Brixham Environmental Laboratory study number 09-0025/C 

This report replaces BR0072/B – See Appendix 1 for details 

Guideline OECD 202 - Acute Immobilisation Test 

Performing laboratory Brixham Environmental Laboratory 

AstraZeneca UK Limited 

Brixham 

Devon, TQ5 8BA 

UK 

Sponsor Honeywel

Author M Webley 

July 2010 

Report No BR0255 Copy number 

Acute toxicity to Daphnia magna

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Acute toxicity to Daphnia magna

Brixham Environmental Laboratory study number : 09-0025/C 

CONFIDENTIAL (CATEGORY B) 

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

trade secret to 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 . 

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Acute toxicity to Daphnia magna

Brixham Environmental Laboratory study number : 09-0025/C 

CONTRIBUTORS 

Study Director 

Study Director for the in-life phase 

Test Facility 

M J Webley 

A L Macken 

Brixham Environmental Laboratory 

AstraZeneca UK Limited 

Brixham 

Devon, TQ5 8BA 

UK 

Principal Scientist; Ecotoxicology A L Macken 

Principal Scientist; Analytical Chemistry R I Cumming 

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Acute toxicity to Daphnia magna

Brixham Environmental Laboratory study number : 09-0025/C 

CONTENTS 

page 

TITLE PAGE.............................................................................................................................1

CONFIDENTIAL (CATEGORY B) ......................................................................................... 2

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT....................................3

QUALITY ASSURANCE STATEMENT ................................................................................4

CONTRIBUTORS.....................................................................................................................5

CONTENTS...............................................................................................................................6

1 SUMMARY .....................................................................................................................7

2 INTRODUCTION............................................................................................................8

3 MATERIALS ...................................................................................................................8

3.1 Test substance ....................................................................................................8

3.2 Test organism.....................................................................................................8

3.3 Dilution water ....................................................................................................9

4 TEST METHOD AND CONDITIONS ........................................................................... 9

4.1 Test procedure and apparatus.............................................................................9

4.2 Preparation of test solutions.............................................................................10

4.3 Chemical analysis ............................................................................................10

4.4 Observation of effects......................................................................................10

4.5 Physical and chemical parameters ...................................................................10

5 RESULTS.......................................................................................................................11

5.1 Analytical data .................................................................................................11

5.2 Biological data .................................................................................................11

5.3 Physical and chemical data ..............................................................................11

6 REFERENCES...............................................................................................................12

TABLE 1 Analytical results..........................................................................................13

TABLE 2 Daphnia magna response .............................................................................14

TABLE 3 Dissolved oxygen concentrations and pH values .........................................15

TABLE 4 Water quality analysis of routine parameters, metals, and pesticides in 

Daphnia magna media ...................................................................................................16

APPENDIX 1 Changes introduced in BR0255/B.......................................................... 17

APPENDIX 2 Dilution water........................................................................................18

APPENDIX 3 Analytical method for the determination of in aqueous samples19

APPENDIX 4 Study Protocol and Amendments....................................................................22

APPENDIX 5 GLP Certificate ...............................................................................................31

circulation ................................................................................................................................32

The last page of this report is...................................................................................................32 

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Acute toxicity to Daphnia magna

1 SUMMARY 

Sponsor Honeywell

Contact International telephone: 

Location of study, raw 

data and final report Brixham Environmental Laboratory, AstraZeneca UK Limited, 

Brixham, Devon, TQ5 8BA, UK 

Test Facility contact M J Webley International telephone: (44) 1803 884316 

Test substance 

common name 

Subject Acute toxicity (immobilisation) to Daphnia magna

Guideline OECD Guideline 202. Acute immobilisation test to Daphnia magna

(Ref 1). METI Guidelines for Testing of Chemicals. Test Guideline 

2-7-2-2, Daphnia spp (adult daphnids) acute immobilization studies 

(Ref 2). 

Test species Daphnia magna

Source of organisms Continuous laboratory cultures at Brixham Environmental 

Laboratory; parental stock 15 ± 1 days old

Stage of organisms at 

the initiation of study 

Less than 24 hours old

Exposure 

concentrations 

Control and nominal concentrations of 4.38, 8.75, 17.5, 35 and 

70 %. 

Length of test 48 hours 

Results Based on mean 

measured 

concentrations of 

Time

24 hrs 

48 hrs 

EC50

150 mg l-1 

82 mg l-1

95% Confidence 

intervals 

120 - 200 mg l-1

66 - 100 mg l-1

Calculation 

method 

Moving average angle 

Moving average angle

48 hour No Observed Effect Concentration (NOEC) = 26 mg l-1

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2 INTRODUCTION 

At the request of Honeywell

 the acute toxicity of to Daphnia magna was 

determined. 

The study was carried out under the study number 09-0025/C between 7 and 10 April 

2009. All original data, together with other relevant records, are filed in the Brixham 

Environmental Laboratory archive. 

The test procedure used in this study was based on the relevant OECD Guideline (Ref 1) 

and METI guideline (Ref 2). 

3 MATERIALS 

3.1 Test substance 

The test substance was supplied by Honeywell

 

Compound name: 

CAS number: 

Molecular structure: 

 

The test substance was sent to Brixham Environmental Laboratory on 6 April 2009 and 

on receipt was assigned the Brixham test substance number 09-0066. The test substance 

(Supplier Lot No. 41024-32) was supplied as a liquefied gas. 

A certificate of analysis (dated 10 April 2009) stated the sample had a purity of 99.97 

area % ( 

The sample was stored, at ambient temperature, in the container in which it was received 

until required for testing. 

3.2 Test organism 

The test organism was the freshwater crustacean, D. magna, obtained from continuous 

laboratory cultures held at Brixham Environmental Laboratory. 

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The stock cultures of D. magna were maintained in a reconstituted water medium, 

identical to the test dilution water, at a temperature of 20 ± 2 °C. The cultures were 

maintained in 2 l glass vessels with a working volume of 1.6 l. A photoperiod of 

16 hours light: 8 hours dark, with 20 minute transition periods was provided. 

The D. magna cultures were fed on a mixed algae diet of Chlorella vulgaris, strain 

CCAP 211/12 and Pseudokirchneriella subcapitata, strain ATCC 22662. The D. magna 

cultures were fed daily ad libitum depending on age and density of the culture. Culture 

conditions were such that the D. magna reproduction was by diploid parthenogenesis. 

D. magna < 24 hours old, obtained from a single culture vessel, were used for testing. 

The parent animals were 15 ± 1 days old and had been maintained with a twice weekly 

renewal of reconstituted water medium since birth. The test organisms and the culture 

from which they were obtained showed no evidence of disease before the test period. 

3.3 Dilution water 

The reconstituted water medium used for testing (and maintenance of stock cultures) was 

Elendt's M4 D. magna medium (Ref 4) and is described in Appendix 1. 

The pH, conductivity, alkalinity, total hardness, free and residual chlorine, ammoniacal 

nitrogen as N, solids-suspended at 105 °C, dissolved organic carbon (DOC) and 

chemical oxygen demand (COD) are determined for representative batches of dilution 

water used at the testing facility throughout the year, along with a range of metals, 

pesticides and PCBs. Results of these analyses for the batch of media used in this study 

are presented in Table 4.

4 TEST METHOD AND CONDITIONS 

4.1 Test procedure and apparatus 

The test vessels employed were glass bottles of 50 ml nominal capacity, filled 

completely, with airtight, disc/crimp closures. The positions of the treatments were 

randomly allocated within the test area. The nominal test solution temperature was 20 ±

1 °C, and a photoperiod of 16 hours light:8 hours dark, with 20 minute dusk and dawn 

transition periods, was provided. 

The test was initiated by the addition of five randomly selected D. magna, in a minimum 

volume of dilution water, to each test vessel. The test was run with 4 replicates of the 

control and each test treatment with 5 daphnids per replicate. Each treatment contained a 

total of 20 D. magna. 

The test solutions were not aerated and the D. magna were not fed during the course of 

the study. 

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4.2 Preparation of test solutions 

The study was run with a dilution water control (DWC) and nominal exposure 

concentrations of 4.38, 8.75, 17.5, 35, 70 %. 

All stock solutions of the test substance were prepared in dilution water. Each test 

solution was prepared in excess of that required for the replicate vessels, to allow for 

initial pH and dissolved oxygen (DO) measurements. The highest concentration (70 %) 

was prepared by bubbling through 3500 ml of media for approximately 

60 minutes via a sintered glass diffuser to obtain a saturated solution of the gas (100 %). 

This was then diluted to 70 % saturation with media that had been diffused with oxygen 

for 30 minutes. All other concentrations were prepared from the 70 % stock. The 

control consisted of dilution water only. All final solutions were observed to be clear 

and colourless and contained nutrients as specified in Appendix 1. Mixing, dilution and 

addition of solutions to test vessels was carried out with minimal agitation to maintain 

the volatile gaseous test substance in solution. 

4.3 Chemical analysis 

The concentration of in the test solutions was measured at 0 and 48 hours using 

Purge and Trap injection with Gas Chromatography and Mass Spectrometry detection. 

Samples were compared to standards of test substance of known concentration in water. 

The method is outlined in Appendix 2. 

At the start of the test, a quantity of each of the excess test solutions were placed in 

volatile bottles with sealed lids and taken away for analysis. At the end of the test, one 

replicate of the dilution water control and each of the test concentrations were sampled. 

4.4 Observation of effects 

An assessment of the response of the D. magna was made 24 and 48 hours after the 

commencement of the test. Each D. magna was viewed by eye and was defined as 

affected if showing no whole body movement, relative to the water, within a period of 

15 seconds even if movement of individual appendages was visible. D. magna so 

affected were termed immobile. Any other overt symptoms of toxicity were also 

recorded. 

The median effect concentration (EC50) was defined as the concentration resulting in 

50 % immobilisation of the D. magna in the time period specified. 

4.5 Physical and chemical parameters 

Measurements were undertaken at the beginning and end of the test for pH and DO 

concentration using calibrated meters. At the start of the test the DO concentration and 

pH of the dilution water control and test substance concentrations were measured using 

the excess solution remaining after filling the test vessels. At the end of the test, the DO 

and pH of one replicate of the dilution water control and all the exposure concentrations 

were measured. Dissolved oxygen and pH were measured in the top concentration after 

24 hours as 100 % mortality was observed. 

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Temperature values were determined daily, in an additional test vessel, using a mercuryin-glass thermometer calibrated to BS593. Hourly temperature measurements were also 

recorded automatically in the additional test vessel using an electronic recording system. 

5 RESULTS 

5.1 Analytical data 

The concentrations of determined in the test solutions are given in Table 1. The 

limit of quantification of in this study was 0.003 mg l-1. Typical chromatograms 

are shown in Appendix 2, Figure 1. All analytical values are quoted to two significant 

figures and values greater than 100 to the nearest whole number. As the initial 

concentration series was made on percentages of an unknown stock and on the basis of 

the analytical data mean measured concentrations were used for the calculation and 

reporting of results.

5.2 Biological data 

The numbers of D. magna immobilised after 24 and 48 hours are given in Table 2. 

The results obtained (based on the mean measured concentration of were: 

Time EC50 95% confidence interval Calculation method 

24 hour 150 mg l-1 120 - 200 mg l-1 Moving average angle 

48 hour 82 mg l-1 66 - 100 mg l-1 Moving average angle 

The EC100 is defined as the lowest test concentration that caused 100 % immobilisation 

within the period of the test, therefore, 

48 hour EC100 = 405 mg l-1

The No Observed Effect Concentration (NOEC) is defined as the highest tested 

concentration in which there was no observed effect on the D. magna within the period 

of the test, therefore, 

48 hour NOEC = 26 mg l-1 

There was no immobility observed in the dilution water control. 

No other symptoms of toxicity were observed. 

5.3 Physical and chemical data 

Dissolved oxygen concentrations ranged from 9.0 to 9.8 mg l-1 (99.2 to 108.0 % of the 

air saturation value (ASV) – Ref 4) and the pH values ranged from 7.8 to 8.3. Individual 

data are shown in Table 3. At 24 h 100 % immobilisation was observed at 405 mg l-1

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Acute toxicity to Daphnia magna

(70 % saturation). At this time pH and DO values were 7.8 and 10.4 mg l-1 (114.7 % of 

ASV) respectively. 

Temperature values taken at 0, 24 and 48 hours were 20.0, 20.4 and 20.4 oC, with the 

values recorded automatically over the test period remaining within 20.0 ± 1 oC. 

6 REFERENCES 

1 OECD Guidelines for the Testing of Chemicals. Test Guideline 202, Daphnia

sp., Acute Immobilisation Test. Adopted 4 April 2004. 

2 METI Guidelines for Testing of Chemicals. Test Guideline 2-7-2-2, Daphnia spp 

(adult daphnids) acute immobilization studies. 

3 Elendt B P and Bias W R (1990). Trace Nutrient Deficiency in Daphnia magna 

Cultured in Standard Medium for Toxicity Testing. Effects of the Optimisation 

of Culture Conditions on Life History Parameters of D. magna. Water Research, 

24, 1157-1167. 

4 National Institute of Oceanography of Great Britain and UNESCO (1973). 

International Oceanographic Tables, 2. 

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TABLE 1 Analytical results 

Measured conc of 

(mg l-1)

a 

Nominal conc of 

(%) 0 hour 48 hour

Mean 

measured 

conca 

(mg l-1) 

Dilution water control < 0.003 < 0.003 - 

4.38 33 20 27 

8.75 39 37 38 

17.5 95 86 91 

35 180 170 175 

70 480 330 405 

a

All analytical measurements are quoted to 2 significant figures and values greater than 100 to the 

nearest whole number. 

The limit of quantification in the study was 0.003 mg l-1 

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TABLE 2 Daphnia magna response 

Time Mean 

measured 

conc of 

Number 

immobilised 

per replicate 

Total 

number 

tested 

Total 

number 

immobilised 

Percentage 

immobilised 

(hour) (mg l-1)

a A B C D 

DWC 0 0 0 0 20 0 0 

27 0 0 0 0 20 0 0 

38 0 0 0 0 20 0 0 

91 0 0 0 0 20 0 0 

175 5 3 4 5 20 17 85 

24 

405 5 5 5 5 20 20 100 

DWC 0 0 0 0 20 0 0 

27 0 0 0 0 20 0 0 

38 0 0 1 0 20 1 5 

91 3 3 4 3 20 13 65 

175 5 5 4 5 20 19 95 

48 

405 5 5 5 5 20 20 100 

DWC = dilution water control 

a

 Mean measured concentrations 

No other symptoms of toxicity were observed. 

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TABLE 3 Dissolved oxygen concentrations and pH values 

Dissolved oxygen conc (mg l-1)

a pHa 

0 hour 24 hourb 48 hour 0 hour 24 hourb 48 hour 

Mean measured 

conc of 

(mg l-1)

a Test 

solution Rep C Rep D Test solution Rep C Rep D 

Dilution water 

control 9.2 - 9.4 7.9 - 8.0 

27 9.2 - 9.4 8.1 - 7.8 

38 9.2 - 9.6 8.3 - 8.0 

91 9.2 - 9.6 8.3 - 8.2 

175 9.0 - 9.6 8.2 - 8.0 

405 9.0 10.4 9.8 8.3 7.8 7.9 

a all measurements are quoted to 2 significant figures 

b parameters measured at 24 hours in concentrations showing 100 % mortality only 

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TABLE 4 Water quality analysis of routine parameters, metals, and pesticides in Daphnia 

magna media 

Parameter Batch number: 

M42008-10

* pH 8.00 

* Conductivity @ 25°C (μS cm-1) 648 

* Hardness as CaCO3 (mg l-1) 244.0 

Solids Suspended @ 1050C (mg l-1) <3.00 

* Alkalinity as CaCO3 (mg l-1) 33.4 

Dissolved Organic carbon (mg l-1) 0.730 mg l-1 

Chemical Oxygen Demand (mg l-1) <12.0 mg l-1 

Ammoniacal nitrogen as N 0.01890 mg l-1 

Highest OP Pesticide <0.0100 μg l-1

Highest OC Pesticide 0.0780 μg l-1

Highest PCB <0.00500μg l-1 

Aluminium <10.0 μg l-1

Arsenic <1.00 μg l-1

Boron 491 μg l-1

Cadmium <0.100 μg l-1

Chromium <0.500 μg l-1

Cobalt 2.20 μg l-1

Copper 7.100 μg l-1

Iron 158.0 μg l-1

Lead <2.000 μg l-1

Manganese 91.0 μg l-1

Mercury <0.0100 μg l-1

Nickel <5.00μg l

-1

Silver <1.00 μg l-1

Zinc 21.0 μg l-1

* Analyses conducted at Brixham Environmental Laboratory 

All other analyses conducted by The Environment Agency, National Laboratory Service Head Office: Olympia 

House, Gelderd Lane, Gelderd Road, Leeds, LS12 6DD. The EA is a non-GLP compliant facility, but is 

accredited to UKAS (ISO 17025) 

All reports archived at Brixham Environmental Laboratory 

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Acute toxicity to Daphnia magna

APPENDIX 1 Changes introduced in BR0255/B 

The report, BR0072/B, was withdrawn because there was a client comment on the report that 

required a report amendment. The client comment has been incorporated in the new version 

of the report, BR0255/B. A description of the change introduced into BR0255/B is shown 

below: 

• In Table 1, the nominal concentration of in the first column has been amended 

from 4.83 to 4.38%. 

• As a result of this change, an extra page has been added to the study plan 

(Appendix 4) as the Study Director was changed as the existing Study Director is no 

longer employed by AstraZeneca. Also, on Page 3 “M J Webley” has been added as 

the new Study Director and on Page 5 “M J Webley” is noted as being the new Study 

Director and “A L Macken” as the Study Director for the in-life phase. 

Note: The report BR0072/B was a reissue of BR0002/B 

The original report, BR0002/B, was withdrawn because there were client comments on the 

report that had not been completed. The client comments were incorporated in the reissued 

version of the report, BR0072/B. A description of the changes introduced into BR0072/B as 

shown below: 

• The relevant METI guideline reference has been added to the Summary and the 

Introduction sections; 

• The GLP statement and the study plan have been added to the Appendices.

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Acute toxicity to Daphnia magna

APPENDIX 2 Dilution water 

Elendt and Bias (Ref 4). The final nominal concentrations in the media were as follows: 

Macronutrients

Component Final nominal concentration (mg l-1) 

 NaHCO3 64.8 

 CaCl2.2H2O 293.8 

 MgSO4.7H2O 123.3 

 KCl 5.8 

 Na2SiO3.5H2O 0.747 

 NaNO3 0.0274 

 KH2PO4 0.0143 

 K2HPO4 0.0184 

Micronutrients 

Component Final nominal concentration (mg l-1) 

 B 0.5 

 Fe 0.2 

 Mn 0.1 

 Li 0.05 

 Rb 0.05 

 Sr 0.05 

 Mo 0.025 

 Br 0.0125 

 Cu 0.0063 

 Zn 0.0063 

 Co 0.0025 

 I 0.0025 

 Se 0.001 

 V 0.0003 

 Na2EDTA.2H2O 2.5 

Vitamin Solution 

Component Final nominal concentration (μg l-1) 

 Thiamine hydrochloride 75.0 

 Vitamin B12 1.0 

 Biotin 0.75 

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APPENDIX 3 Analytical method for the determination of in aqueous samples 

Samples of daphnia medium are diluted in water, then gas extracted with helium (purge and 

trap) and analysed by Gas Chromatography with Mass spectrometric detection GC/MS. 

Quantification is by comparison with known standards of in water using an internal 

standard of 1,2-dichloroethane-d4. 

Gas Chromatograph 

Column 60 m × 0.25 mm (id) DB-VRX (df = 1.4 μ) 

Injector temperature 220 °C 

 Injector Split @ 15ml min-1

Carrier gas Helium @1.0 ml min-1

 Temperature Program Initial temp 40 °C; hold for 5 mins then 

 Ramp at 30 °C min-1 to 250 °C; hold for 1 min 

Mass Spectrometer 

 Start time 3 mins 

 Scan mode Full scan (+ve ion) 

 First mass 50 Da 

 Last mass 300 Da 

 Source temperature 200 °C 

 Transfer line 250 °C 

Purge and trap 

 Sample loop volume 5 ml 

 Line temperature 220 °C 

 Valve temperature 180 °C 

 Mount temperature 75 °C 

 MCS line temperature 40 °C 

 Purge time 12 min 

 Purge gas Helium @ ~20 ml min-1 

 Desorb preheat temperature 220 °C 

 Desorb time 3 min 

 Desorb temperature 220 °C 

 Sample Drain ON 

 Bake time 7 min 

 Bake temperature 230 °C 

 Pressurise time 0.25 min 

 Fill IS time 0.25 min 

 Xfer IS time 0.15 min 

 Rinse line time 0.2 min 

 Purge line time 0.15 min 

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 Bake rinse time 0.3 min 

 Bake transfer time 0.3 min 

 Rinse cycles 3 

 Fill IS ON 

Using the above conditions the retention times of and 1,2-dichloroethane-d4 were 

approximately 5.4 minutes and 9.2 minutes respectively. A 306 μg l-1 standard of in 

water produced a peak area of approximately 6.0 x 106 (arbitrary units) using Xcalibur 1.4. 

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APPENDIX 3, FIGURE 1 Typical chromatograms 

(A) 306 μg l-1 standard of 

RT: 4 00 - 11 00 SM: 7G

4 5 6 7 8 9 10 11

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

HFO

RT: 5.44

Internal Standard

RT: 9.18

NL:

7 98E5

m/z= 

94.5-95 5+

129 5-130 5 

F: MS 

Genesis 

09-

0025BC_111

NL:

4 23E4

m/z= 

64.5-65 5+

66.5-67 5 F: 

MS Genesis 

09-

0025BC_111

(B) 8.75 % of stock of (x 140 dilution) 

RT: 4.00 - 11.00 SM: 7G

4 5 6 7 8 9 10 11

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

HFO

RT: 5.43

Internal Standard

RT: 9.18

NL:

7.05E5

m/z= 

94 5-95.5+

129 5-130.5 

F: MS 

Genesis 

09-

0025BC_113

NL:

3.85E4

m/z= 

64 5-65.5+

66 5-67.5 F: 

MS Genesis 

09-

0025BC_113

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APPENDIX 4 Study Protocol and Amendments 

 

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APPENDIX 5 GLP Certificate 

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CIRCULATION 

Copy number 

1 

(double sided bound)

2 

(electronic copy)

GEL locator Brixham Environmental Laboratory, AstraZeneca UK

Limited 

GEL locator M. Webley, Brixham Environmental Laboratory,

AstraZeneca UK Limited 

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