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### REPORT

2 - b romo - 3,3,3 -trifluoropropene : Assessment of Ready Biodegradability - Sealed -Vessel Carbon Dioxide Evolution Test

HLS study number: WA G0021 Version ID: Final Report Issue date: 30 April 2013H untingdon Life Sciences WA G0021

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## Sponsor , T est Facility and T

est Site Details Sponsor American Pacific Corporation 3883 Howard Hughes Parkway Suite 700 Las Vegas NV 89169 USA Test facility Huntingdon Life Sciences Eye Research Centre Eye Suffolk IP23 7PX UKH untingdon Life Sciences WA G0021

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## Ta ble of Content

s Sponsor, Test Facility and Test Site Details ................................ ................................ .......... 2 Compliance with Good Laboratory Practice ................................ ................................ .......... 5 Quality Assurance Statement ................................ ................................ ................................ 6 C ontributing Scientists ................................ ................................ ................................ .......... 7 Summary ................................ ................................ ................................ .............................. 8 1. Introduction ................................ ................................ ................................ .................. 9 2. Test Substance ................................ ................................ ................................ .......... 10 3. Experimental Procedure ................................ ................................ ............................. 11 3.1 Reagents ................................ ................................ ................................ ........ 11 3.2 Inoculum ................................ ................................ ................................ ......... 11 3.3 Test procedure ................................ ................................ ................................ 11 3.4 Total inorganic carbon (TIC) analysis ................................ .............................. 12 3.5 Calculation of results ................................ ................................ ....................... 13 3.6 Validity criteria ................................ ................................ ................................ 13 3.7 Deviations from protocol ................................ ................................ ................. 14 4. Maintenance of Records ................................ ................................ ............................. 15 5. Results ................................ ................................ ................................ ....................... 16 5.1 Calculation of the theoretical organic carbon content ................................ ...... 16 5.2 Sealed - vessel carbon dioxide production test ................................ ................. 16 6. Discussion and Conclusion ................................ ................................ ........................ 18 

## List of Figures Figure 1 Degradation of the test and reference substances ................................ ....... 19 

## List of T ables Table 1 Inorganic carbon measurements in the control cultures ............................... 20 Table 2 Inorganic carbon measurements in the cultures containing the test substance

................................ ................................ ................................ .................... 20 Table 3 Inorganic carbon measurements in the cultures containing the reference

substance ................................ ................................ ................................ .... 21 Table 4 Inorganic carbon measurements in the cultures containing the test and

reference substance (inhibition assay) ................................ ......................... 21 Table 5 Percentage biodegradation of the test and reference substances ................ 22H untingdon Life Sciences WA G0021

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## List of Ap pendices Appendix 1 Certificate of analysis ................................ ................................ ................... 23 Appendix 2 Preparation of mineral salts medium ................................ ............................ 24 Appendix 3 Compliance statement ................................ ................................ ................. 25H untingdon Life Sciences WA G0021

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## Contributing Scientist s 2 - b romo - 3,3,3 -trifluoropropene : Assessment of Ready Biodegradability - Sealed -Vessel Carbon Dioxide Evolution Test 

### Study management Aquatic Ecotoxicology and Biodegradation Robert A Dickinso n BSc Study Di rectorH untingdon Life Sciences WA G0021

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## Summary The ul timate biodegradabilit y of 2 - bro mo - 3,3,3 - trifluoropropene was assessed in the Sealed Vessel CO 2 Evo lut io n test using U.S. Envi ronmental

Protection Agency (EPA), Office of Prevent io n, Pesticides and Toxic Substances, (OPPTS) Method 835.3140, ‘Ready Biode gradabilit y - CO 2 in Sealed Vessels (Headspace Test) adopted October 2008 and Test Guideline OECD 310, ‘CO 2 in sealed vessels (Headspace Test)’ adopted March 2006. It includes aspects of International Standard ISO 14593, ‘Evaluat io n of ult imate aerobic biodegradabili

ty of organic com

pounds in aqueous medium

– Method by analysis o f inorganic carbon in sealed vessels (CO 2 headspace test)’ adopted March 1999. The test consisted of two con trol groups, two test groups, a positive control group containing a biodegradable reference substance and a group that was used to assess the inhibitory effect of the test substance (test and reference substance together

– inhibit io n assay). The control groups consisted of mineral salts medium (MSM) and act ivated sludge (4 mg/L suspended solids); the test groups contained inoculated mineral salts medium plus 2 - bromo - 3,3,3 - trifluoropropene at a concentration of 10 mg C/L. The positive control group contained inoculated MSM and sodium benzoate (10 mg C/L) and the inhibit io n a ssay contained inoculated MSM, the test substance and sodium benzoate (both at 10 mg C/L). Test, control and reference mixtures were incubated for 28 days on a reci procating shaker at a tem perature of 22  2  C. On each analysis occasion, the appropriate v ials were injected with 1 ml of NaOH (nominally 7M) and shaken for at least one hour. The total inorganic carbon content of each sample was then determined ( using a carbon analyser) . After 28 days, the mean carbon dio xide (CO 2 ) production by the controls was 13.4% of the applied organic carbon. Sodi um b enzoate alone had been biodegraded by 78.1 % by Day 7 and , at most, by 81.8 % on Day 14 . In the presence of 2 - bro mo - 3,3,3 - trifluoropropene , s odi um b enzoate had been degraded by 78.4% by

Day 7 which confirme d that 2 - bromo - 3,3,3 - trifluoropropene was not inhibitory to the activit y of

the microbial inoculum. These results confirm ed that the inoculum was viable and that the test was valid. Production of

CO 2 by mixtures containing 2 - bromo - 3,3,3 - trifluoropropene w as equivalent to , at most, 1.1 % of the theoretical maximum by Day 2 1 . Substances are considered to be readily biodegradable in this test if CO 2 production is equal to or greater than 60% of the theoretical value within ten days o f th e level achieving 10% . Therefore, 2 - bromo - 3,3,3 - trifluoropropene was considered not to be readily biodegradable under the condit io ns of this test .H untingdon Life Sciences WA G0021

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## 1. Introduction The obj ect ive o f thi s study was to assess the ult imate biodegradabilit y of 2 - bro mo - 3,3,3 - trifluoropropene . Substanc es are considered to be readily biodegradable if the CO 2 production exceeds 60% of the theoretical maximum wit hin 28 days and if this level o f CO 2 production i s attained wit hin ten days of degradat ion exceeding 10%. It was conducted in accordance wit h U. S. Environmental Protection Agency (EPA), Office of Prevent io n, Pesticides and Toxic Substances, (OPPTS) Method 835.3140, ‘Ready Biodegradabilit y - CO 2 in Sealed Vessels (Headspace Test) adopted October 2008 and Test Guideline OECD 310, ‘CO 2 in sealed vess els (Headspace Test)’ adopted March 2006. It includes aspects of International Standard ISO 14593, ‘Evaluat io n of ult imate aerobic biodegradabilit y of

organic com

pounds in aqueous medium

– Method by analysis o f inorganic carbon in sealed vessels (CO 2 head sp ace test)’ adopted March 1999. This study

design was considered to be the most appropriate for assessing the ready biodegradabilit y of

vo lat ile liquids with low bo iling points. The parameter determined in this test is the amount of carbon dioxide produc ed in situ by mixtures containing 2 - bromo - 3,3,3 - trifluoropropene , mineral salts medium and act ivated sl udge (4 m

g/L suspended so lids) inoculum over a period of 28 days o f incubat io n at tem peratures in the range 20 to 24  C. The protocol was approved by Hunt ingdon Life Sciences Management and the Study Di rector on 8 August 2012 and by the Sponsor on 9 August 2012 . The experimental start and complet io n dates were 8 January 2013 and 8 February 2013 , respectively . The study was conducted at Huntingdon Life Sciences, Eye Research Centre, Eye, Suffo lk, IP23 7PX.H untingdon Life Sciences WA G0021

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## 2. Te st Substance Name: 2 - bromo - 3,3,3 - trifluoropropene Chemical Name: 2 - bromo - 3,3,3 - trifluoroprop - 1 - ene CAS Number: 1514 - 82 - 5 Use: Fi re ext inguishing agent Molecular Formula: CF 3 CBr=CH 2 Molecular Weight: 174.948 Physical State: Clear liquid wi th a slight yellow tint Source: American Pacific Corp. Batch Number: WN CC - BTP111128 Puri ty : >99.6 % (as received with stabilizers) Specific gravit y: 1.6 5 Storage: Stored free of air and water at room temperature Re - assay date: 12 January 2014 Date received: 2 March 2012 Certificate of analysis: Appendix 1H untingdon Life Sciences WA G0021

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## 3. Experimental Procedure 3.1 Reagents The reagents used in the sealed - vessel carbon dioxide evo lut io n test were: Sodi um b enzoate (AR grade) as the reference substance. Sodi um hydroxide pellets (AR grade) used to prepare the 7M sodium hydroxide solution used fo r the absorption of

inorganic carbon. 

### 3.2 Inoculum A sample of act ivated sludge was obtained fro m th e aeration tank of Worlingwo rth sewage treatment works, which treats predominantly do me stic waste three days before the start of the test. On the day

of collect io n, the sample was passed through a sieve with a mesh of ca . 1 mm

2 . A sub - sample ( ca . 500 m L ) was removed and centrifuged at ca . 3000 rpm for ca . 1 minute and the supernatant removed. The sample was then made up to volume with mineral salts medium (MSM, Appendix 2 ) and centrifuged. This procedure was performed th ree times and the sample was maintained aerated until required. Aliquots (10 m L ) of a ho mo genised sample of the washed activated sludge were filtered through dried (approximately 105  C) and pre - weighed What ma n GF/C filter papers. The filters were dried fo r at least one hour, allowed to cool and re - weighed. The solids level (mixed liquor suspended so lids, MLSS) in the sludge was determined and then an appropriate vo lu me used to inoculate control and test vessels to give a final suspende d solids concentration of 4 mg/L . 

### 3.3 Test procedure Three days before test substance addit io n, u lt rap ure water (Appendix 2) was added to each of two , five - lit re glass vessels , fo llo wed by the vol umes of each of the stock solutions required to prepare five litres of MSM. Each culture bottle was then inocul ated and a magnet ic sti rring bar was added . The bottles were stoppered , then each was placed on an electricallyoperated magnet ic st irrer. The vessels were aerated until the day of test init iation (Day 0) wi th a supply of ai

r that had been treated to rem ove carbon dioxide by

passing i t through cylinders containing fused calcium chloride and soda lime. On Day 0 of the test, the pH of the th

ree - day old inoculated MSM was determined and adj usted wi

th HCl to 7.4  0.2 as required . The reference substance so di um b enzoate was added as an aqueous stock solut io n (1.72 g/L ) to a reservoir of the pre - aerated inoculated MSM labelled reference to gi ve a no minal concentration of 10 mg C /L . Blank - control s contained inoculated MSM alone. Aliquots (100 mL) of each MSM were added to the respective (160 ml) vials. Informat io n from the Sponsor indicated that the test substance was a vo lat ile liquid. Therefore the test substance was added direct ly to vials (160 m L) containing 100 m

L of the inoculated MSM as a vo lu me (3 μL calculated based on specific gravit y) to give a final, no minal test concentration of 10 mg C/L (1 m gC/100 mL , Protocol

deviat io n, see Section 3.7 ). The liquid vo lu me to headspace ratio was approximately 1 : 1.7.H untingdon Life Sciences WA G0021

12 An inhibit io n assay

was prepared by

adding aliquots (3 μL ) of the test substance to vi als (160 mL) containing 100 mL of the inoculated MSM plus the reference substance. The no minal concentration f

or both the test and reference substance was 10 mg C/L (1 mg C/100 mL). The vials were immediately sea led with silicone and crimp seals. The vials were incubated horizontally

on an enclosed reciprocating shaker . The mixtures prepared for the test are summarised in the fo llo wing table: Vial number Contents 1 - 23 Controls - mineral salts medium plus inoculum (4 mg solids /L ) 1 - 17 Reference - inoculated mineral salts medium plus sodium benzoate (10 mg C/L ) 1 - 23 Test - inoculated mineral salts medium plus test substance (10 mg C/L ) 1 - 9 Test plus Reference - inoculated mineral salts medium plus sodium benzoate (10 mg C/L ) plus

test substance (10 mg C /L ) The minimum and maximum temperature of the test area and of a vo lu me of water ( ca . 100 mL ) held in a culture vial under test conditions was recorded, at intervals, using a thermometer . The tem

perature of the 100 mL vo lu me of water is considered representative of tem perature con

trol f or thi s test system . 

### 3.4 Total inorganic carbon (TIC) a nalysis On Days 0, 7, 1 4 , 2 1 and 28 , designated cultures were injected (through the septum) wi th concentrated so dium hydroxide ( 1 m L, no minally 7M) and shaken for a further period of approximately

one hour. The content of each treated culture then carefully transferred from the cul ture vi als to carbon analyser via ls and these were immediately sealed. TIC analysi s was conduct ed on th ree cultures f

rom each group on each sampling occasion except on the last day

of the test, whe n five cultures were analysed. The TIC content of each culture was determined using an OI Analyt ical Model 1010 Total Organic Carbon Analyse r and autosampler fit ted wi th a septum piercing apparatus and associ ated software (WinToc V5.2TM). In thi s analysis, inorganic carbon concentrations were determined by m easuring the amount of carbon dio xide released by acidificat io n of a sample, measured by a sample loop ( no minally 5 mL ). To release carbon dio xide, phosphoric acid (400  L , 5% v/v) was automat ically injected, the sample was purged and the gas released was then concentrated by trapping. It was desorbed and carried to a non - di spersive infrare d detector (NDIR) fo r detection . A so lut io n of sodium carbonate was used as a calibration standard on each analysis occasio n.H untingdon Life Sciences WA G0021

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### 3.5 Calculation of results Theoretical inorganic carbon Assuming that there was 100% mineralisat io n of the test substance, the theoret ic al amount of inorganic carbon (ThIC) produced by test or reference mixtures, after blank - correction by the control cul tures, is equal to the amount of Total Organic Carbon (TOC) added, as the test substance, at the start of the test: i. e., ThIC = TO C By adding concentrated NaOH to the test cultures and shaking, gaseous CO 2 evo lved during biodegradat io n was trapped as carbonate or other forms of inorganic carbon in the liquid phase. The total mass of inorganic carbon in the test vessel (TIC) was, the refore, calculated in the fo llo wing way: TIC = (V 1 x C 1 ) where: V 1 = the volume (in litres) of liquid in the test vessel C 1 = the concentration of TIC in the liquid phase (m gC/L) Calculation of percentage biodegradation The biodegradat io n was e xpressed as a percentage of the ThIC based on the amount of test com pound that was added init ially . It was calculated in the fo llo wing way: %D = (TIC t – TIC b ) x 100

TOC where: TIC t = the TIC concentration ( mg) in the test vial at time t. TIC b = the overall mean TIC concentration (mg) in the blank - control vials at time t. TOC = Total

organic carbon concentration (mg) added to the test vial at the start of the test. 

### 3.6 Validity criteria The test was considered valid if:  The biodegradat io n o f the ref erence substance was ≥60% by Day 14.  The CO 2 evo lved by the blank controls was ≤15% of the applied organic carbon (10 mgC/L ) . This is consistent with the validit y cri teria outlined in ISO14593.H untingdon Life Sciences WA G0021

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### 3.7 Deviations from p rotocol The minimum temperature of the shaker used to incubate the test was slight ly below the minimum temperature (20oC) on two consecut ive days during the study (Days 5 and 6). The maximum temperature of the shaker was slightly above the maximum (24oC) on Day 7 of the test. These deviat ion s were not considered significant or to have affected the integrity of the study . The protocol specified that the test concentration to be used was 20 mgC/L. In error, the test concentration used was 10 mgC/L. As the guidelines used for the study speci fy a concentration range of 2 to 40 mgC/L and there was no significant biodegradation of the test substance or inhibit io n o f th e reference substance (in the inhibit io n assay ) observed during the test, this deviat io n was not considered significant or to hav e affected the integrit y of the study .H untingdon Life Sciences WA G0021

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## 4. Ma intenance of Records This report has been compiled fro m original data, which will be stored, together with a copy of the final report, a copy

of the protocol

and amendments in Hunt ingdon Life Sciences’ Archives, Hu nt ingdon Life Sciences Ltd, Eye, Suffol k, UK. Records will be retained for a minimum period of at least one year from the date of issue of the final report. At the end of the one year retention period the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any i tem be di scarded wi thout the Sponsor’s knowledge. Hunt ingdon Life Sciences will retain the Qualit y Assurance records relevant to this study f or twenty y ears, and a copy

of the final report in its arch ives indefinitely .H untingdon Life Sciences WA G0021

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## 5. Results 5.1 Calculation of the theoretical organic carbon content 2 - b romo - 3,3,3 - trifluoropropene The theoretical organic carbon content of 2 - bro mo - 3,3,3 - trifluoropropene was calculated fro m it s em

pirical form

ula to be : CF 3 CBr=CH 2 MW = 1 74. 95 Carbon content = 20.6 % Sodium benzoate The theoretical organic carbon content of sodium benzoate was calculated fro m it s em pi ri cal fo rm ula to be : C 7 H 5 O 2 Na MW = 144 Carbon content = 58.3% 

### 5.2 Sealed - vessel carbon dioxide production test The levels o f inor ganic carbon in control, test and reference mixtures at intervals during the test are given in Tables 1

– 4 . The percentage biodegradation values against time are given in in Table 5 . Biodegradation of the test and reference substances are illustrated graphically in Figure 1 . After 28 days, the mean carbon dio xide (CO 2 ) production by the controls was 13.4% of the applied organic carbon (10 mgC/L ) . Sodi um b enzoate alo ne had been biodegraded by 7 8 . 1 % by Day 7, and , at most, 81.8% on Day 14 ( Table 5 ). In the presence of 2 - bromo - 3,3,3 - trifluoropropene , s odi um b enzoate (10 mgC/L) had been degraded by 78.4% by

Day 7 which confirmed that 2 - bromo - 3,3,3 - trifluoropropene was not inhibitory to the activit y of the microbial inoculum ( Table 5 ) . These results confirmed that the inocul um was viable and that the test was valid .H untingdon Life Sciences WA G0021

17 Production of

CO 2 by mixtures containing 2 - bromo - 3,3,3 - trifluoropropene was equivalent to , at most, 1.1 % of the theoretical maximum by Day 21. Substances are considered to be readily biodegradable in this test if CO 2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. Therefore, 2 - bromo - 3,3,3 - trifluoropropene was considered not to be readily biodegradable under the condit io ns of this test . The pH of the MSM at the start of the test ranged from 7.5 2 to 7. 53. The tem perature of a 1 00 mL vo lu me of water held under test condit io ns ranged from 19. 7  C to 24.2  C during the test period (Protocol deviat io n, see Section 3.7 ).H untingdon Life Sciences WA G0021

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## 6. Discussion and Conclusion Production of

CO 2 by mixtures containing 2 - bromo - 3,3,3 - trifluoropropene was equivalent to , at most, 1.1 % of the theoretical maximum by Day 21. Substances are considered to be readily biodegradable in this test if CO 2 production is equal to or greater than 60% of the theoretical value within ten days o f th e level achieving 10%. Therefore, 2 - bromo - 3,3,3 - trifluoropropene was considered not to be readily biodegradable under the condit io ns of this test . Carbon dio xide evo lut io n by the controls was <15% of t he applied organic carbon load by Day 28. Sodium benzoate alone had bee n biodegraded by 78.1% by Day 7. The biodegradat io n of sodium benzoate in the presence of the test substance was mo nit ored in order to assess whether any inhibitory effects were exerted on the activit y of the microbial inoculum. None were observed. Th ese resul

ts confi rm that the inocul

um was viable and that the test was valid.H untingdon Life Sciences WA G0021

19 Figure 1 Degradation of the test and reference substances

0 10 20 30 40 50 60 70 80 90 100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29Percentage Degradation

Time in Days

2-bromo-3,3,3-trifluoropropene (10 mgC/L) Reference substance (10 mgC/L) Reference substance (10 mgC/L) in the inhibition assay

Pass level (60%)H untingdon Life Sciences WA G0021

20 Table 1 Inorganic carbon measurements in the c ontrol cultures Day Analysis

Replicate Control culture (mg IC/L)

1 2 3 4 5 0 1 1.827 2.197 2.078 - -

2 1.776 2.2 00 2.111 - - Mean 1.802 2.198 2.094 - - 7 1 2.713 2.864 2.684 - -

2 2.755 2.847 2.695 - - Mean 2.734 2.855 2.690 - - 1 4 1 2.943 2.827 3.06 0 - -

2 2.844 2.9 00 3.068 - - Mean 2.894 2.864 3.064 - - 2 1 1 3.034 3.182 3.125 - -

2 3.1 00 3.232 3.174 - - Mean 3.067 3.207 3.150 - - 28 1 3.279 3.147 3.198 3.546 3.577

2 3.345 3.19 0 3.26 0 3.571 3.617 Mean 3.312 3.168 3.229 3 .558 3.597

Table 2 Inorganic carbon measurements in the cultures containing the

test substance Day Analysis

Replicate Control culture (mg IC/L)

1 2 3 4 5 0 1 2.193 2.262 2.010 - -

2 2.201 2.225 2.083 - - Mean 2.197 2.243 2.046 - - 7 1 3.041 2.699 2.784 - -

2 3.051 2.770 2.809 - - Mean 3.046 2.735 2.797 - - 14 1 2.994 3.079 2.962 - -

2 3.046 3.079 2.946 - - Mean 3.020 3.079 2.954 - - 21 1 3.403 3.231 3.346 - -

2 3.349 3.210 3.463 - - Mean 3. 376 3.220 3.405 - - 28 1 3.497 3.260 3.563 3.429 3.290

2 3.506 3.302 3.588 3.394 3.310 Mean 3.502 3.281 3.575 3.412 3.300H untingdon Life Sciences WA G0021

21 Table 3 Inorganic carbon measurements in the cultures containing the

reference substance Day Analysis

Replicate Control culture (mg IC/L)

1 2 3 4 5 0 1 1.900 2.154 2.004 - -

2 1.996 2.157 2.115 - - Mean 1.948 2.156 2.060 - - 7 1 10.469 10.435 10.591 - -

2 10.495 10.745 10.663 - - Mean 10.482 10.590 10.627 - - 14 1 11.310 10.8 72 10.957 - -

2 11.350 11.129 11.084 - - Mean 11.330 11.001 11.020 - - 21 1 11.250 11.138 11.105 - -

2 11.219 11.138 11.275 - - Mean 11.235 11.138 11.190 - - 28 1 10.891 11.170 11.151 11.362 10.759

2 11.218 11.302 11.451 10.075 11.118 Mean 11 .055 11.236 11.301 10.719 10.938

Table 4 Inorganic carbon measurements in the cultures containing the

test and reference substance (inhibition assay) Day Analysis

Replicate Control culture (mg IC/L)

1 2 3 0 1 2.015 2.174 2.174

2 1.990 2.199 2.194 Mean 2.003 2.186 2.184 7 1 10.642 10.793 10.409

2 10.790 10.883 10.703 Mean 10.716 10.838 10.556 14 1 11.397 11.228 11.327

2 11.441 11.176 11.218 Mean 11.419 11.202 11.273H untingdon Life Sciences WA G0021

22 Table 5 Percentage biodegradation of the test and reference substances Group Culture

replicate Day number and percentage biodegradation

7 14 21 28 % degradation Mean % degradation Mean % degradation Mean % degradation Mean Reference substance - Sodium benzoate (10 mg C /L ) 1 77.2 78.1 83.9 81.8 80.9 80.5 76.8 76.8

2 78.3 80.6 80.0 78.6 3 78.7 80.8 80.5 79.3 4 - - - 73.5 5 - - - 75.7

Test substance - 2 -bromo 3,3,3 - trifluoropro pene (10 mg C /L ) 1 2.9 1.1 0.8 0.8 2.3 1.9 1.3 0.4

2 0 1.4 0.8 0 3 0.4 0.1 2.6 2.0 4 - - - 4 5 - - - 0 Inhibition assay – sodium benzoate (10 mg C /L ) 1 78.6 78.4 84.0 82.8 -

- -

-

2 79.8 81.8 - - 3 77.0 82.5 - -Huntingdon Life Sciences WA G0021

23 Appendix 1 Certificate of analysisHuntingdon Life Sciences WA G0021

24 Appendix 2 Preparation of mineral salts medium Dilution water The dilut io n water used to prepare stock and test solut io ns of mineral salts and the a queous sol ut io n of the reference substance was tap water that had been softened and treated by reverse osmosis (Elgastat, Prima 4 reverse osmosis unit) and then purified (Elgastat UHP unit); no minal resist ivit y,  18 MegOhm.cm. This water complies with the relevant standards [ Bri tish Standard (BS) 3978, 1987; American Society f or Testing and Materials (ASTM) D 1193 - 06] fo r classificat io n as Grade 1 or Ty pe 1 water for laboratory

use. Mineral salts medium The mineral salts medium (MSM) for the sealed - vessel CO 2 evo lut io n test was prepared by mixing 10 ml o f stock sol ution 1 wi

th 800 ml dilution water and then adding 1 ml o f sol ut io ns 2 to 4 to each litre of water required for the test. The pH of this media was 7.4 ± 0.2. Stock 1 g/L Potassi um dihydroge n phosphate 8.50 di - Potassi um hydrogen phosphate 21.75 di - Sodi um monohydrogen phosphate dihydrate 33.40 Ammo nium chloride 0.50 Stock 2 Magnesium sulphate heptahydrate 22.50 Stock 3 Calcium chloride dihydrate 36.40 Stock 4 Iron (III) chloride hexahydrate 0.25 Minor variat io ns in the weights taken are not considered to be significant.Huntingdon Life Sciences WA G0021

25 Appendix 3 C ompliance statement