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 subcapitataReport No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata
Study Number: 09-0025/B

2 CONFIDENTIAL (CATEGORY B)

Not to be photocopied or microfilmed. This doc ument contains information confidential and trade secret to It should not be released in any form to an outside party, nor should information contained he rein be used by a registration authority to support registration of any other product w ithout the written per mission ofReport No BR0027/B 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 CummingReport No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata
Study Number: 09-0025/B

6 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.......................................................... 19Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata
Study Number: 09-0025/B

7 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............................................................................37Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

8 

### 1 SUMMARY

Sponsor

Sponsor contact Internationa l 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 Internationa l 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 Inhib ition 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 (E y C50) = 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 wa s observed during the test at 13.5, 32.0, 60.5 mg l -1

. The test substance was dosed as a gas.Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

9 

### 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 releva nt 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 E nvironmental Laboratory on 6 April 2009 and on receipt was assigned the Brixham test substa nce 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.Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

10 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. 12 86 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 pha se was used as inoculum for the test. The culture was grown in the medium, and under th e environmental conditi ons, 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 te st 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 in to 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 ther e were 6 replicates for the control and each test concentration in total. One blank ve ssel (without algal inoculum) was incubated concurrently for the control and each test con centration to be used to determine ‘background’ particle density at each samp ling 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 C oulter counter model Z1 and counting at a lower threshold equivalent spherical diameter of approximately 2.3 μm. E ach replicate test vessel was inoculated with 0.35 ml of the inoculum cu lture to give a nominal cell density of 0.100 x 10 4 cells ml -1 . One 60 ml volume of Coulter elect rolyte, inoculated in the sameReport No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

11 manner, had a cell density of 0.109 x 10 4 cells ml -1 . This value was used for growth calculations.

After 24, 48 and 72 hours, appropriate vessels we re removed for the blank, control and each test concentration. The appropria te blank particle count was subt racted 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 concentr ations 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 appropria te test solutions were dispen sed 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 th e 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 solu tions were measured at 0 and 72 hours using a purge and trap injection with gas chromatogr aphy 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 si ngle 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 continuous ly 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).Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

12 

### 5 RESULTS

5.1 Analytical data

The concentrations of determined in the te st 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 prepara tion 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 data Algal 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 si gnificant 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 E y C50 was calculated via linear interpolation (Ref 5). The E r C50 could not be calculated as <50 % inhibition was seen.

5.2.1 Yield This response is defined as the biomass at th e end of the test minus the starting biomass (Ref 1). For the purposes of calculation, the ce ll 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 signifi cant 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 signif icant 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 inhib itory effect, thus has been discounted.

The results obtained from these yield analyses, based on mean measured test concentrations, were as follows:Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

13 No observed effect concentration (NOEC) = > 215 mg l -1 Lowest observed effect concentration (LOEC) = > 215 mg l -1 Median effective concentration, yield (E y C50) = 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 N 1 = Cell density at start of test N 2 = 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 horme tic effect rather than an inhi bitory 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 exponent ial 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.Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

14 5.2.4 Validity criteria The validity criteria specified in th e OECD 201 guideline (Ref 1) are;

(i) To achieve ≥ 16 fold exponential increase in bioma ss 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 c ontrol 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 ra nge of representative positions, measured once during the study, was 7490 lux (by cosine receptor), with a maximum range of ± 11 %.Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

15 

### 6 REFERENCES

1 OECD Guidelines for Testing of Chemi cals. Test Guideline 201, Alga, Growth Inhibition Test. Adopted 23 March 2006.

2 Official Journal of the European Comm unities, 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: Expe rimental Design, Application and Data Interpretation Protocol. EPA-600/9-78-018, Corvallis, OR.

5 Short-term Methods for Estimating the Chr onic Toxicity of Effluents and Receiving Waters to Freshwater Orga nisms 4th Edition, 2002. US EPA

6 Dunnett C W (1964). New Tables for Mult iple Comparisons with a Control. Biometrics, 20 , 482-491.Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

16 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 24 a 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

.Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

17 TABLE 2 ALGAL CELL PARTICLE DENSITIES

Nominal conc of Mean measured

conc of Replicate Algal cell particle density (x 10 4 ml -1 )

(% saturation) (mg l ) 24 hours 48 hour s 72 hours A 0.756 2.57 10.00 - B 0.965 2.75 10.5 Control 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 × 10 4 cells ml -1Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

18 TABLE 3 MEAN YIELDS OVER THE TEST PERIOD

Nominal conc of Mean measured conc of

(% saturation) (mg l ) Mean yi eld

(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 < 0.05) from the controlReport No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

19 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 8.48Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

20 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
DayParticle Density (x104cells 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
DayParticle Density (x104cells 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 -1Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

21 APPENDIX 1 Nominal Composition of AAP-Med ium with extr a bicarbonate

Component mg l -1

NaHCO 3 115 NaNO 3 25.5 MgCl 2 ·6H 2 O 12.16 CaCl 2 ·2H 2 O 4.41 MgSO 4 ·7H 2 O 14.7 K 2 HPO 4 1.044 FeCl 3 ·6H 2 O 0.160 Na 2 EDTA·2H 2 O 0.300 H 3 BO 3 0.186 MnCl 2 ·4H 2 O 0.416 ZnCl 2 0.00327 CoCl 2 ·6H 2 0 0.00143 Na 2 MoO 4 ·2H 2 O 0.00726 CuCl 2 ·2H 2 O 0.000012Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

22 APPENDIX 2 Analytical Method for the Determinatio n 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 minReport No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

23 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 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 4.7 x 10 6 (arbitrary units) using Xcalibur 1.4.Report No BR0027/B Toxicity to the green alga Pseudokirchneriella subcapitata

24 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 100Relative Abundance 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 100Relative 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 100Relative Abundance 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 100Relative 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 _128Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

25 APPENDIX 3

GLP CERTIFICATEReport No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

26 APPENDIX 4

TEST PROTOCOLReport No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

27Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

28Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

29Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

30Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

31Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

32Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

33Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

34Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

35Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

36Report No BR0027/B HFO- Toxicity to the green alga Pseudokirchneriella subcapitata

37 CIRCULATION
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