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 

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CONFIDENTIAL (CATEGORY B) 

Not to be photocopied or microfilmed. This document contains information confidential and 

trade secret to U.S. Environmental Protection Agency. It should not be released in any form 

to an outside party, nor should information contained 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 

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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 Money 
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 

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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 .....................................................................................................17
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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 

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5 RESULTS ..............................................................................................................17

5.1 Chemical data.........................................................................................................17

5.2 Biological data .......................................................................................................18

5.2.1 Observation of mortalities and symptoms of toxicity............................................18

5.2.2 Development rate to copepodite 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.......................................................................................................23
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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 

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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 develop 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 Analysis for lindane ..................................................................42

Appendix 2: Analytical method for the determination of lindane in seawater ..................43

Appendix 3 Calculation of time-weighted mean ................................................................45

The last page of this report is....................................................................................................45 
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Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure 

with individually contained animals) 

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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 ‘Harpacticoid copepod development and 

reproduction test with Amphiascus tenuiremis’ (Ref 1) 

Test species Amphiascus tenuiremis 

Source of organisms Laboratory cultures held at Brixham 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°C 
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Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure 

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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 1st brood Not Determined Not Determined 

Time to 2nd brood Not Determined Not Determined 

Interbrood 

duration 

Not Determined Not Determined 

Number of 

offspring – 1st

brood 

17 μg/L >17 μg/L 

Number of 

offspring – 2nd

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 

Results based on 

concentrations of 

lindane 

Comments 

* 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 

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.
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2 INTRODUCTION 

At the request of the U.S. Environmental Protection Agency, EPA East Building, 

1201 Constitutional Avenue, NW Room 4106-G, Mail Code 7203M, Washington, DC20004 

the chronic toxicity of lindane to the marine copepod Amphiascus tenuiremis was determined. 

This study was conducted based on the test method outlined in the draft OECD guideline, 

‘Harpacticoid copepod development and reproduction test with A. tenuiremis’ (Ref 1). The 

procedures used in this study were based on this guideline. 

The experimental start and completion dates were 06 May 2011 and 20 June 2011, 

respectively, and the study number was 11-0030/B. 

All original data, together 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 Sciences Laboratory, 1529 West 

Sequim Bay Road, Sequim WA 98382 at the request 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: 1α,2α,3β,4α,5α,6β-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 certificate of analysis dated May 2003 stated that 

the sample had a purity of 99.6 % (Appendix 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 subsamples 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 Environmental Laboratory. The copepods originated 

from laboratory cultures established by Dr Tom Chandler, Professor of Environmental Health 
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and Marine Sciences, University of South Carolina, 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 before 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 plates (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 nominal 25 ± 2°C by housing the test vessels in a 

temperature-controlled room. The microplate well plates were placed in ‘humidity boxes’ to 
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reduce evaporative losses. The potential for evaporative loss was measured prior to exposure 

by placing seawater filled microplate well plates in the humidity boxes for 72 hours and 

measuring the salinity change over this period. 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 controlled 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 lindane. 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 triethyleneglycol to give a nominal stock solution containing 

0.2 g/L lindane. The resulting stock solution was observed to be clear and colourless. 

New test solutions were prepared every three days 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 were 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 obtained as described in Section 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 wells of the plate were left empty. 

The positions of the microplate well plates were randomally allocated within the humidity 

chamber. Addition of nauplii, subsequent handling, loading, observation and feeding was 

performed in an irregular plate order. 

The copepods were observed daily and the survival and development from a first stage 

nauplius to a first stage copepodite and reproductively 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. 
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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 within 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 test 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 egg sacs. The numbers of successfully hatched 

nauplii (i.e., total viable clutch, or fecundity) were recorded by direct microscopic observation 

for the first and second successive clutches of eggs 

All mating pairs producing two viable 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 6th 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 104 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 using 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) resuspended in filtered, natural 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 additional feed. As all copepods received the same amount of 

food and all test concentrations were treated identically, this was not expected to have 

detrimentally influenced the overall outcome of the study. 
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4.7 Physical and chemical parameters 

The pH, dissolved oxygen and salinity of the 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 solutions 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 filled with water and 

placed inside the humidity chamber. 

4.8 Chemical analysis 

The concentrations of lindane in the test solutions 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 the concentration of lindane. Samples from the 

corresponding ‘off’ control and exposure concentrations were analysed on the following 

renewal day. Triplicate analyses were performed on the nominal 5.0 μg/L exposure 

concentration on six sampling occasions (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 copepods 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 their 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 each 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 the exposure concentrations. Figure 1 shows 
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with individually contained animals) 

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which statistical procedures were chosen based upon the normality and homogeneity of 

variances of the data. 

The development rate (in days) was calculated from nauplii to copepodite and from copepodite to 

adult. Individual development rates, were calculated as follows: 

day (1 2)

1

i i

x − =

where: 

x : mean development rate per plate 

dayi : inspection day (day of development to next stage) 

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

The differences between treatments for the number of offspring in the 1st and 2nd broods and 

the average number of nauplii per pair were analysed. Pairs were excluded from statistical 

analysis if a female mortality (both from exposure 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 reproduction. In addition, pairs that were incorrectly 

paired with 2 individuals of the same sex were also excluded from statistical analysis. 

The endpoints ‘number of successful pairings’; ‘times to 1st and 2nd 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 biological reasons and those that were 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 large 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 adults 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 using the US EPA program ICPIN (version 2, June 

1993), Ref 5. 
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4.10 Validity Criteria 

The following validity criteria should be achieved 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 quantification 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 showed good consistency (Table 1). The time 

weighted mean measured concentration of lindane was calculated as described in Appendix 3. 

The time weighted mean measured concentrations ranged between 72 and 84% of the nominal 

test concentration. 
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Based on these analytical results the time weighted 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 triple 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 mortality (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 attempts to identify sex and pairing on day 15, the 

endpoints of ‘survival to day 15’ and ‘development to copepodite’ are not thought to have 

been affected. However, the remaining endpoints that use data from day 16 onwards are not 

thought to be as reliable. All 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 pairing the adult copepods that were directly attributed to 

operator handling of the organisms. The survival in the controls by Day 36 (the end of the 
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Determination of the effect of lindane on the survival and reproduction of the marine copepod Amphiascus tenuiremis (semi-static procedure 

with individually contained animals) 

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test) was 54% with 51% of these deaths being due to operator handling. All mortalities 

(regardless of test concentration) that were directly 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 were 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 described 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 died after achieving that stage. 

A summary of the mean time taken in days for the copepods to reach the first copepodite 

stage, to reach adult and the development rates for each are shown in Table 3. The time taken 

to develop to copepodite stage for each individual 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, between 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 treatments were pooled. 

There was a significant (p <0.05) decrease in the development rate from nauplii to copepodite 

at 7.2, 11 and 17 μg/L compared with the pooled controls. There were significant decreases 
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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 from 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 controls 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 identification of when a copepodite became an 

adult copepod and the determination of sex, there were occurrences 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 data have been analysed based on the study 

directors’ interpretation of the results. However, the poor reliability 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 1st and 2nd 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 the dilution water control was 3.5 for the 1st 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 with pairing the copepods. The pairing process is 
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) 

21 (45) 

very labour intensive and there were some misidentifications of males and females which 

meant the animals needed to be paired again. This also resulted in excessive handling of the 

animals which may have caused 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 1st or 2nd broods or average number of offspring per 

brood between the control and the solvent control were found; therefore the DWC and SC 

were pooled. 

No significant effects (p <0.05) were found between the pooled controls and test 

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

of offspring in the 1st brood was 17 μg/L and the LOEC was > 17 μg/L. 

No significant effects (p <0.05) were found between the pooled controls and test 

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

of offspring in the 2nd brood was 17 μg/L and the LOEC was > 17 μg/L. 

No significant effects (p <0.05) were found 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 total 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 account, 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 air 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. 
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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 each 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 ± 2o

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 1st brood Not Determined Not Determined 

Time to 2nd brood Not Determined Not Determined 

Interbrood duration Not Determined Not Determined 

Number of offspring – 

1st brood 17 μg/L >17 μg/L 

Number of offspring – 

2nd 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 
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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 development to copepodite. 

7 REFERENCES 

1 OECD Draft Guidelines for the Testing of Chemicals. Harpacticoid Copepod 

Development and Reproduction 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. Cambridge University Press, London. 

3 Stephan C E (1977). Methods for calculating 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 estimating the chronic toxicity of effluents 

and receiving waters to marine and estuarine organisms. US Environmental Protection 

Agency, Office of Research and Development. EPA-600/4-87/028. 

6 Gardiner J and Hughes J (1981). Oxygen solubility tables. Water research centre. 

Process Evaluation Report, 44-45. 
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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 73 
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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 concentration to be <0.5 μg/L. 

* mean of triplicate values 
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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 lindane 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 integer 
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with individually contained animals) 

27 (45) 

Table 2 Cumulative mortality 

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

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 68 
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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 results 
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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 

Time weighted Day turned to adult Development rate 

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 controls 
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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 concentration 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 4.4 
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with individually contained animals) 

31 (45) 

Table 4 continued 

Time weighted mean measured concentration 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 3.5 
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with individually contained animals) 

32 (45) 

Table 4 continued 

Time weighted mean measured concentration 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 figures 
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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 concentration 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 4.4 
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with individually contained animals) 

34 (45) 

Table 5 continued 

Time weighted mean measured concentration 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 3.2 
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with individually contained animals) 

35 (45) 

Table 5 continued 

Time weighted mean measured concentration 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 figures 
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) 

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 found 
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) 

37 (45) 

Table 7 Number of offspring per brood 

Time weighted 

mean measured 

concentration of 

lindane (μg/L) 

Rep 

Mean size 1st

brood 

Mean size 

2nd 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 

controls 
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) 

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 figures
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) 

39 (45) 

Table 9 Summary of water quality measurements 

Dissolved oxygen 

Minimum Maximum 

(mg/L) (mg/L) Time weighted mean measured 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 measured 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 31.81 
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) 

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 ASW06.05.11 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

unequal
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) 

42 (45) 

Appendix 1 Certificate of Analysis for lindane 
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) 

43 (45) 

Appendix 2: Analytical method 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 containing 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 transferred 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 extraction procedure was considered to afford 

nominal recoveries of lindane from sea water and therefore no corrections for extraction 

efficiency were employed. 

The sample extracts were analysed by GC-MS and quantified against standards of lindane in 

hexane using phenanthrene-d10 as an internal standard.

Column 30 m x 0.25 mm (id) 0.25 μm DB-5MS 

Temperature profile 

Ramp (o

C/min) Temperature (oC) Hold (mins)

70 1.0 

15 290 5.0 

Carrier gas Helium (1.0 mL/min) 

Injection Volume 2.0 μL 

Injection mode Splitless 

Purge flow 100 mL/min

Purge time 0.75 min 

Injector temperature 280o

C 

Transfer temperature 275oC 

MS Source 230o

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-->

Abundance

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 lindane 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-->

Abundance

Ion 181.20 (180.90 to 181.90): 11-0030B_SRN101_023.D\data.ms

11.146
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) 

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 calculation 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 Ln (Conc 0)- Ln (Conc1)

Conc 0 -Conc1 Area = ×

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