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 magnaBR0255/B, issued as a replacement for BR0072/B Page 2 of 32

Acute toxicity to Da phn ia magna

Brixham Environmental Laboratory study number : 09-0025/C
CONFIDENTIAL (CATEGORY B)

Not to be photocopied or microfilmed. This doc ument contains inform ation confidential and trade secret to It s hould 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

.BR0255/B, issued as a replacement for BR0072/B Page 5 of 32

Acute toxicity to Da phn ia 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 CummingBR0255/B, issued as a replacement for BR0072/B Page 6 of 32

Acute toxicity to Da phn ia 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 che mical 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 de termination of in aqueous samples 19 APPENDIX 4 Study Protocol and Amendments.................................................................... 22 APPENDIX 5 GLP Certificate ............................................................................................... 31 circulation ................................................................................................................................ 32

The last page of this report is...................................................................................................32BR0255/B, issued as a replacement for BR0072/B Page 7 of 32 Acute toxicity to Daphni a 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 EC 50

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 -1BR0255/B, issued as a replacement for BR0072/B Page 8 of 32 Acute toxicity to Daphni a magna

2 INTRODUCTION

At the request of Honeywell the acute toxicity of to Daphnia magna was determined.

The study was carried out under the stud y 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 Ho neywell

Compound name:

CAS number: Molecular structure:

The test substance was sent to Br ixha m 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 Brix ham Environmental Laboratory.BR0255/B, issued as a replacement for BR0072/B Page 9 of 32 Acute toxicity to Daphni a magna

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 mi nute transition periods was provided.

The D. magna cultures were fed on a mixed algae diet of Chlorella vulgaris , strain CCAP 211/12 and Pseudokirchnerie lla 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 main tained with a twice weekly renewal of reconstituted water medium since birth. The test organisms and the culture from which they were obtained showed no eviden ce 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 hard ness, 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.BR0255/B, issued as a replacement for BR0072/B Page 10 of 32 Acute toxicity to Daphni a magna

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 obt ain 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 wa ter only. All final solutions were observed to be clear and colourless and contained nutrients as sp ecified 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 solu tions was measured at 0 and 48 hours using Purge and Trap injection with Gas Chroma tography 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 concentr ations 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 i ndividual appendages was visible. D. magna so affected were termed immobile. Any other overt symptoms of toxicity were also recorded.

The median effect concentration (EC 50 ) 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 begi nning 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 s ubstance 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 wate r control and all the exposure concentrations were measured. Dissolved oxygen and pH we re measured in the top concentration after 24 hours as 100 % mortality was observed.BR0255/B, issued as a replacement for BR0072/B Page 11 of 32 Acute toxicity to Daphni a magna

Temperature values were determined daily, in an additional test vessel, using a mercuryin-glass thermometer calibrated to BS593. Hour ly 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 de termined 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 unknow n stock and on the basis of the analytical data mean measured concen trations 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 int erval 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 EC 100 is defined as the lowest test conc entration that caused 100 % immobilisation within the period of the test, therefore,

48 hour EC 100 = 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 -1BR0255/B, issued as a replacement for BR0072/B Page 12 of 32 Acute toxicity to Daphni a 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 o C, with the values recorded automatically over th e test period remaining within 20.0 ± 1 o C.

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 Chemical s. 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 Li fe 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.BR0255/B, issued as a replacement for BR0072/B Page 13 of 32 Acute toxicity to Daphni a magna

TABLE 1 Analytical results

Measured conc of (mg l -1 ) a Nominal conc of

(%) 0 hour 48 hour Mean measured

conc a

(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 -1BR0255/B, issued as a replacement for BR0072/B Page 14 of 32 Acute toxicity to Daphni a magna

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.BR0255/B, issued as a replacement for BR0072/B Page 15 of 32 Acute toxicity to Daphni a magna

TABLE 3 Dissolved oxygen concentrations and pH values

Dissolved oxygen conc (mg l -1 ) a pH a

0 hour 24 hour b 48 hour 0 hour 24 hour b 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 onlyBR0255/B, issued as a replacement for BR0072/B Page 16 of 32 Acute toxicity to Daphni a magna

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 CaCO 3 (mg l -1 ) 244.0 Solids Suspended @ 105 0 C (mg l -1 ) <3.00 * Alkalinity as CaCO 3 (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 LaboratoryBR0255/B, issued as a replacement for BR0072/B Page 17 of 32 Acute toxicity to Daphni a magna

APPENDIX 1 Changes introduced in BR0255/B

The report, BR0072/B, was withdrawn because th ere 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 chan ged 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 withdraw n 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 descripti on 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.BR0255/B, issued as a replacement for BR0072/B Page 18 of 32 Acute toxicity to Daphni a 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 )

NaHCO 3 64.8 CaCl 2 .2H 2 O 293.8 MgSO 4 .7H 2 O 123.3 KCl 5.8 Na 2 SiO 3 .5H 2 O 0.747 NaNO 3 0.0274 KH 2 PO 4 0.0143 K 2 HPO 4 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 Na 2 EDTA.2H 2 O 2.5

Vitamin Solution

Component Final nominal concentration ( μ g l -1 )
Thiamine hydrochloride 75.0 Vitamin B 12 1.0 Biotin 0.75BR0255/B, issued as a replacement for BR0072/B Page 19 of 32 Acute toxicity to Daphni a magna

APPENDIX 3 Analytical method for the determin ation 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 minBR0255/B, issued as a replacement for BR0072/B Page 20 of 32 Acute toxicity to Daphni a magna

Bake rinse time 0.3 min Bake transfer time 0.3 min Rinse cycles 3 Fill IS ON

Using the above conditions the retention time s 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 10 6 (arbitrary units) using Xcalibur 1.4.BR0255/B, issued as a replacement for BR0072/B Page 21 of 32 Acute toxicity to Daphnia m agna

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 100Relative Abundance 0 10 20 30 40 50 60 70 80 90 100Relative 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 100Relative Abundance 0 10 20 30 40 50 60 70 80 90 100Relative 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_113BR0255/B, issued as a replacement for BR0072/B Page 22 of 32 Acute toxicity to Daphni a magna

APPENDIX 4 Study Protocol and AmendmentsBR0255/B, issued as a replacement for BR0072/B Page 23 of 32 Acute toxicity to Daphni a magnaBR0255/B, issued as a replacement for BR0072/B Page 24 of 32 Acute toxicity to Daphni a magnaBR0255/B, issued as a replacement for BR0072/B Page 25 of 32 Acute toxicity to Daphni a magnaBR0255/B, issued as a replacement for BR0072/B Page 26 of 32 Acute toxicity to Daphni a magnaBR0255/B, issued as a replacement for BR0072/B Page 27 of 32 Acute toxicity to Daphni a magnaBR0255/B, issued as a replacement for BR0072/B Page 28 of 32 Acute toxicity to Daphni a magnaBR0255/B, issued as a replacement for BR0072/B Page 29 of 32 Acute toxicity to Daphni a magnaBR0255/B, issued as a replacement for BR0072/B Page 31 of 32 Acute toxicity to Daphni a magna

APPENDIX 5 GLP CertificateBR0255/B, issued as a replacement for BR0072/B Page 32 of 32 Acute toxicity to Daphni a magna

CIRCULATION

Copy number

1 (double sided bound)

2 (electronic copy)

GEL locator Brixham Environmental Laboratory, AstraZeneca U K Limited

GEL locator M. Webley, Brixham Environmental Laboratory, AstraZeneca UK Limited