Data Analysis ....................................................................................... 15 4.9.1 Survival and sex ratio............................................................................................. 15 4.9.2 Development rates and reproduction ..................................................................... 15 4.10 Validity Criteria ..................................................................................................... 17Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals) Study No. 11-0030/B



### 7 (45) 5 RESULTS .............................................................................................................. 17 5.1 Chemical data......................................................................................................... 17 5.2 Biological data ....................................................................................................... 18 5.2.1 Observation of mortalities a nd symptoms of toxicity............................................ 18 5.2.2 Development rate to co pepodite and to adult......................................................... 19 5.2.3 Sex Ratio................................................................................................................ 20 5.2.4 Reproduction.......................................................................................................... 20 5.3 Overall observed effect concentrations.................................................................. 21 5.4 Physical and chemical data .................................................................................... 21 6 CONCLUSION...................................................................................................... 22 7 REFERENCES....................................................................................................... 23Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals) Study No. 11-0030/B



### 8 (45) LIST OF TABLES Table 1 Analytical results............................................................................................. 24 Table 2 Cumulative mortality ...................................................................................... 27 Table 3 Summary data - development to copepodite and adult ................................... 29 Table 4 Number of days taken to de velop from nauplii to copepodite........................ 30 Table 5 Number of days taken to develop from copepodite to adult........................... 33 Table 6 Sex Ratio......................................................................................................... 36 Table 7 Number of offspring per brood ....................................................................... 37 Table 10 Dilution water parameters............................................................................... 40

LIST OF FIGURES Figure 1 Statistical analysis flow chart for development and reproduction .................. 41

LIST OF APPENDICES Appendix 1 Certificate of Anal ysis for lindane .................................................................. 42 Appendix 2: Analytical method for the deter mination of lindane in seawater .................. 43 Appendix 3 Calculation of time-weighted mean ................................................................ 45

The last page of this report is................................................................................................ ....45Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 9 (45) 

## 1 SUMMARY

Sponsor 

### U.S. Environmental Protection Agency, EPA East Building, 1201 Constitutional Avenue, NW Room 4106-J, Mail Code 7203M, Washington, DC20004, USA
Sponsor contact 

### Dr P Browne International telephone: 202-564-8474
Sponsor’s Agent 

### U.S. Environmental Protection Agency, EDSP/OCSPP/Office of Science Coordination and Policy 1200 Pennsylvania Ave. NW (7203M) Washington, DC 20460
Agent contact 

### Dr H Navarro International telephone: 919-541-6148
Test facility, location of raw data and final report 

### Brixham Environmental Laboratory, AstraZeneca UK Limited, Freshwater Quarry, Brixham, Devon, TQ5 8BA, UK

Test facility contact 

### R J Brown International telephone: +44 (0) 1803 882882 International fax: +44 (0) 1803 882974 Test substance

common name 

### Lindane

Subject 

### Chronic toxicity to the copepod Amphiascus tenuiremis

Guideline 

### Draft OECD Test Guideline ‘Harpa cticoid copepod development and reproduction test with Amphiascus tenuiremis ’ (Ref 1)
Test species 

### Amphiascus tenuiremis
Source of organisms 

### Laboratory cultures held at Br ixham Environmental Laboratory established March 2011
Stage of organisms at the initiation of study 

### Larvae less than 24 hours old

Test concentrations 

### Control, solvent control and nominal test substance concentrations: 2.5, 5.0, 10, 15 and 20 μg/L
Length of test 

### 36 days
Exposure test dates 

### 10 May 2011 to 15 June 2011

Nominal test temperature 

### 25 ± 2 ° CReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 10 (45) Endpoint No observed effect conc Lowest observed effect

conc Day 15 survival 11 μg/L 17 μg/L

Day 36 survival 7.2 μg/L 11 μg/L

Development (to copepodite) 3.6 μg/L 7.2 μg/L Development

(to adult) 2.1 μg/L* 3.6 μg/L*

Time to 1 st brood Not Determined Not Determined

Time to 2 nd brood Not Determined Not Determined

Interbrood

duration Not Determined Not Determined

Number of offspring – 1 st brood 17 μg/L >17 μg/L

Number of offspring – 2 nd brood 17 μg/L >17 μg/L

Average number of offspring 17 μg/L >17 μg/L

Number of successful mating pairs Not Determined Not Determined

Overall 3.6 μg/L 7.2 μg/L R esults based on concentrations of lindane

Comments * The endpoint for development time to adu lt is considered unreliable because of the misidentification of copepodites as adults as discussed in the report and therefore the overall NOEC and LOEC for this study has been based on development to copepodite.

Due to practical difficulties carrying out the study (see section 5.2), the consistency of data recording and reliability of the data are not considered entirely satisfactory. The data up until day 15 occurred prior to pairing of adults and therefore there were less handling related problems. The ‘survival to day 15’ and ‘development to copepodite’ endpoints are considered to be relatively robust. The data from day 16 to day 36 and the corresponding endpoints are not considered to be as reliable. All reasonable attempts have been made to ensure the data is as robust as possible; however, caution should be applied when interpreting the results.Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 11 (45) 

## 2 INTRODUCTION At the request of the U.S. Environmenta l Protection Agency, EPA East Building, 1201 Constitutional Avenue, NW Room 4106- G, Mail Code 7203M, Washington, DC20004 the chronic toxicity of linda ne to the marine copepod Amphiascus tenuiremis was determined. This study was conducted based on the test me thod outlined in the draft OECD guideline, ‘Harpacticoid copepod developmen t and reproduction test with A. tenuiremis ’ (Ref 1). The procedures used in this study were based on this guideline. The experimental start and completi on dates were 06 May 2011 and 20 June 2011, respectively, and the study number was 11-0030/B. All original data, togeth er with other relevant records, will be repatriated to the sponsor; copies will be retained at Brixham Environmental Laboratory.



## 3 MATERIALS
3.1 Test substance The test substance, Lindane, was supplied by Battelle Marine Scien ces Laboratory, 1529 West Sequim Bay Road, Sequim WA 98382 at the reque st of R Sternberg, U.S Environmental Protection Agency, EPA East Building, 1201 Constitutional Avenue, NW Room 4106-J, Mail Code 7203M, Washington, DC20004, USA.

IUPAC name: 1 α ,2 α ,3 β ,4 α ,5 α ,6 β -hexachlorocyclohexane

CAS Number: 58-89-9

The test substance was received at Brixham Environmental Laboratory on 22 March 2011 and assigned the Brixham test substance number 11-0030. The test substance (Lot: 14419EB) was supplied as a white to off white powder. A cer tificate of analysis dated May 2003 stated that the sample had a purity of 99.6 % (Appendi x 1), this was confirmed in December 2010. The sample was stored at ambient temperature, in the container in which it was received, until required for testing when appropriate subsam ples were provided for the test operators. 

## 3.2 Test organism The test organism was the marine harpacticod copepod, A. tenuiremis , obtained from laboratory cultures held at Brixham Environm ental Laboratory. The copepods originated from laboratory cultures established by Dr Tom Chandler, Professor of Environmental HealthReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 12 (45) and Marine Sciences, University of South Ca rolina, Columbia, SC 29208 who confirmed the taxonomic identity of this species.

The stock cultures of A. tenuiremis were maintained in natural seawater filtered to 1μm at a temperature of 25 ± 2°C. The cultures were maintained in 6 L plastic vessels with a working volume of approximately 3 L. The cultures were originally maintained in a flow-through system but after initial problems in maintaining the salinity level, overlying water was replaced by semi-static renewal. The salinity of the overlying water used for the semi-static renewals was within 30 ± 3‰. A photoperiod of 15 hours light: 8 hours dark, with 30 minute transition periods was provided.

The A. tenuiremis cultures were fed on a mixed algae diet of Rhinomonas reticulata, strain CCAP 995/2, Instant Algae ® 

### Isochrysis 1800 strain CCMP 1324 / T-150 and Dunaliella tertiolecta strain 19/27. The A. tenuiremis cultures were fed twice per week. Twenty four hours before the start of the test gravid females from a single culture vessel were separated into petri-dishes (labelled with time and date of separation). The A. tenuiremis nauplii released by these females were used to initiate the test within a 24 hours period. Therefore, animals <24 hours old, from parents obtained from a single culture vessel, were used for testing. The test organisms and the culture from which they were obtained appeared healthy and showed no evidence of disease before the test period. 

## 3.3 Dilution water Natural seawater with a nominal salinity of 30 ± 3‰ and filtered to 0.2 μm was used as the dilution water for the test. The pH, dissolved oxygen and salinity were determined for the dilution water used in the test. Ammonical nitrogen as N, solids-suspended at 105°C, dissolved organic carbon (DOC) and chemical oxygen demand (COD) were also determined for a recently analysed batch of seawater, along with a range of metals, pesticides and PCBs.

The water was aerated for >24 hours befo re use whereupon the dissolved oxygen concentration was above 80% of the air saturation value and the pH was 8.0 ± 0.3.



## 4 TEST METHOD AND CONDITIONS 4.1 Test apparatus and conditions Disposable polystyrene 96 well micro-well plat es (nominal total volume 300 μL) with loose fitting lids were used as test vessels. The test design incorporated 3 rows of 10 replicates per plate, and 3 plates per test concentration. Each micro-well contained a 250 μL working volume of test solution.

The test solutions were maintained at a nomin al 25 ± 2°C by housing the test vessels in a temperature-controlled room. The microplate well plates were placed in ‘humidity boxes’ toReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 13 (45) reduce evaporative losses. The potential for evaporative loss was measured prior to exposure by placing seawater filled microplate well pl ates in the humidity boxes for 72 hours and measuring the salinity change over this peri od. As no salinity change was observed over a three day period evaporative loss was considered to be low and the humidity chambers appropriate for conducting the test. The photoperiod was contro lled to provide 12 h light: 12 h dark with 20 minute transition periods. The test was initiated by the addition of 30 impartially selected A. tenuiremis nauplii (< 24h old), individually added to each micro-well plate. The plates were loaded in an irregular order. Each treatment contained a nominal total of 90 A. tenuiremis

. The copepods were microscopically observed daily. 

## 4.2 Preparation of test solutions This study was run with a dilution water control (DWC), solvent control (SC) and nominal concentrations of 2.5, 5, 10, 15 and 20 μg/L of li ndane. The solvent used in this study was triethyleneglycol. Every other test solution preparation day, a new stock solution was prepared, by dissolving 0.02 g of lindane in 100 mL of triethyleneglyco l to give a nominal stock solution containing 0.2 g/L lindane. The resulting stock solution wa s observed to be clear and colourless. New test solutions were prepared every three da ys by the addition of appropriate volumes of the nominal 0.2 g/L stock solution to dilution water. All test solutions were shaken vigorously and given 10 minutes stirring before use and we re observed to be clear and colourless. An equivalent volume of triethyleneglycol was added to the solvent control. All test solutions and the solvent control contained 0.1 mL/L triethyleneglycol. 

## 4.3 Test procedure Nauplii (< 24 hours old) were obt ained as described in Secti on 3.2. The nauplii were exposed in individual microplate wells of a 96 microplate well plate ( 30 animals per microplate well plate) with three microplate well plates per treatment (total of 90 animals) for the DWC, SC and each test concentration. The outer we lls of the plate were left empty. The positions of the microplate well plates we re randomally allocated within the humidity chamber. Addition of nauplii, subsequent handling, loading, observation and feeding was performed in an irre gular plate order. The copepods were observed daily and the su rvival and development from a first stage nauplius to a first stage copepodite and reproductiv ely mature adult, were recorded. The sex of each adult was recorded after sexual dimorphism was achieved. Observations were also made for any abnormal behaviour (relative to the controls), including uncoordinated swimming, loss of equilibrium, change in pigmentation and atypical quiescence.Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 14 (45) 

## 4.4 Creation of sexed pairs From day 15, mature virgin males and females were removed from their respective treatment or control microplate well plates and were paired impartially with an opposite sex individual (i.e., from the same microplate well plate with in the same treatment) in a new or unused microplate well. Animals were paired as they were identified to be sexually mature. On occasions a number of animals were misidentified as sexually mature and their gender was incorrectly assigned. These copepods were re-paired as appropriate. Newly mated pairs were transferred to a new te st well with minimal transfer of solution. New test solution (250 μL) was then added to this well. 

## 4.5 Offspring production After pairing of male: female mating pairs, observations were made daily for the presence of females carrying their first and subsequent eg g sacs. The numbers of successfully hatched nauplii (i.e., total viable clutc h, or fecundity) were recorded by direct microscopic observation for the first and second succe ssive clutches of eggs All mating pairs producing two vi able clutch’s was considered to be a “successful mating”. 

## 4.6 Feeding The copepods were fed on Day 0 and at every other renewal day (every 6 th 

### day of the test) with algae. Each microplate well received a 1:1:1 algal cell suspension of each of the chlorophyte, Dunaliella tertiolecta, strain CCAP 19/27, the chrysophyte, Isochrysis sp and the cryptophyte, Rhinomonas reticulata strain CCAP 995/2 

### . The algal cell density added to each microplate well was a nominal 2 x 10 4 

### cells / microplate well. The algae ( Dunaliella and Rhinomonas ) were cultured, under unialgal conditions, and were harvested and centrifuged and the algal cells resuspended in filtered, natural seawater. The algal stocks were measured for cell density usin g a Coulter counter, stored in a refrigerator in the dark, and used within one week. The Isochrysis was an algal concentrate Instant algae® Isochrysis 1800 (strain CCMP 1324 / T-150) resuspe nded in filtered, natura l seawater to the appropriate algal concentration. For practical reasons the algae was added directly to the prepared volume of the test solution rather than to individual test chambers. On Day 18 of the study, the feeding was not correctly recorded in the study book. It was therefore assumed that the copepods had not been fed on this occasion so they were fed during the Day 21 renewal instead. Subsequent feeding occurred every six days after Day 21. It was later confirmed that the animals had received food on Day 18 of the test and therefore over the test the copepods received on a dditional feed. As all copepods received the same amount of food and all test concen trations were treated identically, this was not expected to have detrimentally influenced the overall outcome of the study.Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 15 (45) 

## 4.7 Physical and chemical parameters The pH, dissolved oxygen and salinity of th e dilution water were measured prior to preparation of each new set of test solutions. The pH and dissolved oxygen concentration of the control, solvent control and the highest test concentration (nominal 20 μg/L) were measured for each newly prepared ‘on’ test so lutions and for the ‘off’ test solutions using the excess test solution remaining from the previous renewal. Measurement of these parameters in the actual ‘off’ test solutions was difficult due to the small volumes of test solution used (250μL per replicate). Temperature was monitored throughout the test in an additional vessel fi lled with water and placed inside the humidity chamber. 

## 4.8 Chemical analysis The concentrations of lindane in the test so lutions were measured on the ‘on’ and ‘off’ solutions using the gas chromatography mass spectroscopy (GC-MS) method, outlined in Appendix 2. On exposure Days 0, 6, 15, 21, 27 and 33 samples of the ‘on’ (freshly prepared) control and exposure concentrations were analysed for th e concentration of linda ne. Samples from the corresponding ‘off’ control and exposure concen trations were analysed on the following renewal day. Triplicate analyses were performed on the nominal 5.0 μg/L exposure concentration on six sampling occa sions (Days 0, 6, 15, 21, 27 and 33). 

## 4.9 Biological Data Analysis 4.9.1 Survival and sex ratio Fisher’s Exact Test (Ref 2) was used to identify significant differences in survival between the control(s) and each treatment after 15 and 36 days. Day 15 was chosen as this occurred just prior to pairing of the adult copepods when co pepods should have moulted to adult. After pairing there were a number of mortalities that were directly attributed to operator handling of the test organisms. Any mortality that was attributed to handling by the operator was excluded from the analysis. For days 15 and 36, the concentration that caused 50, 25 and 10 % mortality (Lethal concentration (LC50, 25 and 10)) values and th eir 95% confidence intervals were calculated using Stephen’s method (Ref 3) Fisher’s exact test was used to identify significant differences in sex ratio between treatments. 4.9.2 Development rates and reproduction The development and reproduction data for e ach individual copepod was entered into electronic data files and statistically analysed using Minitab (Ref 4). The data were tested for normality and homogeneity of variances, before appropriate procedures were applied to test for significant differences (p <0.05) between the DWC and the SC and subsequently between the pooled controls (when appropriate) and th e exposure concentrations. Figure 1 showsReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 16 (45) which statistical procedures were chosen based upon the normality and homogeneity of variances of the data. The development rate (in days) was calculated fro m nauplii to copepodite and from copepodite to adult. Individual development ra tes, were calculated as follows:

) 2 1 ( day 1

i i 

### x

−

= 

### where:

x : mean development rate per plate day i 

### : inspection day (day of development to next stage) 1 i



### : length of inspection interval (1 day in this study)

The differences between treatments fo r the number of offspring in the 1 st 

### and 2 nd 

### broods and the average number of nauplii per pair were analysed. Pairs were excluded from statistical analysis if a female mortality (both from exposur e or operator handling) occurred shortly after pairing. This is because a number of mortalities during the study were directly attributed to operator handling and this will have included females that could have been unaffected by the test substance and have been capable of reprodu ction. In addition, pairs that were incorrectly paired with 2 individuals of the same sex were also excluded from statistical analysis. The endpoints ‘number of successf ul pairings’; ‘times to 1 st 

### and 2 nd 

### broods’; and ‘time between broods’ were not analysed. This is because on a number of occasions during the study, animals were identified as being adult too early, leading to a number of pairs having to be reassigned to form new pairs at a later time. It is therefore difficult to distinguish between pairs that were unsuccessful for bi ological reasons and those that we re incorrectly matched. In addition, due to a delay in pairing (because copepods were initially paired incorrectly) or possibly due to stress of extra handling a la rge proportion of the pairs did not produce two broods during the study. As such, the data required to calculate these endpoints are not considered reliable. This misidentification of a dults was not considered to be dose dependent.
EC50, EC20 and EC10 values for the mean total brood size and the associated 95% confidence intervals were determined usin g the US EPA program ICPIN (version 2, June 1993), Ref 5.Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 17 (45) 

## 4.10 Validity Criteria The following validity criteria should be achie ved for the test to be considered valid.

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 microplate well plates survival should be lower than 70% at the end of the exposure period.
Dissolved Oxygen Concentration : Expressed as % of the air saturation value. Percentage The dissolved oxygen concentration must have been at least 70% of the air saturation value (ASV) throughout the exposure period. Temperature : Low variation in exposure temperature. needed o C The temperature should not vary more than 4 o C during the entire test with a 36 day mean temperature of 25 ± 2 o C.

pH : Low variation in exposure pH needed. pH units pH must not vary more than 0.5 units from the average control pH with a 36 day mean pH of 8.0 ± 0.4. Conductivity/salinity : Low variation in conductivity/salinity needed. % Conductivity/salinity must not vary more than 10% from the control start value of 30‰

Sex Ratio: Number The sex ratio in F0 controls (females/males) should on the average range between 0.4 to 2.0.

Viable Offspring : Number The average number of viable offspring produced through two clutches in the control(s) should not be less than 8 individuals.

Average Number of Days to the First Clutch Number The average number of days to the first clutch in the control(s) should at a room temperature of 25oC not exceed the number of days representing 70% of the total exposure period or 25 days, whichever is less.



## 5 RESULTS
5.1 Chemical data The measured concentrations in the ‘on’ and ‘off’ test solutions are given in Table 1. It details the individual means and ranges for the ‘on’ and ‘off’ test solutions together with the overall mean measured concentrations. The limit of qu antification of lindane in this study was 0.5 μg/L. Typical chromatograms are shown in Appendix 2, Figure 1. All analytical results are quoted to 2 significant figures and percentages to the nearest integer. The mean measured concentrations in the ‘on’ test solutions ranged from 66 to 216 % of the nominal value (although most of the high variability is at the lowest test concentration). The ‘off’ values ranged from 30 to 96 % of nominal values. The analysis of the triplicate samples, taken from the nominal 5 μg/L solutions s howed good consistency (Table 1). The time weighted mean measured concentration of linda ne was calculated as described in Appendix 3. The time weighted mean measured concentrations ranged between 72 and 84% of the nominal test concentration.Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 18 (45) Based on these analytical results the time wei ghted mean measured lindane concentrations were used for the calculation and reporting of the results. On Day 6 of the test the solvent control plate 1 was accidently dosed with the 20 μg/L test solution. This was noted immediately and a trip le water change with solvent control test solution was performed to remove any test compound (within 45 minutes of the addition of the incorrect test solution). The ‘off’ concentration of lindane in the solvent control plate 1 was analysed separately to plates 2 and 3 (which were correctly dosed) on Day 9 of the study. The concentration of lindane measured on Day 9 was below the limit of detection of 0.5 μg/L in both plate 1 and in plates 2 and 3 combined (Table 1). No statistically significant differences occurred between the solvent control replicates in any of the endpoints measured, therefore this is not considered to have affected the validity of the replicate and it was used for all statistical analyses. 

## 5.2 Biological data The Lowest Observed Effect Concentration (LOEC) is defined as the lowest tested concentration at which the test substance is observed to have a statistically significant effect on reproduction and parent mortalit y (p <0.05) when compared with the control. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. The No Observed Effect Concentration (NOEC) is defined as the test concentration immediately below the LOEC which, when compared with the control, has no statistically significant effect (p <0.05). It should be noted that the differences between an adult copepod and a large copepodite are very subtle (especially between the adult female and copepodite as the swollen geniculate segments are not there to help identification) and are not easy to distinguish. Moreover, identification between these stages/sexes was hampered due to inexperience with the test organism, due to the organism moving and the edge effects of the well obscuring the operator’s view of the animal. In this study there have been many misidentifications of sex and development stage due to these reasons. Since these issues did not occur until attemp ts to identify sex and pairing on day 15, the endpoints of ‘survival to day 15’ and ‘developm ent to copepodite’ are not thought to have been affected. However, the remaining endpoi nts that use data from day 16 onwards are not thought to be as reliable. A ll reasonable attempts have been made to identify these mistakes and correct them before the analysis of the data. However, due to these reasons caution should be applied when interpreting these results. 5.2.1 Observation of mortalities and symptoms of toxicity The cumulative mortality of the copepods to Day 15 and Day 36 is shown in Table 2. The survival in the control up to Day 15 was 80%, which exceeds the validity criteria of 75% for the test as outlined in the draft OECD guideline. After Day 15 there were a number of mortalities that occurred while pa iring the adult copepods that we re directly attributed to operator handling of the organisms. The survival in the controls by Day 36 (the end of theReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 19 (45) test) was 54% with 51% of these deaths being due to operator handling. All mortalities (regardless of test concentration) that were di rectly attributed to handling were excluded from the analysis. Fisher’s exact test was carried out to check for a significant difference between the control and solvent control. No significant difference (p <0.05) was found, therefore the controls were pooled. Fisher’s exact tests we re then used to compare the pooled controls and each test concentration. There was a significant effect (p <0.05) on survival at 17 μg/L lindane compared with the pooled controls at Day 15; and at 11 and 17 μg/L on Day 36. Therefore, the NOEC for survival on Day 15 was 11 μg/L and the LOEC 17 μg/L. The NOEC for survival on Day 36 was 7.2 μg/L and the LOEC 11 μg/L.
The LC50, 25 and 10 were calculated as describe d in section 4.9.1 and the results are given in the table below:
Day 15 Day 36 Lindane (μg/L) 95% confidence limits Lindane (μg/L) 95% confidence limits LC10 1.4 * 0.51 * LC25 11 * 1.9 * LC50 120 * 8.6 * * The results were estimated by extrapolation and as such, reliable confidence limits were unable to be calculated. Results given to 2 significant figures No unusual behaviour of the copepods was recorded at any test concentration. 5.2.2 Development rate to copepodite and to adult All data were included in calculating average time to achieve a particular development stage, irrespective of whether the copepod di ed after achieving that stage. A summary of the mean time taken in days fo r the copepods to reach the first copepodite stage, to reach adult and the development rate s for each are shown in Table 3. The time taken to develop to copepodite stage for each indi vidual copepod is shown in Table 4 and the time taken to develop from copepodite to adult is shown for each individual copepod in Table 5. The normality and homogeneity of variances, betw een replicates and treatments were assessed and the appropriate statistical tests were applied according to figure 1. No significant difference (p <0.05) in the development rate from nauplii to copepodite or the development rate from copepodite to adult between the control and the solvent control was found, therefore the DWC and SC treatme nts were pooled. There was a significant (p <0.05) decrease in th e development rate from nauplii to copepodite at 7.2, 11 and 17 μg/L compared with the pooled controls. There were significant decreasesReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 20 (45) (p <0.05) in development rate from copepodite to adult at the 3.6, 7.2, 11 and 17 μg/L lindane concentrations compared with the controls. Therefore, the NOEC for development rate fr om nauplii to copepodite was 3.6 μg/L and the LOEC was 7.2 μg/L. The NOEC for development rate from copepodite to adult was 2.1μg/L and the LOEC was 3.6 μg/L. 5.2.3 Sex Ratio The number of male and female A. tenuiremis and the ratio of males to females at each test concentration are shown in Table 6. The ratio of females to males in the DWC is 0.73 and in the solvent control is 0.95. These are both within the range 0.4 to 2.0 specified in the draft OECD guideline (Ref 1) and would fulfil the validity criteria for the test. This statement, however, does assume that there was no sexual differentiation in the number mortalities caused by handling which is not known. Fisher’s exact test was carried out to detect a significant difference between the control and solvent control. No significant difference was found therefore the controls were pooled. Fisher’s exact tests were then used to compare the pooled contro ls and each test concentration. There were no significant differences (p <0.05) observed in any of the test concentrations compared with the pooled controls. Therefore, the NOEC for sex ratio was 17 μg/L and the LOEC was > 17 μg/L. 5.2.4 Reproduction Due to the aforementioned issues with the id entification of when a copepodite became an adult copepod and the dete rmination of sex, there were occu rrences where pairs consisted of two adults of the same sex and/ or one copepodite and one adult. Attempts were made to repair the organisms once they were confirmed to be an adult and their sex identified correctly. Moreover, several animals were lost or killed accidentally during the pairing process reducing the number of organisms for pairing. The da ta have been analysed based on the study directors’ interpretation of th e results. However, the poor re liability and problems regarding the quality of the data have been acknowledged and for these reasons, particular care should be taken when drawing conclusions from these reproduction data. For each pair, the mean number of offspring in the 1 st 

### and 2 nd 

### broods and average number of offspring per brood is shown for each replicate in Table 7. The number of offspring per pair is shown in Table 8, with the number of broods produced by each given in brackets. The mean number of offspring per brood in th e dilution water control was 3.5 for the 1 st 

### brood and 2.6 for the second brood. This is less than the mean brood size of >8 offspring per brood stipulated in the draft OECD guideline (Ref 1). The average number of days to produce the first clutch was 25.6 days which also exceeds the draft guideline criteria of 25 days. This reflects the difficulty that the operators had w ith pairing the copepods. The pairing process isReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 21 (45) very labour intensive and there were some mis identifications of ma les and females which meant the animals needed to be paired again. This also resulted in excessive handling of the animals which may have cau sed additional stress. The normality and homogeneity of variances, between replicates and treatments were assessed and the appropriate statistical tests were applied according to figure 1. No significant differences in number of offspring in the 1 st 

### or 2 nd 

### broods or average number of offspring per brood between the control and the solvent cont rol were found; therefore the DWC and SC were pooled. No significant effects (p <0.05) were f ound between the pooled controls and test concentrations in the number of offspring in the 1 st 

### brood. Therefore, the NOEC for number of offspring in the 1 st 

### brood was 17 μg/L and the LOEC was > 17 μg/L. No significant effects (p <0.05) were f ound between the pooled controls and test concentrations in the number of offspring in the 2 nd 

### brood. Therefore, the NOEC for number of offspring in the 2 nd 

### brood was 17 μg/L and the LOEC was > 17 μg/L. No significant effects (p <0.05) were f ound between the pooled controls and test concentrations in the average number of offspring per brood. Therefore, the NOEC for average number of offspring per brood was 17 μg/L and the LOEC was > 17 μg/L. The EC50, 20 and 10 were calculated using the tota l number of offspring per pair. The results are given in the table below:

Lindane conc. (μg/L) 95% Cl (μg/L) EC10 2.4 0.090 – 2.7 EC20 2.7 0.68 – 3.4 EC50 7.8 * * reliable confidence limits were unable to be calculated 

## 5.3 Overall observed effect concentrations Taking all biological parameters into ac count, based on time weighted mean measured concentrations, the overall NOEC of lindane was 2.1 μg/L and the LOEC was 3.6 μg/L. 

## 5.4 Physical and chemical data A summary of the water quality parameters of the test solutions measured during the study are shown in Table 9. Overall, the measured dissolved oxygen concentrations ranged from 6.60 – 7.63 mg/L, equivalent to a range of 92 to 107% of the ai r saturation value for seawater at a nominal temperature of 25°C (where 100% saturation is equal to 7.15 mg/L, Ref 6), this fulfils the validity criteria of >70% specified in the draft OECD guideline. The pH values ranged from 7.9 to 8.4 which are within the levels (8.0 ± 0.4) specified in the draft OECD guideline.Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 22 (45) The salinity of the seawater used to prepare e ach batch of test solutions ranged from 28.0 to 32.6‰ and remained within the limits 30 ± 3‰ specified in the draft OECD guideline. The temperature was recorded daily by thermometer and ranged from 22.7 to 24.6°C. The temperature did not fluctuate by more than 2.0°C over the 36 day duration of the test; however, on one occasion the temperature did fall below the 25.0 ± 2 o 

### C specified in the draft OECD test guideline but this is not expected to have affected the outcome of the study. Water quality parameters for the recent seawater batch to those used in this study are given in Table 10.



## 6 CONCLUSION

Endpoint No observed effect

conc Lowest observed effect

conc Day 15 survival 11 μg/L 17 μg/L
Day 36 survival 7.2 μg/L 11 μg/L

Development (to copepodite) 3.6 μg/L 7.2 μg/L Development

(to adult) 2.1 μg/L * 3.6 μg/L *

Time to 1 st brood Not Determined Not Determined

Time to 2 nd brood Not Determined Not Determined

Interbrood duration Not Determined Not Determined Number of offspring – 1 st brood 17 μg/L >17 μg/L Number of offspring – 2 nd brood 17 μg/L >17 μg/L Average number of offspring 17 μg/L >17 μg/L

Number of successful mating pairs Not Determined Not Determined

Overall 3.6 μg/L 7.2 μg/LReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 23 (45) * The endpoint for development time to adult is considered unreliable because of the misidentification of copepodites as adults as discussed in the report and therefore the overall NOEC and LOEC for this study has been based on devel opment to copepodite.



## 7 REFERENCES 1 OECD Draft Guidelines for the Testing of Chemicals. Harpacticoid Copepod Development and Repr oduction Test with Amphiascus tenuiremis . Post June 2009. 2 Finney D J, Latscha R, Bennett B M and Hsu P (1963). Tables for testing significance in a 2x2 contingency table. Ca mbridge University Press, London. 3 Stephan C E (1977). Methods for calcula ting an LC50 In: Aquatic Toxicology and Hazard Evaluation. Mayer F L, Hamelink J L, Editors. Proceedings 1st Annual Symposium on Aquatic Toxicology, ASTM, 1977, STP 634 65-8. 4 Minitab (Release 16.1.1.0) - Minitab Inc. Quality Plaza, 1829 Pine Hall Rd, State College PA 16801-3008, USA. 5 US EPA (1988). Short-term methods for estima ting the chronic toxicity of effluents and receiving waters to marine and estuarine organisms. US Environmental Protection Agency, Office of Research a nd Development. EPA-600/4-87/028. 6 Gardiner J and Hughes J (1981). Oxygen solubility tables. Water research centre. Process Evaluation Report, 44-45.Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 24 (45) Table 1 Analytical results

Nominal conc of lindane Day Measured conc of lindane

(μg/L) Percentage of nominal value Overall time weight mean measured

concentration (μg/L) On Off On Off (μg/L) Time weighted mean measured conc as percentage of nominal 0 <0.50 - 3 <0.50 - 6 <0.50 - 9 <0.50 - 15 <0.50 - 18 <0.50 - 21 <0.50 - 24 <0.50 - 27 <0.50 - 30 <0.50 - 33 <0.50 - Control

36 <0.50 - N/A N/A

0 <0.50 - 3 <0.50 - 6 <0.50 - 9 # <0.50 - 15 <0.50 - 18 <0.50 - 21 <0.50 - 24 <0.50 - 27 <0.50 - 30 <0.50 - 33 <0.50 - Solvent control

36 <0.50 - N/A N/A

0 1.9 76 3 0.83 33 6 3.7 148 9 1.8 72 15 5.4 216 18 2.4 96 21 2.5 100 24 1.2 48 27 2.1 84 30 1.5 60 33 2.3 92 2.5

36 0.80 32 2.1 84

0 3.4* 68 3 1.5 30 6 5.7* 114 9 3.3 66 15 5.5* 110 18 2.4 48 21 5.1* 102 24 2.4 48 27 5.0* 100 30 3.3 66 33 4.3* 86 5.0

36 3.2 64 3.6 73Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 25 (45) Table 1 Analytical results continued Nominal conc of lindane Day Measured conc of lindane

(μg/L) Percentage of nominal value Overall time weight mean measured

concentration (μg/L) On Off On Off (μg/L) Time weighted mean measured conc as percentage of nominal 0 6.8 68 3 3.3 33 6 9.8 98 9 5.9 59 15 13 130 18 5.4 54 21 9.9 99 24 4.6 46 27 10 100 30 7.8 78 33 8.7 87 10

36 4.0 40 7.2 72

0 11 73 3 4.5 30 6 14 93 9 7.3 49 15 17 113 18 8.8 59 21 14 93 24 6.4 43 27 16 107 30 12 80 33 15 100 15

36 13 87 11 75

0 21 105 3 8.3 42 6 21 105 9 12 60 15 21 105 18 12 60 21 20 100 24 11 55 27 23 115 30 18 90 33 20 100 20

36 18 90 17 84



### # Day 9 solvent control analysis. Plate 1 was analysed separately to plates 2 & 3 and both showed the lindane concentr ation to be <0.5 μg/L. * mean of triplicate valuesReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 26 (45) Table 1 Analytical results continued Triplicate analyses

Nominal conc of lindane Triplicate values Mean ( ± SD)
(μg/L) Exposure Day (μg/L) (μg/L)

0 3.4 3.3 3.6 3.4 (0.15) 6 5.9 5.7 5.4 5.7 (0.16) 15 5.3 6.0 5.3 5.5 (0.36) 21 5.0 4.9 5.4 5.1 (0.25) 27 5.1 5.1 4.9 5.0 (0.10) 5.0

33 4.3 4.2 4.3 4.3 (0.05) 

### The limit of quantification of li ndane in the control throughout this study was 0.50 μg/L All analytical and calculated results given to 2 significant figures and percentages to the nearest integerReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 27 (45) Table 2 Cumulative mortality Time weighted mean measured co ncentration of lindane (μg/L) Exposure Day Control SC 2.1 3.6 7.2 11 17

0 0 0 0 0 0 0 0 1 0 1 1 0 1 0 0 2 1 3 1 1 2 3 2 3 1 3 1 3 3 3 2 4 1 4 1 4 3 4 2 5 2 5 2 4 3 4 3 6 5 6 3 6 6 4 3 7 7 7 10 6 7 5 10 8 12 8 16 10 9 6 12 9 13 8 16 10 9 8 16 10 15 8 19 11 10 9 18 11 16 12 20 11 12 9 23 12 16 12 20 11 12 11 34 13 16 12 20 11 12 12 35 14 16 12 20 11 12 12 36 15 18 12 20 11 12 14 39 16 24 14 21 13 14 17 42 17 24 15 25 13 14 18 56 18 25 15 26 14 14 19 56 19 25 19 29 17 15 23 58 20 27 21 29 17 17 23 59 21 29 22 31 18 17 25 59 22 31 23 32 20 17 25 59 23 32 24 33 21 22 27 60 24 34 25 34 24 25 28 61 25 36 31 35 27 27 31 63 26 36 31 36 29 28 31 63 27 36 33 36 32 31 31 65 28 36 33 36 33 33 34 65 29 36 33 36 33 33 34 65 30 39 34 38 38 35 38 65 31 41 34 38 41 36 39 66 32 41 34 38 41 36 39 66 33 41 35 38 44 40 43 68 34 41 36 38 47 41 46 68 35 41 36 38 47 42 48 68 36 41 36 38 47 42 48 68Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 28 (45) Table 2 continued Time weighted mean measured concentration of lindane (μg/L) Control SC 2.1 3.6 7.2 11 17 n 90 90 90 90 90 90 90 Total alive 49 54 52 44 48 42 22 % mortality due to natural

/ chemical exposure Day 15 20 12 20 11 13 13 43*

% mortality due to natural

/ chemical exposure Day 36 22 19 26 29 30 33* 73*

% of total mortalities

due to handling at

Day 15 0 1 2 1 0 2 0

% of total mortalities

due to handling at

Day 36 23 21 17 23 17 20 2

Total number

of sexual

pairs 24 27 19 22 16 13 2



### Percentages given to the nearest integer * Indicates a significant increase (p<0.05) in survival compared to the controls No mortalities that could be directly attributed to operator handling were included in the statistical analysis of the resultsReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 29 (45) Table 3 Summary data - development to copepodite and adult
Day turned to

copepodite Day turned to adult Development rate Time weighted mean measured concentration of lindane (μg/L) Rep

Mean (Days) SD Mean (Days) SD Rate from nauplii to copepodite Rate from copepodite

to adult 1 10.0 2.62 17.4 2.89 0.111 0.156 2 9.79 2.89 16.8 2.81 0.115 0.171 3 10.9 3.22 17.9 3.27 0.104 0.175 Control

mean 10.2 2.91 17.6 2.99 0.110 0.170 1 10.2 2.36 17.0 2.52 0.109 0.169 2 9.50 2.00 16.0 1.46 0.116 0.173 3 9.11 1.91 16.3 0.87 0.121 0.148 Solvent control

mean 9.59 2.09 16.4 1.62 0.120 0.160 1 10.4 2.52 18.4 2.81 0.105 0.143 2 9.52 1.72 17.9 2.26 0.114 0.135 3 9.89 1.53 17.4 1.69 0.109 0.167 2.1

mean 9.94 1.92 16.0 2.26 0.110 0.149 1 10.2 1.87 17.8 2.40 0.107 0.148 2 10.6 2.16 19.6 2.70 0.103 0.125 3 9.37 1.28 17.2 1.59 0.115 0.141 3.6

mean 10.1 1.77 18.2 2.23 0.110 0.140* 1 11.6 3.25 19.7 4.08 0.096 0.134 2 10.4 1.32 19.0 2.47 0.103 0.128 3 12.4 4.12 21.2 5.32 0.092 0.120 7.2

mean 11.5 2.90 20.0 3.96 0.100* 0.130* 1 11.5 2.63 21.4 5.36 0.0963 0.120 2 12.3 3.69 22.0 6.09 0.0933 0.114 3 14.8 5.79 26.0 4.99 0.0797 0.0887 11

mean 12.9 4.04 23.1 5.48 0.0898* 0.107* 1 15.3 4.40 23.3 5.75 0.0719 0.135 2 16.1 3.53 26.8 4.02 0.0668 0.0974 3 17.5 4.87 27.0 5.39 0.0640 0.101 17

mean 16.3 4.27 25.7 5.05 0.0676* 0.110* 

### Results given to 3 significant figures * Indicates a significant increase (p<0.05) in development times compared to the controlsReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 30 (45) Table 4 Number of days taken to develop from nauplii to copepodite Time weighted mean measured co ncentration of lindane (μg/L) Plate Rep Control Solvent control 2.1 3.6 7.2 11 17

1 M M 19 11 14 10 M 2 9 M M 10 14 M M 3 8 10 M 4 M 12 M 4 M 7 M M 9 11 M 5 M 17 M M 9 12 17 6 9 9 9 9 M 12 M 7 8 7 M 11 M M M 8 8 M M 13 20 M 11 9 M 13 9 8 11 9 M 10 10 12 10 M 8 16 M 11 8 M 9 11 9 - 12 12 16 9 M M 11 15 M 13 9 M M 9 9 17 M 14 M 12 11 11 9 13 M 15 9 11 11 14 11 12 M 16 9 10 9 11 11 12 M 17 16 8 9 10 9 11 M 18 16 11 M 9 11 M 15 19 8 10 8 9 20 9 M 20 8 8 11 11 10 M 17 21 M 9 M 14 M 11 M 22 10 10 8 9 13 9 13 23 10 8 11 8 12 8 26 24 11 11 13 9 17 8 M 25 10 15 9 11 11 9 13 26 M 8 M 8 M 13 17 27 8 11 12 7 13 15 M 28 9 9 9 10 13 8 12 29 11 10 12 9 8 M M 1

30 M 9 9 13 10 M M Mean 10 10 10 10 12 11 15 Std Dev 2.6 2.4 2.5 1.9 3.3 2.6 4.4Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 31 (45) Table 4 continued Time weighted mean measured co ncentration of lindane (μg/L)

Plate Rep Control Solvent control 2.1 3.6 7.2 11 17

1 M M 8 M 12 19 M 2 9 8 9 9 9 11 M 3 11 9 M 10 12 13 M 4 8 M 10 10 11 10 M 5 9 M 9 16 9 13 M 6 M 12 10 10 M 18 M 7 12 11 M 11 M 17 M 8 9 9 M 9 10 17 15 9 7 8 12 10 10 9 M 10 11 14 M 13 10 15 12 11 10 13 M 10 M M 17 12 M 13 M 10 14 19 M 13 M 8 12 11 10 15 13 14 7 M 10 9 11 M M 15 8 12 14 8 11 11 M 16 21 9 M 9 M 10 15 17 10 M 9 10 M 13 M 18 10 11 10 17 10 9 M 19 10 8 9 10 12 10 17 20 12 8 12 10 10 15 M 21 8 8 M M 9 8 M 22 9 7 10 8 9 17 M 23 M 8 M 13 8 9 12 24 9 8 8 12 10 13 17 25 13 M 8 M 10 9 M 26 8 9 8 11 11 9 M 27 8 8 8 11 10 10 25 28 M 8 8 9 11 7 19 29 9 10 8 9 9 8 16 2

30 7 9 8 M 12 9 15 Mean 9.8 9.5 9.5 11 10 12 16 Std Dev 2.9 2.0 1.7 2.2 1.3 3.7 3.5Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 32 (45) Table 4 continued Time weighted mean measured co ncentration of lindane (μg/L)

Plate Rep Control Solvent control 2.1 3.6 7.2 11 17

1 15 12 10 9 M 9 M 2 18 10 12 9 12 14 M 3 13 9 9 9 16 M M 4 16 9 M 8 16 M M 5 11 8 9 M 10 11 M 6 10 7 12 12 M 14 M 7 17 8 14 M 12 M 25 8 8 M 8 9 13 17 M 9 12 9 9 10 M M 20 10 8 9 9 13 16 M M 11 M 8 M 9 22 15 M 12 16 10 11 10 17 11 16 13 8 9 8 8 14 15 M 14 10 9 12 10 14 13 M 15 8 8 9 11 8 26 M 16 8 8 9 10 24 9 M 17 8 7 M 8 10 8 M 18 11 8 9 9 10 13 21 19 10 7 11 8 9 20 M 20 10 11 11 10 12 9 10 21 M 9 9 10 M 12 M 22 10 10 10 9 9 31 M 23 9 8 9 7 12 10 M 24 7 16 9 8 10 M 12 25 M M 12 10 9 21 M 26 M 7 9 9 9 13 17 27 12 9 9 9 9 17 M 28 8 8 8 M 8 9 19 29 9 12 9 10 13 22 M 3

30 M 10 11 9 9 17 M Mean 11 9.1 9.9 9.4 12 15 18 Std Dev 3.2 1.9 1.5 1.3 4.1 5.8 4.9 

### M indicates mortality Results given to 2 significant figuresReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 33 (45) Table 5 Number of days taken to develop from copepodite to adult Time weighted mean measured co ncentration of lindane (μg/L) Plate Rep Control Solvent control 2.1 3.6 7.2 11 17

1 M M 7 8 14 M M 2 11 M M 6 10 M M 3 7 5 M M 8 11 M 4 M 8 M M 6 10 M 5 M 5 M M 10 10 8 6 7 6 7 7 M 8 M 7 11 12 M 8 M M M 8 7 M M 7 M M 6 9 M 5 7 8 6 9 M 10 6 8 9 M 10 9 M 11 7 M M 11 7 M M 12 5 6 M M 6 13 M 13 6 M M 7 8 16 M 14 M 5 9 9 9 10 M 15 10 9 9 5 7 M M 16 6 5 7 11 7 9 M 17 7 7 7 6 12 10 M 18 8 6 M 7 7 M M 19 7 5 11 7 10 M M 20 7 7 9 9 M M 15 21 M 6 10 M 7 M 22 6 5 8 7 9 6 5 23 5 7 6 8 9 7 M 24 10 9 4 7 10 M M 25 6 8 10 7 M 6 8 26 M 7 M 8 M M M 27 11 9 8 9 5 15 M 28 6 7 7 6 M 7 15 29 6 6 11 7 8 M M 1

30 M 8 7 5 8 M M Mean 7.4 6.8 7.9 7.6 8.5 9.6 9.5 Std Dev 1.9 1.7 1.8 1.6 2.1 2.9 4.4Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 34 (45) Table 5 continued Time weighted mean measured co ncentration of lindane (μg/L)

Plate Rep Control Solvent control 2.1 3.6 7.2 11 17

1 M M 8 M 9 14 M 2 6 7 7 13 10 13 M 3 8 6 M M 10 11 M 4 7 M 9 6 8 13 M 5 7 M 7 6 9 11 M 6 M 5 7 10 M M M 7 3 6 M 9 M M M 8 10 6 M 11 11 16 9 9 8 7 5 11 11 6 M 10 4 M M 12 7 18 10 11 9 5 M 11 M M 11 12 M 5 M 7 11 M M 13 M 7 9 9 6 11 10 14 8 M 6 8 10 M M 15 7 5 7 12 9 M M 16 5 8 M 7 M 11 17 17 5 M 7 6 M 17 M 18 5 4 7 8 6 11 M 19 9 7 7 6 9 11 15 20 5 7 11 10 8 13 M 21 7 7 M M 7 7 M 22 6 8 10 8 7 M M 23 M 10 M 10 8 7 12 24 10 7 8 M 7 4 7 25 9 M 12 M 7 11 M 26 7 6 8 9 9 8 M 27 7 7 8 7 11 8 M 28 M 7 8 11 10 8 11 29 8 5 12 11 7 7 16 2

30 8 11 12 M 9 9 9 Mean 7.0 6.7 8.3 9.1 8.6 11 12 Std Dev 1.8 1.6 2.0 2.2 1.6 3.6 3.2Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 35 (45) Table 5 continued Time weighted mean measured co ncentration of lindane (μg/L)

Plate Rep Control Solvent control 2.1 3.6 7.2 11 17

1 7 5 6 7 M 13 M 2 6 6 6 7 6 9 M 3 3 7 7 8 8 M M 4 8 7 M 8 M M M 5 6 8 10 M 9 10 M 6 6 9 7 8 M 18 M 7 6 8 2 11 M 8 7 M 9 7 8 13 M 9 3 7 7 8 M M 8 10 7 7 8 10 M M M 11 M 8 8 12 11 M 12 8 8 5 6 11 12 8 13 7 7 9 9 9 18 M 14 5 7 4 8 18 9 M 15 7 8 7 7 8 M M 16 7 8 7 6 M 12 M 17 7 9 M 8 M M M 18 5 8 8 9 7 11 15 19 6 9 5 10 14 15 20 9 6 9 8 M M 14 21 M 7 8 7 M 10 M 22 6 6 9 9 8 M M 23 10 8 7 M 9 11 M 24 12 M 10 9 8 M 11 25 M M 10 6 9 11 M 26 M 9 7 7 7 10 M 27 8 7 8 M 7 15 M 28 12 8 9 M 9 12 M 29 7 8 8 7 14 M M 3

30 M 6 10 7 8 13 M Mean 7.0 7.4 7.5 7.8 9.5 12 11 Std Dev 2.2 1.1 1.9 1.1 2.9 2.6 3.3 

### M indicates mortality Results given to 2 significant figuresReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 36 (45) Table 6 Sex Ratio Time weighted mean measured concentration of lindane (μg/L) Females Males Sex ratio

Control 30 41 0.73 Solvent control 36 38 0.95 2.1 40 26 1.5 3.6 41 33 1.2 7.2 27 41 0.66 11 27 31 0.87 17 15 7 2.1 

### Results given to 2 significant figures No significant differences foundReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 37 (45) Table 7 Number of offspring per brood Time weighted mean measured concentration of lindane (μg/L) Rep Mean size 1 st brood Mean size 2 nd brood Mean total offspring per brood

1 3.8 2.8 4.0 2 3.3 2.0 2.8 3 3.4 2.9 4.2 Control

Mean 3.5 2.6 3.7 1 4.3 1.6 3.9 2 4.1 3.0 4.2 3 4.0 3.5 4.2 Solvent control

Mean 4.1 2.7 4.1 1 2.5 1.8 2.2 2 3.4 4.9 4.4 3 4.0 3.2 3.9 2.1

Mean 3.3 3.3 3.5 1 2.7 2.1 2.4 2 1.4 0.0 1.4 3 3.0 1.3 3.0 3.6

Mean 2.4 1.1 2.3 1 2.4 1.8 2.3 2 3.8 2.0 3.8 3 0.67 0.0 0.67 7.2

Mean 2.3 1.3 2.2 1 1.3 1.8 1.9 2 1.8 0.40 1.7 3 1.0 0.0 1.0 11

Mean 1.4 0.72 1.5 1 - - - 2 0.0 0.0 0.0 3 4.0 2.0 3.0 17

Mean 2.0 1.0 1.5 

### Results given to 2 significant figures There was no significant differences (p<0.05) in any reproductive endpoint compared to the controlsReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 38 (45) Table 8 Total offspring per mating pair

Time weighted mean measured concentration of lindane (μg/L)

Plate Pair Control Solvent control 2.1 3.6 7.2 11 17 1 9 (2) 6 (2) 7 (2) 3 (2) 19 (2) 0 (0) - 2 16 (2) 4 (1) 7 (2) 0 (0) 2 (1) 9 (2) - 3 8 (1) 5 (1) 12 (2) 0 (0) 0 (0) 3 (1) - 4 7 (2) 11 (2) 0 (0) 4 (2) 0 (0) 0 (0) - 5 0 (0) 15 (2) 0 (0) 4 (2) 0 (0) - - 6 0 (0) 0 (0) 0 (0) 4 (2) - - - 7 - 3 (1) - 19 (2) - - - 1

8 - 3 (1) - - - - - Mean 6.7 5.9 4.3 4.9 4.2 3.0 - 1 3 (1) 8 (2) 3 (1) 0 (0) 0 (0) 0 (0) 0 (0) 2 0 (0) 7 (2) 8 (2) 5 (1) 0 (0) 2 (1) - 3 6 (2) 12 (2) 9 (2) 0 (0) 11 (2) 0 (0) - 4 7 (2) 8 (2) 5 (2) 0 (0) 4 (1) 6 (2) - 5 0 (0) 9 (2) 9 (2) 0 (0) 2 (1) 0 (0) - 6 6 (2) 11 (1) 15 (2) 5 (1) 3 (1) 5 (1) - 7 13 (2) 7 (2) 9 (2) 0 (0) 7 (1) - - 8 7 (2) 1 (1) - - 17 (2) - - 9 - 0 (0) - - - - - 2

Mean 5.3 7.0 8.3 1.4 5.5 2.2 0.0 1 8 (2) 3 (1) 7 (2) 2 (1) 2 (1) 3 (1) 6 (2) 2 16 (2) 9 (2) 4 (2) 0 (0) 0 (0) 0 (0) - 3 0 (0) 6 (1) 10 (2) 0 (0) 0 (0) 0 (0) - 4 5 (1) 8 (2) 4 (1) 0 (0) - - - 5 10 (2) 8 (2) 10 (2) 7 (1) - - - 6 0 (0) 14 (2) 8 (2) 11 (2) - - - 7 12 (2) 6 (2) - 7 (2) - - - 8 3 (1) 8 (2) - 6 (1) - - - 9 4 (2) 5 (2) - - - - - 10 5 (1) 8 (2) - - - - - 3

Mean 7.0 7.5 7.2 4.1 0.67 1.0 6.0 

### Number of broods given in brackets Means calculated to 2 significant figuresReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 39 (45) Table 9 Summary of water quality measurements Dissolved oxygen
Minimum Maximum (mg/L) (mg/L) Time weighted mean me asured concentration of lindane (μg/L) On Off On Off Control 6.74 6.60 7.52 7.63 Solvent Control 6.98 6.86 7.41 7.63 17 6.84 6.84 7.56 7.44


### pH Minimum Maximum

Time weighted mean me asured concentration of lindane (μg/L) On Off On Off
Control 7.86 7.87 8.23 8.34 Solvent Control 7.95 8.04 8.23 8.16 17 7.97 7.94 8.23 8.39


### Temperature Minimum (°C) Maximum (°C) Additional Temperature Replicate 22.7 24.6 

### Media before preparation of test solutions

Seawater batch Study Day pH Dissolved oxygen (mg/L) Salinity 

### (‰)
Day 0 8.17 7.72 28 Day 3 8.10 7.11 29 Day 6 8.13 7.31 30.76 Day 9 8.07 7.28 29.54 Day 12 8.14 7.06 31.01 Day 15 8.04 7.45 31.56 Day 18 8.26 7.22 31.56 Day 21 8.17 7.18 31.06 Day 24 8.15 7.08 32.14 ASW06/05/2011

Day 27 8.12 7.19 32.55 Day 30 8.11 7.09 31.41 ASW 08/06/2011 Day 33 8.23 6.89 31.81Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 40 (45) Table 10 Dilution water parameters

Parameter Value

*pH 8.17 *Salinity 28 ppt Suspended Solids @105oC 9.50 mg/L Total organic carbon <1 mg/L Ammoniacal nitrogen as N <0.010 mg/L Highest organophosphorous pesticides <0.01 μg/L Highest organochlorine pesticides <0.1 μg/L Highest PCB <0.002 μg/L Aluminium <110 μg/L Arsenic 1.10 μg/L Boron 4.94 mg/L Cadmium <0.04 μg/L Chromium <0.5 μg/L Cobalt <10 μg/L Copper 3.99 μg/L Iron <100 μg/L Lead <0.04 μg/L Manganese <020 μg/L Mercury <0.010 μg/L Nickel 0.714 μg/L Silver <1 μg/L Zinc 1.44 μg/L
* Analyses conducted on seawater batch ASW 06.05.11 at Brixham Environmental Laboratory (BEL)
All samples (from seawater collected on 22 June 2011) sent to The Environment Agency (EA), National Laboratory Service (NLS), Olympia House, Gelderd Lane, Gelderd Road, Leeds, LS12 6DD, for analysis at NLS laboratories. The EA is a non-GLP compliant facility.Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 41 (45) Figure 1 Statistical analysis flow chart for development and reproduction

Exploratory Data Analysis Calculation of mean, standard deviations, CoV

Test for normality

in treatments

Test for normality

in replicates

Bartlett’s test (normal data) or Levene’s test (non-normal data)

for equal variances between treatments

Nested ANOVA + Dunnett’s test to determine

significant differences from control treatment, then 1-way ANOVA test on individual treatments to

determine significant differences between replicates within each treatment One-way ANOVA + Dunnett’s test to determine significant differences from control treatment, then Kruskal Wallis test on individual treatments to determine significant differences between replicates within each treatment Kruskal-Wallis test followed by series of Mann-Whitney tests to determine significant differences from control treatment, then 1-way

ANOVA test on individual treatments to

determine significant differences between replicates Kruskal-Wallis test followed by series of Mann-Whitney tests to determine significant differences from control treatment, then Kruskal Wallis test on individual treatments to determine significant differences between replicates within each treatment Bartlett’s test (normal data) or Levene’s test (non-normal data)

for equal variances between replicates Bartlett’s test (normal data) or Levene’s test (non-normal data)

for equal variances between replicates Treatment variances equal Treatment variances unequal

Replicate variances

equal Replicate variances

equal Replicate variances unequal Replicate variances unequalReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 42 (45) Appendix 1 Certificate of Analysis for lindaneReport No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 43 (45) Appendix 2: Analytical me thod for the determination of lindane in seawater Exactly 5.0 mL of the aqueous samples were taken by pipette and transferred to a 10 mL glass tube and exactly 0.50 mL of hexane containi ng 0.10 mg/L of phenanthrene-d10, used as an internal standard, was added. The sample was then shaken manually for 2.0 mins and allowed to settle. An aliquot of the organic layer was tr ansferred to a glass autosampler vial insert and reserved for analysis by GC-MS using the conditions below. This procedure gives a x 10 concentration factor and an internal standard concentration of 0.10 mg/L. On each day that samples were prepared, the sample extraction procedure was evaluated by fortifying triplicate 5.0 mL aliquots of control media with 50 μL of a 1.0 mg/L stock solution of lindane prepared in acetone, or 20 μL of a 2.5 mg/L stock solution of lindane prepared in acetone
. These 10 μg/L fortification samples were then taken through the analytical procedure described above. The sample extr action procedure was considered to afford nominal recoveries of lindane from sea wate r and therefore no corrections for extraction efficiency were employed. The sample extracts were analysed by GC-MS a nd quantified against sta ndards of lindane in hexane using phenanthrene-d10 as an internal standard. Column 30 m x 0.25 mm (id) 0.25 μ m DB-5MS Temperature profile Ramp ( o 

### C/min) Temperature ( o 

### C) Hold (mins) 70 1.0 15 290 5.0 Carrier gas Helium (1.0 mL/min) Injection Volume 2.0 μ L Injection mode Splitless Purge flow 100 mL/min Purge time 0.75 min Injector temperature 280 o 

### C Transfer temperature 275 o 

### C MS Source 230 o 

### C Solvent delay 5 mins Scan mode SIM Ions Internal standard m/ z 188.0 Test Substance m/z 181.2 Using the above conditions the retention time of the test substance was 11.2 mins and a 100 μg/L gave a response of 3,100 relative abundance. The retention time of the internal standard was 11.4 mins and a 0.10 mg/L concentration gave a response of 31,000 relative abundance.Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 44 (45) Appendix 2 figure 1 Typical Chromatograms (A) 100 μg/L standard of lindane in hexane

10.95 11.00 11.05 11.10 11.15 11. 20 11.25 11.30 11.35 11.40 11.45 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

Time--> A bundance

Ion 181.20 (180.90 to 181.90) : 11-0030B_SRN101_008.D\data.ms 11.150



### (B) Extract of a 10 μg/L sample of li ndane in sea water (x10 concentration)

10.60 10.70 10.80 10.90 11.00 11.10 11.20 11.30 11.40 11.50 11.60 11.70 200 400 600 800 1000 1200 1400 1600 1800 2000 2200

Time--> A bundance

Ion 181.20 (180.90 to 181.90): 11-0030B_SRN101_023.D\data.ms 11.146Report No BR0476/B Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure with individually contained animals)



### 45 (45) Appendix 3 Calculation of time-weighted mean

The time-weighted mean is calculated following ANNEX 6 of OECD 211 Test Guideline. The formula used to make the cal culation is detailed below.
Days is the number of days in the renewal period Conc 0 is the measured concentration at the start of each renewal period Conc 1 is the measured concentration at the end of each renewal period Ln(Conc 0) is the natural logarithm of Conc 0 Ln(Conc 1) is the natural logarithm of Conc 1

Area is the area under the exponential curve for each renewal period. It is calculated by:



### Days 1) (Conc Ln

- 0) (Conc Ln 1 Conc

- 0 Conc Area × =

The time-weighted mean ( TW Mean) is the Total Area divided by the Total Day