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Working for a better future

REPORT

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

Activated Sludge Respiration Inhibition Test

HLS study number: WAG0020

Version ID: Final

Issue date: 21 October 2013
Huntingdon Life Sciences

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Sponsor and Test Facility Details

Sponsor American Pacific Corporation

3883 Howard Hughes Parkway 

Suite 700

Las Vegas 

NV 89169

USA

Test Facility

In Life and Analysis Huntingdon Life Sciences Ltd

Eye Research Centre

Eye

Suffolk

IP23 7PX

UK
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Table of Contents

Sponsor and Test Facility Details.......................................................................................... 2

Compliance with Good Laboratory Practice .......................................................................... 4

Quality Assurance Statement................................................................................................ 5

Contributing Scientists .......................................................................................................... 6

Summary .............................................................................................................................. 7

1. Introduction .................................................................................................................. 8

2. Test Substance ............................................................................................................ 9

3. Experimental Procedure............................................................................................. 10

3.1 Dilution water.................................................................................................. 10

3.2 Preparation of solutions of the test substance................................................. 10

3.3 Test methods.................................................................................................. 10

3.4 Stability of test concentrations ........................................................................ 10

3.5 Toxicity Test ................................................................................................... 10

3.6 Deviations from Protocol................................................................................. 10

4. Maintenance of Records............................................................................................. 11

5. Results....................................................................................................................... 12

6. Conclusion ................................................................................................................. 13

List of Tables

Table 1 Measured concentrations.............................................................................14

List of Appendices

Appendix 1 Certificate of analysis...................................................................................15

Appendix 2 The determination of 2-bromo-3,3,3-trifluoropropene in water......................16

Appendix 3 Compliance statement .................................................................................24
exposure phase.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 3106, as

amended by Statutory Instrument 2004 No. 994).

OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98) 17.

EC Commission Directive 2004/10/SC of 11 Februan’ 2004 (Official Journal No.L 50/44).

These principles of Good Laboratory Practice are accepted by the regulatory authorities ofthe United States of America and Japan on the basis of intergovernmental agreements.

Z.i Oycjher Z.cj3

Jordan Davies BSc MSc Date

Study Director

Huntingdon Life Sciences

4
Protocol Amendment No.2

Protocol Amendment No.3

Report AuditProtocol Amendment No.4

08 May 2013

29 Jul 2013

22 Aug 2013—23 Aug 2013

02 October2013

08 May2013

29 Jul 2013

23 Aug 2013

02 October2013

Process based inspections: At or about the time this study was in progress inspections ofprocedures employed on this type of study were carried out. These were conducted andreported to appropriate Company Management as indicated below:Process Based InspectionsDose FormulationSampling of test media for

chemical analysisEnvironmental measurements &monitoring/Ammonia analysis

Set-up oftest or reference

compound/inoculum mixture

Study management & conductResults Processing

Chromatography

Sample Extraction and Clean UpSample Preparation (to include

sample management & tracking)

Standard preparation &

Fortification procedures

Date(s) of Inspection

15 Jan 2013

04 Feb 2013

07 Mar 2013

24 Jan 2013

26 Mar 2013-27 Mar2013

06 Feb 2013

12 Feb 2013

27 Feb 2013-28 Feb 2013

27 Feb 2013

05 Aug 2013-06 Aug 2013

Date of Reporting to

Management

16 Jan 2013

04 Feb 2013

07 Mar 2013

28 Jan 2013

27 Mar 2013

06 Feb 2013

12 Feb 2013

28 Feb 2013

01 Mar2013

07 Aug 2013

where this study was conducted

were reported to Company Management.

was carried out on an

T Pulhnger MRQA Date

Group ManagerDepartment of Quality Assurance

Huntingdon Life Sciences

5
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Contributing Scientists

2-bromo-3,3,3-trifluoropropene : Activated Sludge Respiration Inhibition 

Test

Study management 

Aquatic Ecotoxicology and Biodegradation

Jordan Davies BSc MSc

Study Director (replacement)

Robert A Dickinson BSc 

Study Director (original)

Expert department

Environmental Analysis

Simon Tate Higher National Certificate (Applied Chemistry)

Senior Study Manager
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Summary

A preliminary investigation was conducted to assess if test concentrations of 2-bromo-3,3,3-

trifluoropropene could be adequately maintained under the conditions of the activated sludge 

respiration inhibition test (OCSPP 850.3300: Modified activated sludge, respiration inhibition 

test, January 2012).

Samples of 2-bromo-3,3,3-trifluoropropene were prepared in reverse osmosis water (500 mL) 

at nominal concentrations of 10 and 1000 mg/L. The test substance was added directly to the 

water in bottles (500 mL) housed in a Coordinated Environmental Services (CES) ltd 

automated respirometer. Mixtures were aerated at ca.1 L/minute and stirred for three hours. 

Samples of the mixtures were taken before the instrument was initiated (0 hours) and after 

three hours of aeration.

Results of the analytical measurements show that at the start of the test, recoveries of 2-

bromo-3,3,3-trifluoropropene were between 42% and 71% of nominal. At the end of the 

incubation phase measured test levels had decreased to <1% of nominal. As the analytical 

method was not fully validated, no claim of compliance for GLP is made for these results. 

This was not validated as during the preliminary investigation, loss of the test material was so 

significant, that if the test substance was used in a definitive test, concentrations would not 

have been maintained during the exposure phase.

These results indicate that under conditions of the test it is not possible to maintain test 

concentrations of 2-bromo-3,3,3-trifluoropropene and therefore, it would not be feasible to 

conduct a toxicity assay using the activated sludge respiration inhibition test as the test 

substance would be lost through volatility. Due to the volatile nature of the test compound

and the losses observed during the preliminary investigations it would not be expected that 

sewage micro-organisms would be impacted by this compound. 
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1. Introduction

The objective of this study was to assess the effects of 2-bromo-3,3,3-trifluoropropene on 

sewage micro-organisms by measuring the rate of oxygen uptake of activated sludge at 20 

2C in its presence at a range of concentrations. 

This type of test is most appropriate for pure substances, mixtures or commercial 

formulations that are soluble in water. As the test substance was known to be volatile, the 

study included an analytical trial to assess the feasibility of using the activated sludge 

respiration inhibition test to determine the toxicity of the test substance to activated sludge. 

The analytical method used during the trial was not fully validated. This would have been 

completed if the trial had indicated that further testing was feasible. Since further testing was 

not practicable, the assay was not validated, so no claim of compliance to GLP is made for 

the analytical results although the work followed the principles of GLP. 

The investigation was designed to meet the requirements of OCSPP 850.3300: Modified 

activated sludge, respiration inhibition test, January 2012.

The protocol was approved by the Study Director, by Huntingdon Life Sciences Management 

and by the Sponsor on 14 August 2012.

The experimental start and completion date was on 31 January 2013.
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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)

Stabilisers: 

Specific gravity: 1.65

Boiling point: 34 oC at 760 mmHg

Water solubility: 1.0 g/L at 20oC

Storage: Stored free of air and water at room temperature

Re-assay date: 12 January 2014

Sample received: 2 March 2012

The Sponsor’s Certificate of analysis is given in Appendix 1.
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3. Experimental Procedure

3.1 Dilution water

The dilution water used to prepare solutions of test mixtures was reverse osmosis (RO) water.

3.2 Preparation of solutions of the test substance

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

Therefore 2-bromo-3,3,3-trifluoropropene (3.1 μL and 313 μL calculated based on a specific 

gravity of 1.6) was added directly to bottles (500 mL) containing 500 mL of RO water to 

give final, nominal test concentrations of 10 mg/L and 1000 mg/L. The mixtures were 

shaken until the test substance was no longer visible. 

3.3 Test methods

In this test, a Co-ordinated Environmental Services (CES) Ltd automated respirometer and 

associated software was used to incubate and aerate the test mixtures in the analytical trial. 

Mixtures were incubated in 500 mL bottles fitted with a sinter to deliver air and a magnetic 

stirrer bar for mixing. Aliquots of RO water (500 mL) were added to duplicate bottles at each 

concentration. The test substance was then added as a volume (3.1 and 313 μL) to give the 

final nominal concentrations of 10 and 1000 as mg/L respectively. 

Samples were taken immediately after dosing for chemical analysis. The instrument was then 

initiated and aeration (ca. 1 L/min) and stirring was employed on the mixtures for 3 hours. 

At the end of the incubation period, the mixtures were re-sampled for chemical analysis.

3.4 Stability of test concentrations

The test concentrations of 2-bromo-3,3,3-trifluoropropene were quantified using gas 

chromatography with Mass Spectrometric detection (GC-MS, see Appendix 2). Two samples 

(20 mL) of media were taken from the test vessels at 0 and 3 hours. Each sample was added 

to a polypropylene tube (50 mL) containing 5 mL of ethyl acetate and ca. 5 g sodium 

chloride.

On each occasion, one of the samples was analysed and the other was stored in a freezer in 

case further analysis was required.

3.5 Toxicity Test

Not performed as the results of the analytical trial indicated that it was not possible to 

maintain exposure concentrations of the test substance due to its volatility. 

3.6 Deviations from Protocol

There were no deviations from the protocol.
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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.
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5. Results

Results of the analytical chemistry are given in Table 1 and methods are given in Appendix 2.

Analytical measurements show that at the start of the test recoveries of 2-bromo-3,3,3-

trifluoropropene were between 54% and 71% of nominal at 10 mg/L and between 42% and 

48% of nominal at 1000 mg/L. At the end of the incubation phase, test levels had decreased

to <1% of nominal in all mixtures.

These results indicate that under conditions of the test it is not possible to maintain test 

concentrations of 2-bromo-3,3,3-trifluoropropene and therefore, it would not be feasible to 

conduct a toxicity assay using the activated sludge respiration inhibition test as 2-bromo3,3,3-trifluoropropene would be lost through volatility.

Therefore no further testing was carried out and the results of the preliminary investigation 

were reported.
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6. Conclusion

After 3 hours of aeration and stirring, analytical measurements of test samples showed that 

<1% of the nominal test concentrations of 2-bromo-3,3,3-trifluoropropene remained.

These results indicate that under conditions of the test it is not possible to maintain test 

concentrations of 2-bromo-3,3,3-trifluoropropene and therefore, it would not be feasible to 

conduct a toxicity assay using the activated sludge respiration inhibition test as the test 

substance would be lost through volatility.

Due to the volatile nature of the test compound and the losses observed during the 

preliminary investigations it would not be expected that sewage micro-organisms would be 

impacted by this compound.
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Table 1 Measured concentrations

Nominal

2-bromo-3,3,3-

trifluoropropene conc.

(mg/L)

Measured 2-bromo-3,3,3-trifluoropropene concentrations (mg/L)

0 hours %N 3 hours %N

10 7.06 71 nd -

10 5.43 54 nd -

1000 420 42 <0.5 <1

1000 483 48 1.57 <1

nd Not detected above the limit of detection of the analytical method (<LOD 0.005 mg/L).
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Appendix 1 Certificate of analysis
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Appendix 2 The determination of 2-bromo-3,3,3-trifluoropropene in water

1. Method

1.1 General principle

Samples were extracted with ethyl acetate, prior to quantitation by GC-MS.

Materials Grade 

Acetone AR

Ethyl acetate HPLC

Hexane Glass distilled

1.1.1 Test article fortifying and calibration solutions

An appropriate amount of 2-bromo-3,3,3-trifluoropropene was accurately weighed (corrected 

for purity) and dissolved in acetone to give a 1 mg/mL stock standard solution. Appropriate 

dilutions of the stock standard solution were made with hexane to give fortification standard 

solutions. An appropriate fortification standard solution was progressively diluted with ethyl 

acetate in order to produce a calibration line in the range 1 to 50 ng/mL.

1.2 Procedure

An aliquot of water (20 mL) was taken and fortified if required. An aliquot of ethyl acetate 

(5 mL) was added and the samples were shaken vigorously. The phases were centrifuged at 

3500 rpm for 3 minutes and an aliquot of the ethyl acetate layer was diluted by a factor of 20 

with ethyl acetate, prior to analysis by GC-MS. Any further dilutions, where required, were

performed using ethyl acetate.
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1.2.1 Instrumentation

GC-MS conditions

Instrument: Voyager

Data system: MassLab v1.3

Ionisation mode: CI –

Ion monitoring details: m/z 79

Column: Rtx-1 (60 m x 0.32 mm x 5μm film thickness)

Temperature programme: 40°C held for 4 min then ramped at 30°C/min to 250°C and 

held for 3 min

Injector temperature: 200°C

Injection volume: 2 μL

Retention time: approx. 5.9 minutes

LOD: 1 ng/mL (equivalent to 0.005 mg/L in aqueous media)

1.2.2 Calculation of results

Test samples were quantified using the following equation:

Residue found (mg/L) = 

1000

1

 x D x 

M

1

x x 

Where x (residue concentration in final solution) was calculated using the linear regression:

y = m x + c where x (concentration in ng/mL) = 

y  c

m

c = intercept

m = slope

y = peak area of sample

M = matrix concentration (mL/mL)

D = dilution factor
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2. Results and Discussion

2.1 Calibration

The chromatographic response to 2-bromo-3,3,3-trifluoropropene was shown to be linear 

over the range of concentrations 1 to 50 ng/mL. Typical calibration data is presented in 

Table 1 and Figure 1. Typical chromatograms of calibration standards are presented in 

Figures 2 and 3.

2.2 Limit of detection (LOD)

The limit of detection of the method is defined as the value of the lowest calibration standard 

chromatographed that gave a peak height to baseline noise ratio 3. For this study the limit 

of detection was 1 ng/mL (equivalent to 0.005 mg/L in water).

2.3 Samples

Samples of water from the test were analysed in a suitably sized batch along with a control

sample fortified with 2-bromo-3,3,3-trifluoropropene which acted as a procedural recovery 

sample. The procedural recovery performed on control HPLC water was in the range 70 to 

110% confirming the validity of the methodology on the day of analysis. Typical 

chromatograms of untreated, untreated fortified with 2-bromo-3,3,3-trifluoropropene and 

treated extracts are presented in Figures 4 to 7.
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Table 1 Typical standard calibration for 2-bromo-3,3,3-trifluoropropene by 

GC-MS

Standard concentration (ng/mL) Peak area

0 0

1 104.415

2 182.686

4 273.549

8 622.550

10 773.833

20 1422.779

30 2103.317

40 2721.646

50 3404.771

Calibration curve: y = 67.5580 x + 46.2481 (batch 1)

r = 0.9996

Where x = concentration

y = peak area

r = regression coefficient
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Figure 1 Typical standard chemical calibration for 

2-bromo-3,3,3-trifluoropropene by GC-MS
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Figure 2 Typical chromatogram of a 1 ng/mL 

2-bromo-3,3,3-trifluoropropene calibration standard

ID

E:\STUDYDATA\WAG_0020\Voyager_WAG_0020.PRO\Data\020B107.RAW

4.00 6.00 8.00 10.00 12.00

57 Time

100

%

020B107 Sm (Mn, 2x3) SIR of 1 Channel CI79

5.50e4

Area

5.99

104.415

Figure 3 Typical chromatogram of a 50 ng/mL 

2-bromo-3,3,3-trifluoropropene calibration standard

ID

E:\STUDYDATA\WAG_0020\Voyager_WAG_0020.PRO\Data\020B117.RAW

4.00 6.00 8.00 10.00 12.00

57 Time

100

%

020B117 Sm (Mn, 2x3) SIR of 1 Channel CI79

5.50e4

Area 5.99

3404.771
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Figure 4 Chromatogram of a sample of untreated HPLC water (analytical 

identification H2O Con)

ID

E:\STUDYDATA\WAG_0020\Voyager_WAG_0020.PRO\Data\020B112.RAW

4.00 6.00 8.00 10.00 12.00

57 Time

100

%

020B112 Sm (Mn, 2x3) SIR of 1 Channel CI79

5.50e4

8.33

Figure 5 Chromatogram of a sample of untreated HPLC water fortified 

with 2-bromo-3,3,3-trifluoropropene at 10 mg/L (analytical 

identification H2O F10)

ID

E:\STUDYDATA\WAG_0020\Voyager_WAG_0020.PRO\Data\020B114.RAW

4.00 6.00 8.00 10.00 12.00

57 Time

100

%

020B114 Sm (Mn, 2x3) SIR of 1 Channel CI79

5.50e4

Area

5.96

1160.464
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Figure 6 Chromatogram of a test sample at 10 mg/L of BTFP at 0 hours 

(analytical identification 13/WAG/A0317 D*100)

ID

E:\STUDYDATA\WAG_0020\Voyager_WAG_0020.PRO\Data\020B102.RAW

4.00 6.00 8.00 10.00 12.00

57 Time

100

%

020B102 Sm (Mn, 2x3) SIR of 1 Channel CI79

5.50e4

Area

5.97

999.715

Figure 7 Chromatogram of a test sample at 10 mg/L of BTFP at 3 hours

(analytical identification 13/WAG/A0321)

ID

E:\STUDYDATA\WAG_0020\Voyager_WAG_0020.PRO\Data\020B120.RAW

4.00 6.00 8.00 10.00 12.00

57 Time

100

%

020B120 Sm (Mn, 2x3) SIR of 1 Channel CI79

5.50e4

8.36
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Appendix 3 Compliance statement