Brixham Environmental 

Laboratory study number 

09-0025/B 

Guideline OECD 201, Alga, Growth Inhibition Test 

Test Facility Brixham Environmental Laboratory, AstraZeneca UK Limited, 

Brixham, Devon , TQ5 8BA, UK 

Sponsor 

Authors M Fox, A L Macken 

Issued September 2009 

Report No BR0027/B Copy number 

Toxicity to the green alga Pseudokirchneriella subcapitata

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Toxicity to the green alga Pseudokirchneriella subcapitata 

Study Number: 09-0025/B 

2 

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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Toxicity to the green alga Pseudokirchneriella subcapitata 

Study Number: 09-0025/B 

5 

CONTRIBUTORS 

Study Director 

Test Facility 

A L Macken 

Brixham Environmental 

Laboratory 

AstraZeneca UK Limited 

Brixham 

Devon TQ5 8BA 

UK 

Principal Scientist, 

Ecotoxicology 

A V Ward 

Principal Scientist, 

Analytical Chemistry 

R I Cumming 

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CONTENTS 

page 

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

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

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

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

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

1 SUMMARY.............................................................................................................8

2 INTRODUCTION ...................................................................................................9

3 MATERIALS...........................................................................................................9

3.1 Test substance ..........................................................................................................9

3.2 Test species ............................................................................................................10

3.3 Culture medium......................................................................................................10

4 TEST METHOD AND CONDITIONS.................................................................10

4.1 Test procedure and apparatus.................................................................................10

4.2 Preparation of test solutions...................................................................................11

4.3 Analytical method ..................................................................................................11

4.4 Physical and chemical parameters .........................................................................11

5 RESULTS ..............................................................................................................12

5.1 Analytical data .......................................................................................................12

5.2 Biological data .......................................................................................................12

5.2.1 Yield.......................................................................................................................12

5.2.2 Growth rates...........................................................................................................13

5.2.3 Additional biological data......................................................................................13

5.2.4 Validity criteria ......................................................................................................14

5.3 Physical and chemical data ....................................................................................14

6 REFERENCES.......................................................................................................15

TABLE 1 ANALYTICAL RESULTS....................................................................................16

TABLE 2 ALGAL CELL PARTICLE DENSITIES..............................................................17

TABLE 3 MEAN YIELDS OVER THE TEST PERIOD ......................................................18

TABLE 4 MEAN GROWTH RATES OVER THE TEST PERIOD .....................................18

TABLE 5 TEST SOLUTION pH MEASUREMENTS..........................................................19

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FIGURE 1 EFFECT ON GROWTH OF GREEN ALGA

PSEUDOKIRCHNERIELLA SUBCAPITATA AFTER 72 HOURS ......................20

APPENDIX 1 .........................................................................................................................21

APPENDIX 2 .........................................................................................................................22

APPENDIX 2, FIGURE 1......................................................................................................24

APPENDIX 3 .........................................................................................................................25

APPENDIX 4 .........................................................................................................................26

The last page of this report is............................................................................37 

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1 SUMMARY 

Sponsor

Sponsor 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 882882 

International fax: (44) 1803 882974 

Test substance 

common name 

Subject Toxicity to the green alga Pseudokirchneriella subcapitata (Formally 

known as Selenastrum capricornutum) 

Guideline followed OECD guideline 201, Alga, Growth Inhibition Test (Ref 1), also satisfies 

requirements of the relevant EU guideline (Ref 2) and METI guideline 

(Ref 3) 

Test species Green alga Pseudokirchneriella subcapitata

Source of organisms Brixham Environmental Laboratory culture from strain ATCC 22662 

Test concentrations Control and measured concentrations of 13.5, 32.0, 60.5, 115, 215 mg l-1

(Nominal concentrations: 4.38, 8.75, 17.5, 35, 70 % saturation) 

Length of test 72 hours shaken 

Test dates 6 to 9 April 2009 

Nominal 

test temperature 

24 ± 2 °C 

Results based on 

mean measured test 

concentrations of 

Based on yield at the start and end of the test, the results obtained were:

No Observed Effect Concentration (NOEC) = >215 mg l-1

Lowest Observed Effect Concentration (LOEC) = >215 mg l-1 

Median effective concentration, yield (EyC50) = 106.7 mg l-1

95% confidence limits = 42.1 – 154.0 mg l-1 

Based on growth rate over the test period, the results obtained were:

No Observed Effect Concentration (NOEC) = 115 mg l-1

Lowest Observed Effect Concentration (LOEC) = 215 mg l-1

A hormetic effect on algae growth was observed during the test at 13.5, 

32.0, 60.5 mg l-1. 

The test substance was dosed as a gas. 

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2 INTRODUCTION 

At the request of 

 the toxicity of to the green alga Pseudokirchneriella 

subcapitata was determined. 

The study was carried out under the study number 09-0025/B between 6 and 10 April 2009. 

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

Laboratory archive. 

The procedures employed were based on the relevant OECD test guideline (Ref 1). The study 

is also considered acceptable when compared to the procedural requirements of the relevant 

EU Guideline (Ref 2) and METI guideline (Ref 3). 

3 MATERIALS 

3.1 Test substance 

The test substance, was supplied by 

Chemical 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 99.98 % 

( ). 

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

required for testing. 

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A separate test substance (CAS 67-56-1) was used for chemical analysis, which was received 

on 6 February 2009 and on receipt it was assigned the Brixham test substance number 

09-0025. The test substance (Supplier Lot No. 1286 ppm [e]) was supplied as a clear 

liquid (>99 % in methanol). 

3.2 Test species 

The test species was the unicellular green alga Pseudokirchneriella subcapitata strain

ATCC 22662 from laboratory cultures maintained under axenic conditions. A 3 day old 

culture of the alga in the exponential growth phase was used as inoculum for the test. The 

culture was grown in the medium, and under the environmental conditions, described for the 

test. 

3.3 Culture medium 

The culture medium used for the test was AAP-medium (Appendix 1) which had additional 

bicarbonate added as a carbon source for the algae as the test was run in sealed vessels with no 

headspace. The medium used for the maintenance of cultures was AAP-medium only. Both 

media are based on information published by Miller et al (Ref 4). 

4 TEST METHOD AND CONDITIONS 

4.1 Test procedure and apparatus 

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

approximately 60 ml of test solution, with airtight, Teflon faced disc/crimp closures. The 

cultures were incubated at 24 ± 2 °C (the nominal test temperature), under continuous 

"cool-white" illumination of approximately 8000 lux, with a nominal orbital shaking at 

160 ± 10 rpm. Small glass beads were placed into each vessel to aid mixing and prevent algal 

clumping. 

Two replicates of the control and each concentration of were employed for each 

sampling occasion (24, 48 and 72 hours), thus there were 6 replicates for the control and each 

test concentration in total. One blank vessel (without algal inoculum) was incubated 

concurrently for the control and each test concentration to be used to determine ‘background’ 

particle density at each sampling occasion (24, 48 and 72 hours). Analytical samples for 0 

hours were taken for excess solutions. One extra vessel for each concentration and the control 

were prepared and sealed for 72 hour analytical analysis. The position of each test replicate 

vessel in the incubator was randomised by rows daily. 

The algal cell densities of the inoculum and test cultures were determined by electronic 

particle counting, using a Coulter counter model Z1 and counting at a lower threshold 

equivalent spherical diameter of approximately 2.3 μm. Each replicate test vessel was 

inoculated with 0.35 ml of the inoculum culture to give a nominal cell density of 

0.100 x 104

 cells ml-1. One 60 ml volume of Coulter electrolyte, inoculated in the same 

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manner, had a cell density of 0.109 x 104

 cells ml-1. This value was used for growth 

calculations. 

After 24, 48 and 72 hours, appropriate vessels were removed for the blank, control and each 

test concentration. The appropriate blank particle count was subtracted from that of the test 

culture to obtain the cell density. 

At the end of the test microscopic observations were made on samples taken from a single 

replicate of the control and each test substance concentration. 

4.2 Preparation of test solutions 

This study was run with a control together with nominal concentrations of 4.38, 8.75, 

17.5, 35, 70 % saturation. 

The test solutions were prepared by additions of stock solution to sterile culture medium. A 

stock solution of the highest concentration was prepared by bubbling through 3500 ml 

of sterile media for approximately 60 minutes to obtain a saturated solution of the gas 

(100 %). This was diluted to 70 % saturation with sterile oxygenated media; this stock was 

used for the top concentration. All other concentrations were made from this, and inverted in 

volumetric flasks to mix. The resulting solutions were clear and colourless. The control 

consisted of sterile culture medium only. In all cases the final solutions contained nutrients as 

specified in Appendix 1. 

Aliquots of 60 ml of the appropriate test solutions were dispensed to each test and blank 

vessel, with the remainder of the test solutions being used for physical and chemical analyses. 

One extra vessel for each concentration and the control was prepared and sealed for 0 hour 

analysis due to the volatility of the test substance.Analytical method 

The concentrations of in the test solutions were measured at 0 and 72 hours using a 

purge and trap injection with gas chromatography and mass spectrometry method outlined in 

Appendix 2. 

Samples for analysis were taken at 0 hours from a specific airtight vessel (to prevent loss of 

volatile test substance) and at 72 hours from a single test replicate solution of each exposure 

(containing alga). 

4.4 Physical and chemical parameters 

The pH of the control and test solutions was measured at the start of the test with a calibrated 

pH meter, using the excess remaining after filling the test vessels. At the end of the test the 

pH of one replicate vessel (containing algae) from each test concentration was determined. 

The temperature of the incubator was continuously monitored using an electronic recording 

system with hourly recording of values. The light intensity was measured once during the 

study, in each of four representative positions, using a photometer reading in lux (cosine). 

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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. Due to the preparation technique of the primary stock solution 

(bubbling) the nominal concentrations of were unknown until analytical analysis was 

conducted. Therefore it is not possible to make a comparison between the nominal and 

measured concentrations. 

The mean measured concentrations to 3 significant figures were used for reporting of 

results. 

5.2 Biological dataAlgal cell particle densities (cell particles per unit volume) were 

measured as a surrogate for biomass. The algal cell particle densities measured at each time 

period are given in Table 2. The means of these values are also shown in Table 2 and are 

plotted as growth curves in Figure 1. 

For the reporting of results, the Lowest Observed Effect Concentration (LOEC) is defined as 

the lowest tested concentration, which is observed to have a significant effect, (p<0.05), when 

compared with the control. However, all test concentrations above the LOEC must have a 

harmful effect equal to or greater than those observed at the LOEC. The No Observed Effect 

Concentration (NOEC) is defined as the test concentration immediately below the LOEC 

(Ref 1). A hormetic effect was seen in the test concentrations (13.5, 32.0, 60.5 mg l-1); as a 

consequence of this hormesis EyC50 was calculated via linear interpolation (Ref 5). The 

ErC50 could not be calculated as <50 % inhibition was seen. 

5.2.1 Yield 

This response is defined as the biomass at the end of the test minus the starting biomass 

(Ref 1). For the purposes of calculation, the cell particle density count (cell particles per unit 

volume) is an acceptable surrogate for biomass. 

These cell particle densities were examined by one-way analysis of variance and Dunnett’s 

procedure (Ref 6) was used to identify significant differences (p<0.05) from the control. The 

test substance concentrations yields are given in Table 3, together with the yields expressed as 

percentages of the control and significant differences from the control. 

A significant difference (p < 0.05) was found at 32.0 mg l-1 (Table 3) although this was due to 

the hormetic effect rather than an inhibitory effect, thus has been discounted. 

The results obtained from these yield analyses, based on mean measured test concentrations, 

were as follows: 

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No observed effect concentration (NOEC) = > 215 mg l-1

Lowest observed effect concentration (LOEC) = > 215 mg l-1

Median effective concentration, yield (EyC50) = 106.7 mg l-1

95% confidence limits = 42.1 – 154.0 mg l-1 

5.2.2 Growth rates 

The growth rate (0 to 72 hours) was calculated for each replicate culture, according to the 

formula. 

t

)N / (NLog rateGrowth 12n =

where N1 = Cell density at start of test 

 N2 = Cell density at end of test 

 t = Time interval (days) 

These data were analysed as described for the yield method. The mean growth rates, with the 

significant difference (p < 0.05) identified, are given in Table 4, together with the rates 

expressed as percentages of the control. 

Three significant differences (p < 0.05) were found at 13.5, 32.0 and 60.5 mg l-1 (Table 4) 

although this was due to the hormetic effect rather than an inhibitory effect, thus has been 

discounted. 

The results obtained from these growth rate analyses, based on mean measured test 

concentrations, were as follows: 

No observed effect concentration (NOEC) = 115 mg l-1

Lowest observed effect concentration (LOEC) = 215 mg l-1

5.2.3 Additional biological data 

The increase in biomass for the parent inoculum culture was monitored over its 3-day growth 

period and was considered to be in an exponential growth phase. As such, the overall factor 

increase in cell particle density (considered equivalent to biomass) was 131 times, an 

approximate daily factor of 5.1, which is deemed acceptable. 

The microscopic observations, made at the end of the test, showed that compared to the 

control the algal cells sampled from the 13.5, 32.0, 60.5 mg l-1 test concentrations appeared 

normal. At 115 and 215 mg l-1 test concentrations algal cells sampled were enlarged, with 

deformed cells only noted in the top concentration (215 mg l-1) where very low cells numbers 

were also observed. 

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5.2.4 Validity criteria 

The validity criteria specified in the OECD 201 guideline (Ref 1) are; 

(i) To achieve ≥16 fold exponential increase in biomass within the 72 hour test period. In 

this test cell particle density increase (measured as a surrogate for biomass) was 94 

times over the 72 hours. 

(ii) The mean coefficients of variation for replicate sectional (daily) specific growth rates 

must not exceed 35 % and in this test was determined to be 31.9 %. 

(iii) The replicate coefficient of variation of average specific growth rates during the whole 

test period in replicate cultures must not exceed 7 % and was calculated as 0.76 %. 

5.3 Physical and chemical data 

At the start of the test the pH ranged from 7.91 to 8.26 and at the end of the test the range was 

from 8.48 to 10.14 (Table 5). 

The increase in pH for the control replicates observed over the test duration was 0.57 units. 

The largest increase observed for the test was 1.94 units. 

The temperature of the incubator was accidentally set at 24 ± 2 °C, however the hourly 

temperature measurements, recorded automatically ranged from 23 - 25 °C. This increased 

temperature, although slightly outside the recommended limits of the guideline, was not 

deemed to affect the outcome of the test 

The mean light intensity calculated from a range of representative positions, measured once 

during the study, was 7490 lux (by cosine receptor), with a maximum range of ± 11 %. 

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6 REFERENCES 

1 OECD Guidelines for Testing of Chemicals. Test Guideline 201, Alga, Growth 

Inhibition Test. Adopted 23 March 2006. 

2 Official Journal of the European Communities, L 383 A, Part C.3, Algal inhibition 

test. 29 December 1992. 

3 METI Guidelines for Testing of Chemicals. Test Guideline 2-7-7, Algae growth 

inhibition studies. 

4 Miller W E, Greene J C and Shiroyama T (1978). The Selenastrum capricornutum

Printz. Algal Assay Bottle Test: Experimental Design, Application and Data 

Interpretation Protocol. EPA-600/9-78-018, Corvallis, OR. 

5 Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving 

Waters to Freshwater Organisms 4th Edition, 2002. US EPA 

6 Dunnett C W (1964). New Tables for Multiple Comparisons with a Control. 

Biometrics, 20, 482-491. 

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TABLE 1 ANALYTICAL RESULTS 

Nominal conc of 

[ Measured conc of 

[

(mg l-1) 

Mean measured 

conc of [

(% saturation) 0 hour 72 hour (mg l-1) 

Control <0.003 <0.003 <0.003 

4.38 17 10 13.5 

8.75 40 24a 32.0 

17.5 71 49 60.5 

35 130 100 115 

70 260 170 215 

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

a

 Mean of triplicates 24, 25, 22 mg l-1. 

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TABLE 2 ALGAL CELL PARTICLE DENSITIES 

Nominal conc 

of 

Mean 

measured 

conc of Replicate Algal cell particle density  (x 104

 ml-1) 

(% saturation) (mg l ) 24 

hours 

48 

hours 

72 

hours 

 A 0.756 2.57 10.00 

Control - B 0.965 2.75 10.5 

 Mean 0.861 2.66 10.3 

 A 0.683 5.07 26.9 

4.38 13.5 B 0.803 5.38 34.0 

 Mean 0.743 5.23 30.5 

 A 1.04 6.99 25.7 

8.75 32.0 B 0.946 5.25 43.3 

 Mean 0.993 6.12 34.5 

 A 0.984 4.99 28.3 

17.5 60.5 B 0.912 5.09 19.5 

 Mean 0.948 5.04 23.9 

 A 0.924 3.03 11.5 

35 115 B 0.856 2.82 9.63 

 Mean 0.890 2.93 10.6 

 A 0.305 0.545 2.03 

70 215 B 0.264 0.637 1.81 

 Mean 0.285 0.591 1.92 

Inoculum (Day 0) cell density = 0.109 × 104 cells ml-1 

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TABLE 3 MEAN YIELDS OVER THE TEST PERIOD 

Nominal conc of Mean measured 

conc of 

(% saturation) (mg l )

Mean yield 

(0-72 hours) 

Percentage 

of control 

Control - 10.1 - 

4.38 13.5 30.3 299 

8.75 32.0 34.4* 339 

17.5 60.5 23.8 235 

35 115 10.5 103 

70 215 1.81 18 

* Significant difference (P < 0.05) from the control 

TABLE 4 MEAN GROWTH RATES OVER THE TEST PERIOD 

Nominal conc of 

(% saturation) 

Mean measured 

conc of 

(mg l ) 

Mean growth 

rate 

(0-72 hours) 

Percentage 

of control 

Control - 1.51 - 

4.38 13.5 1.88* 124 

8.75 32.0 1.92* 127 

17.5 60.5 1.8* 119 

35 115 1.52 101 

70 215 0.956* 63 

* Significant difference (P

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TABLE 5 TEST SOLUTION pH MEASUREMENTS 

pH 

Nominal conc of 

Mean measured 

conc of 

(% saturation) (mg l-1)

0 Hours 72 hours 

Control - 7.91 8.48 

4.38 13.5 8.06 10.00 

8.75 32.0 8.18 9.99 

17.5 60.5 8.21 10.14 

35 115 8.25 9.53 

70 215 8.26 8.48 

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FIGURE 1 EFFECT ON GROWTH OF GREEN ALGA PSEUDOKIRCHNERIELLA 

SUBCAPITATA AFTER 72 HOURS 

NB: Mean measured test concentrations shown 

0.1

1

10

100

Day 0 Day 1 Day 2 Day 3

Day 

Particle Density (x104 cells ml-1)

Con.

13.5 mg l-1

32.0 mg l-1

60.5 mg l-1

115 mg l-1

215 mg l-1

0.1

1

10

100

Day 0 Day 1 Day 2 Day 3

Day 

Particle Density (x104 cells ml-1)

Con.

13.5 mg l-1

32.0 mg l-1

60.5 mg l-1

115 mg l-1

215 mg l-1

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APPENDIX 1 

Nominal Composition of AAP-Medium with extra bicarbonate 

Component mg l-1

NaHCO3 115 

NaNO3 25.5 

MgCl2·6H2O 12.16 

CaCl2·2H2O 4.41 

MgSO4·7H2O 14.7 

K2HPO4 1.044 

FeCl3·6H2O 0.160 

Na2EDTA·2H2O 0.300 

H3BO3 0.186 

MnCl2·4H2O 0.416 

ZnCl2 0.00327 

CoCl2·6H20 0.00143 

Na2MoO4·2H2O 0.00726 

CuCl2·2H2O 0.000012 

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APPENDIX 2 

Analytical Method for the Determination of in aqueous water samples 

Samples of algal culture 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 at 15 ml min-1

Carrier gas Helium at 1.0 ml min-1

 Temperature Program Initial temp 40 °C; hold for 3 min then 

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

Mass Spectrometer 

 Start time 3 min 

 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 at ~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 0.15 min 

 Xfer IS time 0.15min 

 Rinse line time 0.2 min 

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 Purge line time 0.15 min 

 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 4.7 x 106

 (arbitrary units) using Xcalibur 1.4. 

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APPENDIX 2, FIGURE 1 

Typical Chromatograms 

(A) 306 μg l-1 standard concentrations of 

(B) 8.75 % of test solution of (× 84 dilution) 

RT: 4 .00 - 11.0 0 SM: 7 G

4 5 6 7 8 9 1 0 1 1

Tim e (m in)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

100

Relative Abundance

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

100

Relative Abundance

H F O -1233zd

RT : 5.4 4

In te rn a l S ta nd a rd

R T: 9 .18

NL :

6.57 E 5

m /z= 

94.5 -9 5 .5+

129 .5 -1 30 .5 

F : M S 

Genesis 

09-

0025BC _132

NL :

3.77 E 4

m /z= 

64.5 -6 5 .5+

6 6 .5 -6 7 .5 F : 

M S G ene sis 

09-

0025BC _132

RT: 4 .00 - 11.0 0 SM: 7 G

4 5 6 7 8 9 1 0 1 1

Tim e (m in)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

100

Relative Abundance

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

100

Relative Abundance

H F O -1233zd

R T: 5.44

In te rn a l S ta nd a rd

R T: 9 .18

NL :

8.36 E 5

m /z= 

94.5 -9 5 .5+

129 .5 -1 30 .5 

F : M S 

Genesis 

09-

0025BC _128

NL :

4.29 E 4

m /z= 

64.5 -6 5 .5+

6 6 .5 -6 7 .5 F : 

M S G ene sis 

09-

0025BC _128

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APPENDIX 3 

GLP CERTIFICATE 

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APPENDIX 4 

TEST PROTOCOL 

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Report No BR0027/B 

HFO- Toxicity to the green alga Pseudokirchneriella subcapitata 

32 

SANITIZED
Report No BR0027/B 

HFO- Toxicity to the green alga Pseudokirchneriella subcapitata 

33 

SANITIZED
Report No BR0027/B 

HFO- Toxicity to the green alga Pseudokirchneriella subcapitata 

34 

SANITIZED
Report No BR0027/B 

HFO- Toxicity to the green alga Pseudokirchneriella subcapitata 

35 

SANITIZED
Report No BR0027/B 

HFO- Toxicity to the green alga Pseudokirchneriella subcapitata 

36 

SANITIZED
Report No BR0027/B 

HFO- Toxicity to the green alga Pseudokirchneriella subcapitata 

37 

CIRCULATION 

Copy number 

1 

(double sided bound)

A Tveit 

2 

(electronic copy)

A Tveit 

GEL locator M Webley Brixham Environmental Laboratory, AstraZeneca UK 

Limited 

GEL locator Reports Centre M. Webley, Brixham Environmental Laboratory, 

AstraZeneca UK Limited 

SANITIZED