Page 1 of 25

Working for a better future

REPORT

2-bromo-3,3,3-trifluoropropene :

Assessment of Ready Biodegradability -

Sealed-Vessel Carbon Dioxide Evolution Test

HLS study number: WAG0021

Version ID: Final Report

Issue date: 30 April 2013
Huntingdon Life Sciences

WAG0021

2

Sponsor, Test Facility and Test 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

UK
Huntingdon Life Sciences

WAG0021

3

Table of Contents

Sponsor, Test Facility and Test Site Details .......................................................................... 2

Compliance with Good Laboratory Practice .......................................................................... 5

Quality Assurance Statement................................................................................................ 6

Contributing 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 Tables

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 ................22
Huntingdon Life Sciences

WAG0021

4

List of Appendices

Appendix 1 Certificate of analysis...................................................................................23

Appendix 2 Preparation of mineral salts medium............................................................24

Appendix 3 Compliance statement .................................................................................25


Huntingdon Life Sciences

WAG0021

7

Contributing Scientists

2-bromo-3,3,3-trifluoropropene: Assessment of Ready Biodegradability -

Sealed-Vessel Carbon Dioxide Evolution Test

Study management 

Aquatic Ecotoxicology and Biodegradation

Robert A Dickinson BSc 

Study Director
Huntingdon Life Sciences

WAG0021

8

Summary

The ultimate biodegradability of 2-bromo-3,3,3-trifluoropropene was assessed in the SealedVessel CO2 Evolution test using U.S. Environmental Protection Agency (EPA), Office of 

Prevention, Pesticides and Toxic Substances, (OPPTS) Method 835.3140, ‘Ready 

Biodegradability - CO2 in Sealed Vessels (Headspace Test) adopted October 2008 and Test 

Guideline OECD 310, ‘CO2 in sealed vessels (Headspace Test)’ adopted March 2006. It 

includes aspects of International Standard ISO 14593, ‘Evaluation of ultimate aerobic 

biodegradability of organic compounds in aqueous medium – Method by analysis of 

inorganic carbon in sealed vessels (CO2 headspace test)’ adopted March 1999. 

The test consisted of two control 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 – inhibition assay). The control 

groups consisted of mineral salts medium (MSM) and activated 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 inhibition assay 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 reciprocating shaker at a 

temperature of 22  2C. On each analysis occasion, the appropriate vials 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 dioxide (CO2) production by the controls was 13.4% of the 

applied organic carbon.

Sodium benzoate alone had been biodegraded by 78.1% by Day 7 and, at most, by 81.8% on 

Day 14. In the presence of 2-bromo-3,3,3-trifluoropropene , sodium benzoate had been 

degraded by 78.4% by Day 7 which confirmed that 2-bromo-3,3,3-trifluoropropene was not 

inhibitory to the activity of the microbial inoculum. These results confirmed that the 

inoculum was viable and that the test was valid.

Production of CO2 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 CO2 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 conditions of this test.
Huntingdon Life Sciences

WAG0021

9

1. Introduction

The objective of this study was to assess the ultimate biodegradability of 2-bromo-3,3,3-

trifluoropropene. Substances are considered to be readily biodegradable if the CO2

production exceeds 60% of the theoretical maximum within 28 days and if this level of CO2

production is attained within ten days of degradation exceeding 10%. 

It was conducted in accordance with U.S. Environmental Protection Agency (EPA), Office of 

Prevention, Pesticides and Toxic Substances, (OPPTS) Method 835.3140, ‘Ready 

Biodegradability - CO2 in Sealed Vessels (Headspace Test) adopted October 2008 and Test 

Guideline OECD 310, ‘CO2 in sealed vessels (Headspace Test)’ adopted March 2006. It 

includes aspects of International Standard ISO 14593, ‘Evaluation of ultimate aerobic 

biodegradability of organic compounds in aqueous medium – Method by analysis of 

inorganic carbon in sealed vessels (CO2 headspace test)’ adopted March 1999. 

This study design was considered to be the most appropriate for assessing the ready

biodegradability of volatile liquids with low boiling points.

The parameter determined in this test is the amount of carbon dioxide produced in situ by 

mixtures containing 2-bromo-3,3,3-trifluoropropene, mineral salts medium and activated 

sludge (4 mg/L suspended solids) inoculum over a period of 28 days of incubation at 

temperatures in the range 20 to 24C.

The protocol was approved by Huntingdon Life Sciences Management and the Study

Director on 8 August 2012 and by the Sponsor on 9 August 2012.

The experimental start and completion dates were 8 January 2013 and 8 February 2013, 

respectively.

The study was conducted at Huntingdon Life Sciences, Eye Research Centre, Eye, Suffolk, 

IP23 7PX.
Huntingdon Life Sciences

WAG0021

10

2. Test Substance

Name: 2-bromo-3,3,3-trifluoropropene

Chemical Name: 2-bromo-3,3,3-trifluoroprop-1-ene

CAS Number: 1514-82-5

Use: Fire extinguishing agent

Molecular Formula: CF3CBr=CH2

Molecular Weight: 174.948

Physical State: Clear liquid with a slight yellow tint

Source: American Pacific Corp.

Batch Number: WNCC-BTP111128

Purity: >99.6% (as received with stabilizers)

Specific gravity: 1.65

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 1
Huntingdon Life Sciences

WAG0021

11

3. Experimental Procedure

3.1 Reagents

The reagents used in the sealed-vessel carbon dioxide evolution test were:

Sodium benzoate (AR grade) as the reference substance.

Sodium hydroxide pellets (AR grade) used to prepare the 7M sodium hydroxide solution used 

for the absorption of inorganic carbon.

3.2 Inoculum

A sample of activated sludge was obtained from the aeration tank of Worlingworth sewage 

treatment works, which treats predominantly domestic waste three days before the start of the 

test. On the day of collection, the sample was passed through a sieve with a mesh of 

ca. 1 mm2. A sub-sample (ca. 500 mL) 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 

three times and the sample was maintained aerated until required. Aliquots (10 mL) of a 

homogenised sample of the washed activated sludge were filtered through dried 

(approximately 105C) and pre-weighed Whatman GF/C filter papers. The filters were dried 

for at least one hour, allowed to cool and re-weighed. The solids level (mixed liquor 

suspended solids, MLSS) in the sludge was determined and then an appropriate volume used 

to inoculate control and test vessels to give a final suspended solids concentration of 4 mg/L.

3.3 Test procedure

Three days before test substance addition, ultrapure water (Appendix 2) was added to each of 

two, five-litre glass vessels, followed by the volumes of each of the stock solutions required 

to prepare five litres of MSM. Each culture bottle was then inoculated and a magnetic 

stirring bar was added. The bottles were stoppered, then each was placed on an electricallyoperated magnetic stirrer. The vessels were aerated until the day of test initiation (Day 0) 

with a supply of air that had been treated to remove carbon dioxide by passing it through 

cylinders containing fused calcium chloride and soda lime.

On Day 0 of the test, the pH of the three-day old inoculated MSM was determined and 

adjusted with HCl to 7.4  0.2 as required. 

The reference substance sodium benzoate was added as an aqueous stock solution (1.72 g/L) 

to a reservoir of the pre-aerated inoculated MSM labelled reference to give a nominal 

concentration of 10 mg C/L. Blank-controls contained inoculated MSM alone. Aliquots 

(100 mL) of each MSM were added to the respective (160 ml) vials.

Information from the Sponsor indicated that the test substance was a volatile liquid. 

Therefore the test substance was added directly to vials (160 mL) containing 100 mL of the 

inoculated MSM as a volume (3 μL calculated based on specific gravity) to give a final, 

nominal test concentration of 10 mgC/L (1 mgC/100 mL, Protocol deviation, see Section 

3.7). The liquid volume to headspace ratio was approximately 1 : 1.7.
Huntingdon Life Sciences

WAG0021

12

An inhibition assay was prepared by adding aliquots (3 μL) of the test substance to vials 

(160 mL) containing 100 mL of the inoculated MSM plus the reference substance. The 

nominal concentration for both the test and reference substance was 10 mg C/L (1 mg C/100 

mL).

The vials were immediately sealed 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 following 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 volume of water 

(ca. 100 mL) held in a culture vial under test conditions was recorded, at intervals, using a 

thermometer. The temperature of the 100 mL volume of water is considered representative 

of temperature control for this test system.

3.4 Total inorganic carbon (TIC) analysis

On Days 0, 7, 14, 21 and 28, designated cultures were injected (through the septum) with

concentrated sodium hydroxide (1 mL, nominally 7M) and shaken for a further period of 

approximately one hour. The content of each treated culture then carefully transferred from 

the culture vials to carbon analyser vials and these were immediately sealed.

TIC analysis was conducted on three cultures from each group on each sampling occasion

except on the last day of the test, when five cultures were analysed. 

The TIC content of each culture was determined using an OI Analytical Model 1010 Total

Organic Carbon Analyser and autosampler fitted with a septum piercing apparatus and 

associated software (WinToc V5.2TM).

In this analysis, inorganic carbon concentrations were determined by measuring the amount 

of carbon dioxide released by acidification of a sample, measured by a sample loop 

(nominally 5 mL). To release carbon dioxide, phosphoric acid (400 L, 5% v/v) was 

automatically injected, the sample was purged and the gas released was then concentrated by

trapping. It was desorbed and carried to a non-dispersive infrared detector (NDIR) for 

detection.

A solution of sodium carbonate was used as a calibration standard on each analysis occasion.
Huntingdon Life Sciences

WAG0021

13

3.5 Calculation of results

Theoretical inorganic carbon

Assuming that there was 100% mineralisation of the test substance, the theoretical amount of

inorganic carbon (ThIC) produced by test or reference mixtures, after blank-correction by the 

control cultures, is equal to the amount of Total Organic Carbon (TOC) added, as the test 

substance, at the start of the test:

i.e., ThIC = TOC

By adding concentrated NaOH to the test cultures and shaking, gaseous CO2 evolved during 

biodegradation 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, therefore, calculated 

in the following way:

TIC = (V1 x C1) 

where:

V1 = the volume (in litres) of liquid in the test vessel

C1 = the concentration of TIC in the liquid phase (mgC/L)

Calculation of percentage biodegradation

The biodegradation was expressed as a percentage of the ThIC based on the amount of test 

compound that was added initially. It was calculated in the following way:

%D = (TICt – TICb) x 100

 TOC 

where:

TICt = the TIC concentration (mg) in the test vial at time t.

TICb = 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 biodegradation of the reference substance was ≥60% by Day 14.

 The CO2 evolved by the blank controls was ≤15% of the applied organic carbon 

(10 mgC/L). This is consistent with the validity criteria outlined in ISO14593.
Huntingdon Life Sciences

WAG0021

14

3.7 Deviations from protocol

The minimum temperature of the shaker used to incubate the test was slightly below the 

minimum temperature (20oC) on two consecutive 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 deviations 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 specify a concentration range of 2 to 40 mgC/L and there 

was no significant biodegradation of the test substance or inhibition of the reference 

substance (in the inhibition assay) observed during the test, this deviation was not considered 

significant or to have affected the integrity of the study.
Huntingdon Life Sciences

WAG0021

15

4. Maintenance of Records

This report has been compiled from original data, which will be stored, together with a copy

of the final report, a copy of the protocol and amendments in Huntingdon Life Sciences’ 

Archives, Huntingdon Life Sciences Ltd, Eye, Suffolk, 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 item be discarded 

without the Sponsor’s knowledge.

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study for 

twenty years, and a copy of the final report in its archives indefinitely.
Huntingdon Life Sciences

WAG0021

16

5. Results

5.1 Calculation of the theoretical organic carbon content

2-bromo-3,3,3-trifluoropropene

The theoretical organic carbon content of 2-bromo-3,3,3-trifluoropropene was calculated 

from its empirical formula to be:

CF3CBr=CH2

MW = 174.95 Carbon content = 20.6%

Sodium benzoate

The theoretical organic carbon content of sodium benzoate was calculated from its empirical

formula to be:

C7H5O2Na

MW = 144 Carbon content = 58.3%

5.2 Sealed-vessel carbon dioxide production test

The levels of inorganic 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 dioxide (CO2) production by the controls was 13.4% of the 

applied organic carbon (10 mgC/L).

Sodium benzoate alone had been biodegraded by 78.1% by Day 7, and, at most, 81.8% on 

Day 14 (Table 5). 

In the presence of 2-bromo-3,3,3-trifluoropropene, sodium benzoate (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 activity of the microbial inoculum (Table 5). 

These results confirmed that the inoculum was viable and that the test was valid.
Huntingdon Life Sciences

WAG0021

17

Production of CO2 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 CO2 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 conditions of this test.

The pH of the MSM at the start of the test ranged from 7.52 to 7.53. 

The temperature of a 100 mL volume of water held under test conditions ranged from 19.7C 

to 24.2C during the test period (Protocol deviation, see Section 3.7).
Huntingdon Life Sciences

WAG0021

18

6. Discussion and Conclusion

Production of CO2 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 CO2 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 conditions of this test.

Carbon dioxide evolution by the controls was <15% of the applied organic carbon load by 

Day 28. Sodium benzoate alone had been biodegraded by 78.1% by Day 7. 

The biodegradation of sodium benzoate in the presence of the test substance was monitored 

in order to assess whether any inhibitory effects were exerted on the activity of the microbial 

inoculum. None were observed.

These results confirm that the inoculum was viable and that the test was valid.
Huntingdon Life Sciences

WAG0021

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 29

Percentage 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%)
Huntingdon Life Sciences

WAG0021

20

Table 1 Inorganic carbon measurements in the control 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 2.200 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 - -

14

1 2.943 2.827 3.060 - -

2 2.844 2.900 3.068 - -

Mean 2.894 2.864 3.064 - -

21

1 3.034 3.182 3.125 - -

2 3.100 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 3.190 3.260 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 3.300
Huntingdon Life Sciences

WAG0021

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 10.872 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 11.273
Huntingdon Life Sciences

WAG0021

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-bromo3,3,3-

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

WAG0021

23

Appendix 1 Certificate of analysis
Huntingdon Life Sciences

WAG0021

24

Appendix 2 Preparation of mineral salts medium 

Dilution water

The dilution water used to prepare stock and test solutions of mineral salts and the aqueous 

solution 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); nominal resistivity,  18 MegOhm.cm. This water complies with the relevant standards 

[British Standard (BS) 3978, 1987; American Society for Testing and Materials (ASTM) 

D 1193-06] for classification as Grade 1 or Type 1 water for laboratory use.

Mineral salts medium

The mineral salts medium (MSM) for the sealed-vessel CO2 evolution test was prepared by

mixing 10 ml of stock solution 1 with 800 ml dilution water and then adding 1 ml of 

solutions 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

Potassium dihydrogen phosphate 8.50

di-Potassium hydrogen phosphate 21.75

di-Sodium monohydrogen phosphate dihydrate 33.40

Ammonium 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 variations in the weights taken are not considered to be significant.
Huntingdon Life Sciences

WAG0021

25

Appendix 3 Compliance statement