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) 

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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 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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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 Johnson 

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Report 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. ................................................................................................31 

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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 International 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 Protection 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, Winterbourne 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 concentration) 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-1

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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 based 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 (Ref 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 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 that the sample had a 

purity of 99.98 area% ( . 

The sample supplied for the chemical analysis was received on 6 February 2009 and was 

assigned the Brixham test substance number 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. 

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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 April 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 from 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 water, which had been passed through activated 

carbon, coarsely filtered to emo 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 sampling 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, and a glass lid with ground edges were used 

for the dilution water control and the exposure 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. 

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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 periods commencing at 06:00 and 21:40 hours. 

At the start of the test seven fish randomly allocated to each of the test concentrations 

and the dilution water control. The fish were 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 material 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 test material on the bott m of the jar. 

The test solutions were prepared by using 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. 

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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 Laboratory 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 temperature measurements were also recorded 

automatically in the dilution water control using an electronic recording system. 

5 RESULTS 

5.1 Analytical data 

The concentrations of determined 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 LC50 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 results 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 Binomial

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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 concentration that caused 100% mortality within 

the period of the test, therefore: 96 hour LC100 = 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 recovered by the end of the study. 

The 96 hour NOEC (no observed effect concentration) 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 temperatures recorded were within the range 

15 ± 1 °C. Daily dissolved oxygen, pH and temperature measurements are shown in 

Tables 4 and 5. During the course f the study 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 adversely 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 showed 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 CaCO3, and the conductivity of 

the water was 206 μS 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 Agency. 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'. 

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Acute toxicity to rainbow trout (Oncorhynchus mykiss) 

3 Official Journal of the European Communities, 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. 

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Acute toxicity to rainbow trout (Oncorhynchus mykiss) 

TABLE 1 

ANALYTICAL RESULTSa

Nominal conc of Mean measured 

conc over the 

test durationb

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 28c 22d 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 figures 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-1

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Acute toxicity to rainbow trout (Oncorhynchus mykiss) 

TABLE 2 

PERCENTAGE MORTALITIES DURING TEST 

Nominal conc of Mean measured 

conc of 

Cumulative percentage mortality observeda

(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 Cabd Cb Cb

 Cb A* 

60 59 Cbcd Cbd Cbd C† TD 

120 140 Ccd 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 dead 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. 

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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) 

DOa

(%) 

DO 

(mg l-1) 

DOa

(%) 

DO 

(mg l-1) 

DOa

(%) 

DO 

(mg l-1) 

DOa

(%) 

DO 

(mg l-1) 

DOa

(%) 

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 Terminated 

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Acute toxicity to rainbow trout (Oncorhynchus mykiss) 

TABLE 6 

QUALITY OF DECHLORINATED WATER SUPPLY 

Test 

day 

pH Conductivity @ 

25°C 

Alkalinity 

as CaCO3

Hardness 

total as 

CaCO3

Chlorine 

(as Cl2) 

Total 

ammonia 

as NH3-Na

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 Laboratory

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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: 38

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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. 

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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 water, 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 0.15min 

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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 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 1.8 x 106 (arbitrary units) using Xcalibur 1.4. 

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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

100

Relaive Abundance

0

10

20

30

40

50

60

70

80

90

100

Relaive Abundance

RT: 5.43

RT: 9.18

5.44

4.77 9.49 4 26 5.96 6 1 8.06 8.78 9.88 11 95 12 56

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

100

Relaive Abundance

0

10

20

30

40

50

60

70

80

90

100

Relaive Abundance

RT 5.44

RT: 9.18

5.43

3.28 4.924.47 6.02 6.67 7 94 8.79 10 02 10 89 11.38 12 56

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_42

 

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APPENDIX 3 

GLP CERTIFICATE 

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APPENDIX 4 

TEST PROTOCOL 

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Acute toxicity to rainbow trout (Oncorhynchus mykiss) 

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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