Report No BR0016/B page 1 of 31
Brixham Environmental Laboratory study number 09-0025/D

Guideline OECD 203, Fish, Acute Toxicity Test

Test Facility Brixham Environmental Laboratory AstraZeneca UK Limited Brixham Devon TQ5 8BA UK

Sponsor

Author K S Hurd Report No BR0016/B Copy number

Acute toxicity to rainbow trout ( Oncorhynchus mykiss )Report No BR0016/B page 2 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

Brixham Environmental Laboratory study number 09-0025/D 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 wr itten permission of

.Report No BR0016/B page 5 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

Brixham Environmental Laboratory study number 09-0025/D
CONTRIBUTORS

Study Director

Test Facility K S Hurd

Brixham Environmental Laboratory AstraZeneca UK Limited Brixham Devon TQ5 8BA UK

Principal Scientist, Ecotoxicology K S Hurd

Principal Scientist, Analytical Chemistry P A JohnsonReport No BR0016/B page 6 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

Brixham Environmental Laboratory study number 09-0025/D
CONTENTS
page

TITLE PAGE............................................................................................................................. 1 CONFIDENTIALITY STATEMENT....................................................................................... 2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT.................................... 3 QUALITY ASSURANCE STATEMENT ................................................................................ 4 CONTRIBUTORS..................................................................................................................... 5

CONTENTS.......................................................................................................................

........ 6 1 SUMMARY ..................................................................................................................... 7 2 INTRODUCTION............................................................................................................ 8 3 MATERIALS AND METHODS ..................................................................................... 8 3.1 Test substance ........................................................................................................ 8 3.2 Test organism......................................................................................................... 9 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 Analytical method..................................... .... ........ ..................................... 10 4.4 Observations for mortality and symptoms f toxicity ......................................... 11 4.5 Physical and chemical parameters ............. ....... ............................................... 11 5 RESULTS.............................................. .... ....... .. ..................................................

. 11 5.1 Analytical data ....................... ........... .... ....................................................... 11 5.2 Biological data ............................. ..................................................................... 11 5.3 Physical and chemical data . ........... ................................................................ 12 6 REFERENCES.......................... ..... .......................................................................... 12 TABLES ................................... .... ............................................................................. 14 APPENDICES .............................. ........................................................................................ 19 CIRCULATION ................ ... ...... .................................................................................

.. 31

The last page of this report is. ................................................................................................31Report No BR0016/B page 7 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

1 SUMMARY

Sponsor

Sponsor contact International telephone

Test facility, location of raw data and final report Brixham Environmental Laboratory, AstraZeneca UK Limited, Brixham, Devon, TQ5 8BA, UK

Test facility contact M J Webley Internationa l telephone: (44) 1803 882882 International fax: (44) 1803 882974
Test substance

common name Subject Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

Guideline OECD guideline 203, Fish, Acute Toxicity Test (Ref 1), also considered acceptable when compared to the requirements of the relevant US Environmental Protec tion Agency guideline (Ref 2), EU guideline (Ref 3) and METI guid line (Ref 4)

Test species Rainbow trout Oncorhynchus mykiss

. Source of organisms Houghton Springs Fish Farm, Wi nterbourne Houghton, Blandford Forum, Dorset DT11 0PD

Nominal test concentrations Dilution water control and ominal concentrations of 15, 30, 60, 120 and 240 mg l -1

Length of test 96 hours, static, no aeration

Test dates 11 - 15 May 2009

Weight and length of dilution water control fish at end of exposure period Weight: Length r nge 0 85 – 1.20 g, mean 1.03 g r nge 40 - 47 mm, mean 43 mm

Results based on mean measured concentrations of Time

24 hour 48 hour 72 hour 96 hour LC50

59 mg l -1 59 mg l -1 38 mg l -1 38 mg l -1 95% confidence limits 25 – 140 mg l -1 25 – 140 mg l -1 25 – 59 mg l -1 25 – 59 mg l -1 Calculation Method Binomial Binomial Binomial Binomial

The NOEC (no observed effect conc entration) is defined as the highest tested concentration in which there were no mortalities within the period of the test

96 hour NOEC = 25 mg l -1Report No BR0016/B page 8 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

2 INTRODUCTION

At the request of the acute toxicity of to rainbow trout Oncorhynchus mykiss was determined.

The study was carried out under the study number 09-0025/D between 11 to 16 May 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 ba sed on the relevant OECD guideline (Ref 1) and is also considered acceptable when compared to the requirements of the relevant US Environmental Protection Agency guideline (R ef 2), EU guideline (Ref 3) and METI guideline (Ref 4).

3 MATERIALS AND METHODS

3.1 Test substance

The test substance, was supplied by

Active ingredient Chemical Abstracts name:

CAS number:

Molecular structure:

Two test samples were sent to Brixham Environmental Laboratory. The sample used in the exposure was received on 6 April 2009 and wa s assigned the Brixham test substance number 09-0066. The test substance (Supplie r Lot No. 41024-32) was supplied as a liquefied gas. A certificate of analysis (dat ed 10 April 2009) stated that the sample had a purity of 99.98 area% (

. The sample supplied for the chemical anal ysis was received on 6 February 2009 and was assigned the Brixham test substance num ber 09-0025. The test substance (Supplier Lot No. 1286ppm ) was supplied, dissolved in methanol, as a clear liquid. A certificate of analysis (dated 2 March 2009) stated that the sample was a solution of 1,286 mg l -1 in methanol prepared by weight on a NIST traceable calibrated balance and then confirmed by Gas Chromatographic analysis.Report No BR0016/B page 9 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss ) 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 rainbow trout Oncorhynchus mykiss . The fish (batch 68-09) were obtained from Houghton Springs Fish Farm, Winterbourne Houghton, Blandford Forum, Dorset DT11 0PD on 25 March 2009 and held in stock tanks at Brixham Environmental Laboratory for at least 12 days before the start of the test. No mortalities were observed in the batch of fish in the 48 hours prior to the test.

Following their arrival on site the fish were held in 25% seawater and given, twice daily a prophylactic treatment of Formalin (25ppm) and Malachite green (1ml 100l -1 ) for 9 days. The treatment was completed on 3 Apr il 2009, 38 days prior to the test. Prior to the test the fish were acclimatised to the test temperature (15 ± 1 ° C) for a minimum period of 7 days.

During the holding and acclimatisation periods the fish were fed appropriate amounts of a commercial fish food. Food was withheld fro m the fish for at least 24 hours prior to the commencement of the test.

At the end of the exposure period, the fish from the dilution water control were weighed and measured. The range in weight was 0.85 to .20 g with a mean of 1.03 g, and the range in length was 40 to 47 mm with a mean of 43 mm. The loading of fish in the dilution water control was approximately 0.36 g l -1

3.3 Dilution water

The dilution water was dechlorinat d tap wate r, which had been passed through activated carbon, coarsely filtered to em o e particulate material and dechlorinated with sodium thiosulphate. Salts were added as required, to maintain minimum hardness levels, and the treated water was hen passed through an ultraviolet steriliser to a set of 25 and 10 μm filters. The supply was then delivered to a temperature controlled header tank in the test laboratory set to th nominal test temperature ± 1 ° C.

Appendix 1 details the samplin g schedule for all of the dechlorinated water supply analyses.

4 TEST METHOD AND CONDITIONS

4.1 Test procedure and apparatus

The test procedure employed was a static system. Single glass spherical vessels, with an approximate external diameter of 370mm, a nd a glass lid with ground edges were used for the dilution water control and the expos ure solutions. The vessels had a working volume of between 20 – 25 l.

The test was undertaken in a temperature controlled room which was set at the nominal test temperature of 15 ± 1 ° C. The test solutions were not aerated.Report No BR0016/B page 10 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss ) The photoperiod in this study was 16 hours fluorescent light and 8 hours dark with 20 minute dawn and dusk transition peri ods commencing at 06:00 and 21:40 hours.

At the start of the test seven fish randomly a llocated to each of the test concentrations and the dilution water control. The fish we re not fed during the course of the test.

4.2 Preparation of test solutions

This study was run with nominal concentrations of 15, 30, 60, 120, 240 mg l -1 together with a dilution water control.

A single stock solution was prepared by bubbling the gas through 23l of dilution water for approximately 45 minutes after which it was sealed and allowed to stand for 30 minutes. Samples were then taken for chemical analysis to confirm the concentration of the stock. These showed that initial levels of in the stock were approximately 260 mg l -1 so an attempt to get more test mate rial into stock was made by bubbling gas through the solution for a further 15 minutes after which additional samples for analysis were taken. The second set of analytical results showed that the stock solution now contained a nominal 475 mg l -1 of test material and its ppearance was clear with a number of colourless, oily globules of te st material on the bott m of the jar.

The test solutions were prepared by usi ng the foll wing amounts of and dilution
water:

Nominal concentration of

(mg l -1 ) Volume prepared

(L) Nominal amoun of stock soluti n r quired

( l) Actu l amount of stock solution used

(ml) Nominal amount of dilution water required

(ml) Actual amount of dilution water used

(ml) DWC 20 nil nil 20000 20000

15 20 632 630 19368 19370 30 20 1263 1265 18737 18735 60 20 2526 2525 17474 17475 120 2 5053 5055 14947 14945 240 2 12126 12125 11874 11875

Initially 10l of dilution water were added directly to the test vessels followed by the required amount of stock solution. After this the remaining volume of dilution water was then added and the vessels sealed using the glass lids. The resultant solutions were as follows; 15 – 120 mg l -1 solutions were clear and colourless while the 240 mg l -1 solution was clear with some colourless, oily globules on the base of the test vessel

. 4.3 Analytical method

The concentrations of in the test solutions were measured at 0 and 96 hours using the gas chromatography method outlined in Appendix 2.

Samples for analysis were taken from the centre of the test solutions.Report No BR0016/B page 11 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss ) 4.4 Observations for mortality and symptoms of toxicity

Observations for mortalities and symptoms of toxicity were made at 24, 48, 72 and 96 hours. Symptoms of toxicity were also recorded at 3 hours.

The LC50 values, and their 95% confidence limits, were calculated at the various time intervals by the Brixham Environmental La boratory computer program “PCLC50” using Stephan's method (Ref 5).

4.5 Physical and chemical parameters

Daily measurements of the test solutions were undertaken throughout the 96 hour period for pH and dissolved oxygen concentration using calibrated meters. Temperature values were determined daily using a mercury-in-glass thermometer calibrated to 0.1 ° C and conforming to BS593. Hourly temperatur e measurements were also recorded automatically in the dilution water control using an electronic recording system.

5 RESULTS

5.1 Analytical data

The concentrations of de termined in the test solu ions are given in Table 1. The limit of quantification of in this study was 0.077 mg l -1 . Typical chromatograms are shown in Appendix 2, Figure 1. All analytica values are quoted to two significant figures and percentages to the nearest in eger. Th measured concentrations at the start of the test ranged from 93 to 179% of the nominal values. The measured concentrations at the end of the test ranged from 65 to 238% of the nominal values. The mean measured concentrations ranged from 80 to 208% of the nominal values confirming that was in solution.

The high measured con entr tion for the nominal 240 mg l -1 solution could be attributed to the previously n ted pr sence of colourless, oily globules (section 4.2).

On the basis of the ana ytical data the mean measured concentrations were used for the calculation and repo ting of the results.

5.2 Biological data

A summary of fish mortalities is given in Table 2. The LC 50 is defined as the concentration, calculated from the data obtained, resulting in the death of 50% of the fish in the time period specified. The resu lts obtained based on mean measured concentrations of were:

Time LC50 95% confidence limits Calculation method

24 hour 59 mg l -1 25 – 140 mg l -1 Binomial 48 hour 59 mg l -1 25 – 140 mg l -1 Binomial 72 hour 38 mg l -1 25 – 59 mg l -1 Binomial 96 hour 38 mg l -1 25 – 59 mg l -1 BinomialReport No BR0016/B page 12 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

The concentration response graph for the 24, 48, 72 and 96 hour LC50 values were plotted (Fig 1). The 95% confidence limits are shown as horizontal bars.

There were no mortalities in the dilution water control.

The LC100 is defined as the lowest test con centration that caused 100% mortality within the period of the test, therefore: 96 hour LC 100 = 59 mg l -1

. The general symptoms of toxicity, other than mortalities, noted in this study were surfacing, loss of balance, and dark discolouration

. These data are shown in Table 3. In the 25 mg l -1 concentration the fish showed symptoms of toxicity for the first 72 hours but appeared to have recovere d by the end of the study.

The 96 hour NOEC (no observed effect concentra tion) is defined as the highest tested concentration in which there were no mortalities within the period of the test, therefore: 96 hour NOEC = 25 mg l -1

At the end of the exposure period, the fish from the dilution water ontrol were weighed and measured. The range in weight was 0.85 – 1.20g with a me n of 1.03g, and the range in length was 40 – 47mm with a mean of 43mm The loading of the fish in the dilution water control was 0.36 g l -1

5.3 Physical and chemical data

The dissolved oxygen concentrations in he test v ssels ranged from 4.6 to 9.7 mg l -1 , pH values ranged from 6.8 to 7.8, and the temper atures recorded were within the range 15 ± 1 ° C. Daily dissolved oxygen, pH and te mperature measurements are shown in Tables 4 and 5. During the course f the st udy the dissolved oxygen concentration in the dilution water control and 30 mg l 1 t st tanks fell below 60% of the air-saturation value (6.0 mg l -1 ) (Ref 6) and is therefore a deviation from the guideline. This occurred at 72 and 96 hours but was not deem d to have adversel y affected the results of the study. The dissolved oxygen in the remaining test tanks stayed at ≥ 60 % of the air-saturation value throughout the study The continuous record of temperature recorded automatically in the dilution water control during the study s howed that on one occasion the temperature of the dilution water control fell below 14.0 °C for approximately 3 hours. The minimum recorded temperature was 13.9 °C and this was not considered to have adversely affected the results of the study.

The total hardness of the dilution water was 53.3 mg l -1 CaCO 3 , and the conductivity of the water was 206 μ S cm -1 @ 25 ° C. Full water quality parameters of the dechlorinated water supply are shown in Table 6.

6 REFERENCES

1 OECD Guidelines for Testing of Chemicals. Test Guideline 203, Fish, Acute Toxicity Test. Adopted 17 July 1992.

2 OPPTS 'US Environmental Protection Agen cy. Office of Prevention, Pesticides and Toxic Substances. Ecological Effects Test Guideline OPPTS 850.1075, Fish Acute Toxicity Test, Freshwater and Marine “Public Draft”. April 1996'.Report No BR0016/B page 13 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

3 Official Journal of the European Commun ities, L 383 A, Part C.1, Acute toxicity for fish. 29 December 1992.

4 METI Guidelines for Testing of Chemicals. Test Guideline 2-7-1-1, Fish acute toxicity studies.

5 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.

6 National Institute of Oceanography of Great Britain and UNESCO (1973). International Oceanographic Tables, 2

.Report No BR0016/B page 14 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

TABLE 1

ANALYTICAL RESULTS a

Nominal conc of Mean measured conc over the test duration b Mean measured conc as % of nominal Measured conc of

(mg l -1 ) (mg l -1 ) 0 hour 96 hour (mg l -1 )

Dilution water control <0.077 <0.077 <0.077 -

15 14 9.8 12 80

30 28 c 22 d 25 83

60 57 60 59 98

120 120 150 140 117

240 430 570 500 208

a All measurements are quoted to 2 significant figu res and percentages are quoted to the nearest integer

b Calculated using the arithmetic mean of the 0 nd 96 ho r results

c Mean of triplicate analyses: 27, 28, 28 mg l -1

d Mean of triplicate analyses: 22, 22, 21 mg l -1

The limit of quantification in the study was 0.077 mg l -1Report No BR0016/B page 15 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

TABLE 2

PERCENTAGE MORTALITIES DURING TEST

Nominal conc of Mean measured

conc of Cumulative percentage mortality observed a
(mg l -1 ) (mg l -1 ) 24 hour 48 hour 72 hour 96 hour

Dilution water control - 0 0 0 0

15 12 0 0 0 0

30 25 0 0 0 0

60 59 86 86 100 100

120 140 100 100 100 100

240 500 100 100 100 100

a

Seven fish were exposed in each test vessel

TABLE 3

SYMPTOMS OF TOXICITY OBSERVED

Nominal conc of Mean measured conc

of Symptoms observed

(mg l -1 ) (mg l -1 ) 3 hour 24 hour 48 hour 72 hour 96 hour

15 12 A A A A A

30 25 C abd C b C b C b A*

60 59 C bcd C bd C bd C† TD

120 140 C cd C† TD TD TD

240 500 C† C† TD TD TD

Definition of symbols

A = 10% or less of test population dead or exhibiting symptoms of toxicity (considered not to be biologically significant). B = Between 11% and 30% of test population d ead or exhibiting symptoms of toxicity. C = More than 30% of test population dead or exhibiting symptoms of toxicity.
TD = Terminated † = Mortalities only

a = Hyperexcitable c = Loss of balance

b = Surfacing d = Dark-discolured

* condition of these fish improved as the test proceeded.Report No BR0016 page 16 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

TABLE 4

DISSOLVED OXYGEN CONCENTRATIONS IN EXPOSURE VESSELS

Nominal conc of Measured conc of 0 hour 24 hour 48 hour 72 hour 96 hour

(mg l -1 ) (mg l -1 ) DO (mg l -1 ) DO a (%) DO (mg l -1 ) DO a (%) DO (mg l -1 ) DO a (%) DO (mg l -1 ) DO a (%) DO (mg l -1 ) DO a (%)

Dilution

water control Dilution
water control 9.7 96 7.8 77 6.3 62 6.0 59 5.1 50

15 12 9.7 96 8.0 79 7.4 73 7.2 71 7.1 70

30 25 9.6 95 6.9 68 6.1 60 5.0 50 4.6 46

60 59 9.4 93 7.6 75 7.0 69 7.3 72 TD TD

120 140 9.3 92 8.8 87 TD TD TD TD TD TD

240 500 8.4 83 8.3 82 TD TD TD TD TD TD

TD Terminated DO Dissolved oxygen

a Quoted as percentage of the 100% air saturation value of 10.1 mg l -1 at 15°C (Ref 6)

TABLE 5

pH AND TEMPERATURE IN EXPOSURE VESSELS

Nominal conc of Measured conc of 0 hour 24 hour 48 hour 72 hour 96 hour

(mg l -1 ) (mg l -1 ) pH Temp (°C) pH Temp (°C) pH Temp (°C) pH Temp (°C) pH Temp (°C)

Dilution

water control Dilution
water control 7.8 15 2 7.4 15.2 7.2 14.8 7.2 14.8 7.1 14.7

15 12 7.8 15.9 7.3 15.0 7.1 14.7 7.0 14.8 7.0 14.8

30 25 7.8 15.5 7.2 14.9 7.1 14.7 6.9 14.6 6.8 14.9

60 59 7.8 15.4 7.3 15.0 7.2 14.6 7.2 14.7 TD TD

120 140 7.8 15.3 7.5 14.8 TD TD TD TD TD TD

240 500 7.8 14.9 7.7 14.9 TD TD TD TD TD TD

TD TerminatedReport No BR0016 page 17 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

TABLE 6

QUALITY OF DECHLORINATED WATER SUPPLY

Test day pH Conductivity @

25°C Alkalinity as CaCO 3 Hardness total as CaCO 3 Chlorine (as Cl 2 ) Total ammonia as NH 3 -N a Salinity

Free residual Combined residual (μS cm -1 ) (mg l -1 ) (mg l -1 ) (μg l -1 ) (μg l -1 ) (mg l -1 ) (‰)

0 7.7 206 32.4 53.3 <2 <2 <0.00200 0.1

- Not sampled a Indicates data from most recently analysed dilution water batch (18 March 2009). Analysis conducted at Environment Agency, NLS Olympia House, Gelderd Lane, Gelderd Road, Leeds, LS12 9DD

. This is a non GLP compliant facility. All reports archived at Brixham Environmental LaboratoryReport No BR0016 page 18 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

FIGURE 1

CONCENTRATION RESPONSE CURVE

CONCENTRATION RESPONSE RELATIONSHIP Rainbow Trout

STUDY NO. 09-0025D (09-0066)

0 20 40 60 80 100 120

10 100 1000
NOMINAL CONCENTRATION (mgl -1 )Time (Hours) LC50 (mgl-1)
24 Hour: 59
48 Hour: 59
72 Hour: 38
96 Hour: 38Report No BR0016 page 19 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

APPENDIX 1

SCHEDULE FOR WATER QUALITY ANALYSIS OF FRESHWATER

Parameter Sampling frequency pH & Conductivity Daily (mon-fri)
Total Hardness Alkalinity
Free Chlorine
Residual Chlorine Weekly

Ammonia – as N Solids Suspended Total Organic Carbon – as C Chemical Oxygen Demand Monthly

Fluoride, Anions/Cations and Metals Quarterly Organo-chlorine Pesticides Organo-phosphorus Pesticides
PCBs Quarterly

Daily and weekly analyses performed by BEL. All other nalyses performed by The Environmental Agency, NLS Olympia House, Gelderd Lane, Gelderd Road, Leeds, LS12 9DD.Report No BR0016 page 20 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

APPENDIX 2

ANALYTICAL METHOD FOR THE DETERMINATION OF IN AQUEOUS SAMPLES

Samples of dechlorinated medium are diluted in wa ter, then purged 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 m n -1 Temperature Program Initial temp 40 ° C; hold for 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 5ml Line temperature 220 ° C Valve temperature 180 ° C Mount temperature 75 ° C MCS line temperature 40 ° C Purge time 12min Purge gas Helium Desorb preheat temperature 220 ° C Desorb time 3min Desorb temperature 220 ° C Sample Drain ON Bake time 7min Bake temperature 230 ° C Pressurise time 0.25min Fill IS time 0.25min Xfer IS time 0.15min Rinse line time 0.2min Purge line time 0.15minReport No BR0016 page 21 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

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

Using the described conditions the retention t imes 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 1.8 x 10 6 (arbitrary units) using Xcalibur 1.4.Report No BR0016 page 22 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

APPENDIX 2, FIGURE 1

TYPICAL CHROMATOGRAMS

(A) 306 μg l -1 standard of

RT: 0.00 - 13.01 SM: 7G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time (min) 0 10 20 30 40 50 60 70 80 90 100Relaive Abundance 0 10 20 30 40 50 60 70 80 90 100Relaive Abundance RT: 5.43

RT: 9.18

5.44

4.77 9.49 12 56 4 26 5.96 6 1 11 95 9.88 8.78 8.06 NL: 5.08E5 m/z= 94.5-95.5+ 129.5-130.5 F: + c Full ms [ 50.00-300.00] MS Genesis 09-0025D_43

NL: 2.42E4 m/z= 64.5-65.5+66.5-67.5 F: + c Full ms [ 50.00-300.00] MS Genesis 09-0025D_43

(B) 120 mg l -1 of (x 240 dilutio )

RT: 0.00 - 13.01 SM: 7G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time (min) 0 10 20 30 40 50 60 70 80 90 100Relaive Abundance 0 10 20 30 40 50 60 70 80 90 100Relaive Abundance RT 5.44

RT: 9.18

5.43

12 56 11.38 4.924.47 6.02 6.67 3.28 10 89 8.79 10 02 7 94 NL: 7.48E5 m/z= 94.5-95.5+ 129.5-130.5 F: + c Full ms [ 50.00-300.00] MS Genesis 09-0025D_42

NL: 2.47E4 m/z= 64.5-65.5+66.5-67.5 F: + c Full ms [ 50.00-300.00] MS Genesis 09-0025D_42Report No BR0016 page 23 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

APPENDIX 3

GLP CERTIFICATEReport No BR0016 page 24 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

APPENDIX 4

TEST PROTOCOLReport No BR0016 page 25 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )Report No BR0016 page 26 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )Report No BR0016 page 27 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )Report No BR0016 page 28 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )Report No BR0016 page 29 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )Report No BR0016 page 30 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )Report No BR0016 page 31 of 31 Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

CIRCULATION

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GEL locator Brixham Environmental Laboratory, AstraZeneca U K Limited

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