## FINAL REPORT

Study Title



## 96

- HOUR ACUTE TOXICITY STUDY IN FATHEAD MINNOW WITH

1- PROPANESULFONIC ACID , 2

- HYDROXY -3-(2-PROPEN-1-

YLOXY)- , SODIUM SALT (1:1)

(STATIC)

Author

L.M. Bouwman, MSc.

Test Facility

WIL Research Europe B.V.

Hambakenwete ring 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 500677

Substance 204240/A

- Page 1 of 22

-1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 2 - 1. CONTENTS

1. CONTENTS

................................................................................................ ...................................... 2

2. STATEMENT OF GLP COM PLIANCE

............................................................................................. 3

3. QUALITY ASSURANCE ST ATEMENT

............................................................................................ 4

4. SUMMAR Y

................................................................................................ ........................................ 5

5. INTRODUCTION

.............................................................................................................................. 6

5.1. Preface

................................................................................................ ...................................... 6

5.2. Aim of the study

........................................................................................................................ 6

5.3. Guidelines

................................................................................................ ................................ . 6

5.4. Storage and retention of records and materials

........................................................................ 6

5.5. Definitions

................................................................................................ .................................. 6

6. MATERIALS AND METHOD S

.......................................................................................................... 7

6.1. Test Substance

......................................................................................................................... 7

6.1.1. Test substance information

............................................................................................. 7

6.1.2. Study specific test substance information

...................................................................... 7

6.1.3. Reference substance

...................................................................................................... 7

6.2. Test System

.............................................................................................................................. 7

6.3. Holding

................................................................................................ ...................................... 8

6.4. Preparation of test solution s

...................................................................................................... 8

6.5. Limit test

................................................................................................ .................................... 9

6.5.1. Test concentrations

......................................................................................................... 9

6.5.2. Test procedure and conditions

....................................................................................... 9

6.5.3. Sampling for analysis of test concentrations

.................................................................. 9

6.5.4. Measurements and recordings

..................................................................................... 10

6.6. Electronic data capture

........................................................................................................... 10

6.7. Interpretation

........................................................................................................................... 10

6.7.1. Data handlin g

................................................................................................................ 10

6.7.2. Acceptability of the test

................................................................................................ . 10

6.8. List of deviations

..................................................................................................................... 10

6.8.1. List of protocol deviations

............................................................................................. 10

6.8.2. List of standard operating procedures deviations

......................................................... 10

7. RESULTS

................................................................................................ ....................................... 10

7.1. Measured concentrations

........................................................................................................ 10

7.2. Mortality and other effects

....................................................................................................... 11

7.3. Dete rmination of effect concentrations

................................................................................... 11

7.4. Experimental conditions

.......................................................................................................... 11

8. CONCLUSION

................................................................................................ ................................ 12

TABLES

Table 1 Incidence of mortality and total mortality

...................................................................... 11

Table 2 Effect parameters

.......................................................................................................... 11

Table 3 pH

- values and dissolved oxygen concentrations (mg/l)

............................................... 11

Table 4 Temperatures (°C)

........................................................................................................ 11

APPENDICES

APPENDIX 1 REFERENCE TEST

...................................................................................................... 13

APPENDIX 2 CERTIFICATE OF ANALYSIS

...................................................................................... 14

APPENDIX 3 ANALYTICAL REPORT

................................................................................................ 151- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 5 - 4. SUMMARY

96

- Hour Acute Toxicity Study in Fathead minnow with 1- Propanesulfo nic acid, 2

- hydroxy -3- (2

- propen

1- yloxy)

- , sodium salt (1:1)

.

The study procedure described in this report was based on the OPPTS 850.1075

, 1996

. In addition,

the procedures were designed to meet the test methods of the OECD guideline No. 203, 1992

.

The batch of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) tested was a

clear colourless liquid and completely soluble in test medium at the concentration tested.

Preparation of test solutions started with a nominal concentration of 1000 mg/l , containing 386 mg of

the active ingredient (a.i.) Sodium -1- allyloxy -2- hydroxypropane sulphonate. Fifteen minutes of

m agnetic stirring was applied to accelerate the dissolving of the test substance in the test medium.

The final test concentra tion was c lear and colourless.

A limit test was performed exposing fourteen fish (two replicates, seven fish per replicate) to a nominal

concentration of 1000 mg/l. Simultaneously, seven fish were exposed to a control. The total test period

was 96 hours and samples for analytical confirmation of actual exposure concentrations were taken at

the start, after 48 hours of exposure and at the end of the test.

Analysis of the samples taken at the start of the test showed that the actual test concentration was i n

agreement with nominal (102

- 103%). This concentration remained stable during the test period (99

- 101% of initial at the end of the test). Given these results, the effect parameters were based on the

nominal exposure concentration.

The study met the acce ptability criteria prescribed by the protocol and was considered valid.

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) induced no visible effects in

fathead minnow at an analytically confirmed nominal concentration of 1000 mg/l

. This concentration

contained 386 mg of the active ingredient per litre.

Hence, t he 96h

- LC 50 was >1000 mg test substance /l , corresponding to >386 mg a.i./l.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 6 - 5. INTRODUCTION

5.1. Preface

Sponsor Society of Chemical Manufacturers and Affiliates

1850 M Street, N W Suite 700

WASHINGTON, DC 20036

USA

Study Monitor Dr. C. T. Helmes

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD 's

- Hertogenbosch

The Netherlands

Study Director L.M. Bouwman, MSc.

Principal Scientist M.J.C. Brekelmans, M Sc.

Study Plan Start : 13 May 2013

Completion : 17 May 2013

5.2. Aim of the study

The purpose of the study was to evaluate the test substance for its ability to generate acute toxic

effects in Pimephales promelas during an exposure period of 96 hours and, if possible, to determine

the LC 50 at all observation times.

5.3. Guidelines

The study procedure described in this report was based on the EPA Ecological Effects Test

Guidelines, OPPTS 850.1075, Fish Acute Toxicity Test, Freshwater and marine. ‘Public Draft ’,

EPA 712 -C- 96

- 118, April 1996.

In addition, the procedure was designed to meet the test methods prescribed by the following

guideline:

• OECD guidelines for Testing of Chemicals, guideline No. 203: "Fish Acute Toxicity Test", Adopted

17 July, 1992.

5.4. Stora ge and retention of records and materials

Records and materials pertaining to the study including protocol, raw data, specimens (except

specimens requiring refrigeration or freezing) and the limit report are retained in the WIL Research

Europe archives for a period of at least 2 years after finalisation of the report. After this period, the

sponsor will be contacted to determine how the records and materials should be handled. WIL

Research Europe will retain information concerning decisions made.

Those specimens requiring refrigeration or freezing will be retained by WIL Research Europe for as

long as the quality of the specimens permits evaluation but no longer than three months after

finalisation of the report.

WIL Research Europe will retain a test subs tance sample until the expiry date, but no longer than 10

years after finalisation of the report. After this period the sample will be destroyed.

5.5. Definitions

Fish were considered to be dead when no reaction was observed after touching the caudal peduncle

and visible breathing movements were absent. In addition, fish that were convulsing or showing other1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 7 - severe forms of distress not considered transient in nature and likely to become more severe before

the exposure is terminated, will be sacrificed for humane reasons. These fish will be treated as having

died in the test, based on OECD guideline on humane endpoint (ENV/JM/MONO/(2000)7).

The LC 50 is the concentration killing 50% of the fish after a defined period of exposure.

6. MATERIALS AND METHOD S

6.1. Test Su bstance

6.1.1. Test substance information

Identification 1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1)

Synonyms COPS_1 or CS

- AHPS

Structure

O SO 3 -

OH Na +

Molecular formula C 6 H 11 NaO 5 S

Molecular weight 218.22

Description Clear colourless liquid

Batch 12E1101

Purity/Composition

Sodium -1- allyloxy -2-

hydroxypropane sulphonate

3- allyl

- 1,2

- propanediol

Water CAS Number Percent

52556

- 42

- 0 38.64

123

- 34

- 2 2.26

7732

- 18

- 5 59.1

Test sub stance storage At room temperature in the dark

Stability under storage conditions Stable

Expiry date 13 August 2013

See APPENDIX 2 ; Certificate of analysis.

6.1.2. Study specific test substance information

Purity/composition correc tion factor

required No

Hygroscopic No

Volatile No

Specific Gravity / Density 1.17

Stability in water 1 year

6.1.3. Reference substance

This report includes the results of a reference test with pentachlorophenol ( PCP) ( APPENDIX 1 )

.

6.2. Test System

Species Fathead minnow ( Pimephales promelas , Teleostei,

Cyprinidae) Rafinesque

Source In house culture (Batch FM 13

- 02)1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 8 - Mean length 1 1.9 ± 0.1 cm

Mean weight 1 0.14 ± 0.04 g

Reason for selection This system has been selected as an internationally

accepted species.

Total fish used 21

6.3. Holding

Medium Adjusted ISO medium, formulated using R O- water (tap

water purified by reverse osmosis; GEON

Waterbehandeling

, Berkel

- Enschot

, The Netherlands ) with

the following composition:

CaCl 2 .2H 2 O 2 11.5 mg/l

MgSO 4 .7H 2 O 88.8 mg/l

NaHCO 3 46.7 mg/l

KCl 4.2 mg/l

Measurements Conductivity, pH, nitrate, nitrite and ammonia concentration:

once a week. Temperature: continuous. In addition, pH and

temperature were measured before transferring the fish to

the test system.

Water quality parameters Were kept within the optimum limits for the respective fish

species.

Feeding Daily with pelleted fish food ( Cy prico Crumble Excellent

(300

- 500 um), Coppens International bv, Helmond, The

Netherlands )

Validity of batch In the batch of fish used for the test, mortality during the

seven days prior to the start of the test was less than 5%.

6.4. Preparation of test solutions

The standard test procedures required generation of test solutions, which should contain completely

dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The

testing of concentrations that disturb the test system should be prevented (e.g. film of the test

substance on the water surface ). No correction was made for the purity/composition of the test

substance.

The batch of 1- Propanesulfonic a cid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) tested was a

clear colourless liquid and completely soluble in test medium at the concentration tested.

Preparation of test solutions started with a nominal concentration of 1000 mg/l , containing 386 mg of

the active ingredient (a.i.) Sodium -1- allyloxy -2- hydroxypropane sulphonate. Fifteen minutes of

m agnetic stirring was applied to accelerate the dissolving of the test substance in the test medium.

The final test concentration was clear and colourles s.

1 Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 9 - 6.5. Limit test

6.5.1. Test concentrations

1- Propanesulfonic acid, 1000 mg/l, containing 386 mg a.i. per litre

2- hydroxy -3- (2

- propen -1- yloxy) -, sodium salt (1:1)

Control Test medium without test substance or other additives

6.5.2. Test procedure and conditions

Test duration 96 hours

Test type Static

Test vessels 6.5 litres, all

- glass, containing 5 litres of test solution

Test medium Adjusted ISO medium with a hardness of 180 mg CaCO 3 per

litre and a pH of 7. 7 ± 0. 3.

Number of fish 7 for the control and 14 for the test concentration

Number of replicates 1 for the control;

2 for the test concentration, containing 7 fish each

Loading 0.20 g fish/litre, i.e. 7 fish per 5 litres of test med ium

Illumination 16 hours photoperiod daily

Aeration The test media were not aerated during the test.

Feeding No feeding from 24 hours prior to the test and during the

total test period

Introduction of fish Within 40 minutes after preparation of the test media from a

holding tank with comparable water quality parameters and

pH and temperature differences between test and holding

tank media of less than 0.5 unit and 0.5 °C

.

Euthanasia At the end of the test the surviving fish were rapidly killed by

expos ing them to ca. 1.2% ethylene glycol monophenylether

in water.

6.5.3. Sampling for analysis of test concentrations

S amples for possible analysis were taken from the control and both replicates of the test concentration

according to the schedule below. The method of analysis is described in the appended Analytical

Report ( APPENDIX 3 )

.

Frequency at t=0 h, t= 48 h and t= 96 h

Volume 1.5 ml

Storage Samples were stored in a freezer until analysis.

Additionally, reserve samples of 1.5 ml were taken from all test solutions for possible analysis. If not

already used, these samples were stored in a freezer for a maximum of three months after delivery of

the draft report, pending on the decision of the sponsor for additional analysis.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 10

- 6.5.4. Measur ements and recordings

Mortality and other effects At 6 , 24, 48, 72 and 96 hours following the start of exposure.

In addition, every afternoon and morning from day 1, except

for the afternoon at day 2, to observe for any dead or

severely distressed fish.

Dissolved oxygen content, Daily in all vessels with surviving fish, beginning at the

pH and temperature start of the test (day 0).

6.6. Electronic data capture

Temperature during sample storage was monitored continuously using the following programme:

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ,

USA)

6.7. Interpretation

6.7.1. Data handling

No LC 50 could be calculated because the test substance proved to be non

- toxic (LC 50 > maximum

concentration tested ).

6.7.2. Acceptability of the test

1. No mortality was observed in the control at the end of the test.

2. Test conditions were maintained constant throughout the test.

3. The dissolved oxygen concentration has been at least 60% of the air saturation value throughout

the test (> 5 mg/l at 22°C).

4. The concentrations of the substance being tested have been at least 80% of the nominal

concentration throughout the test.

6.8. List of deviations

6.8.1. List of protocol deviations

1. Fish mortality was not recorded at the end of the second day of exposure.

Evaluation: This is a requirement for the Ethical Committee, not for the endpoints of this study.

The study integrity was not adversely affected by th is deviation.

6.8.2. List of standard operating procedures deviations

Any deviations from standard operating procedures were evaluated and filed in the study file. There

were no deviations from standard operating procedures that affected the integrity of the study.

7. RESULTS

7.1. Measured concentrations

The results of analysis of the samples taken during the limit t est are described in T able 2 of the

appe nded Analytical Report.

Ana lysis of the samples taken at the start of t he test showed that the actual test concentration was in

agreement with nominal (102

- 103%). This concentration remained stable during the test period (99

- 101% of initial at the end of the test) . Given these results, the effect parameters were based on the

nominal exposure concentration.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 11

- 7.2. Mortality and other effects

Table 1 shows the mortality data recorded during the limit test. No mortality or other clinical effects

were observed in both the control and the test concentration. The responses recorded in this test

allowed for reliable determination of an LC 50

.

Table 1 Incidence of mortality and total mortality

Concentration

Test substance 1

(mg/l) Vessel

number Initial

number

of fish Cumulative mortality Total

Mortality

(%) 6h 24h 48h 72h 96h

control A 7 0 0 0 0 0 0

1000 A 7 0 0 0 0 0 0 B 7 0 0 0 0 0

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

7.3. Determination of effect concentrations

Table 2 shows the effect parameters based on the analytically confirmed nominal concentration of the

test substance and on the corresponding concentration of the active ingredient

.

Table 2 Effect parameters

Parameter Conce ntration

Test substance 1

(mg/l) Concentration

Active ingredient 2

(mg/l)

24 , 48, 72 & 96 h

- LC 50 >1000 >386

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

2 Active ingredient is Sodium -1- allyloxy -2- hydroxyprop ane sulphonate

7.4. Experimental conditions

The results of measurement of pH and oxygen concentrations are presented in Table 3

. The

temperatures measured during the study in the various test vessels are presented in Table 4

. All test

conditions remained within the ranges prescribed by the protocol (pH: 6.0

- 8.5, constant within 1 unit;

temperature 20

- 24 °C, constant within 2°C; oxygen > 60% of air saturation).

Table 3 pH

- values and dis solved oxygen concentrations (mg/l)

Concentration

Test substance 1

(mg/l) Vessel

number Day 0 Day 1 Day 2 Day 3 Day 4

pH 2 O 2 pH O 2 pH O 2 pH O 2 pH O 2

control A 7.9 9.2 7.8 7.7 7.6 7.2 7.6 7.1 7.8 7.1

1000 A 7.7 9.0 7.7 7.6 7. 6 7.1 7.6 6.9 7.7 6.9

B 7.6 9.1 7.6 7.4 7.5 7.0 7.5 6.8 7.7 6.8

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

2 pH of culture medium was : 7.7

Table 4 Temperatures (°C)

Concentrat ion

Test substance 1

(mg/l) Vessel

Number Day 0 2 Day 1 Day 2 Day 3 Day 4

control A 22.8 22.6 22.7 22.5 22.7

1000 A 22.9 22.8 22.8 22.8 22.7

B 23.0 22.9 22.8 22.8 22.8

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium s alt (1:1)

2 Temperature of culture medium was : 23.3 °C. Therefore, the fish were acclimatized to 22.9°C before the start of the test.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 12

- 8. CONCLUSION

Under the conditions of the present test 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1 :1) induced no visible effects in fathead minnow at an analytically confirmed nominal

concentration of 1000 mg/l. This concentration contained 386 mg of the active ingredient per litre.

Hence, t he 96h

- LC 50 was >1000 mg test substance /l , corresponding to >386 mg a.i./l.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 13

- APPENDIX 1 REFERENCE TEST

96

- hour acute toxicity study in the Fathead minnow with pentachlorophenol (PCP); WIL Research

Europe Project 502823 (Batch FM 13

- 02).

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In

addition, the procedures were designed to meet the test methods of the ISO International Standard

7346

- 1: Static method, 1996.

Start: 13 May 2013

End: 17 May 2013

This reference test was carried out to check the sensitivity of the test system as used by WIL

Research Europe. The reference substance was pentachlorophenol (PCP, SIGMA, Art. P2604, Batch

06324ED

- 405).

Concentrations: 0.10, 0.22 and 0.46 mg/l in ISO

- medium.

Incidence of mortality observed in the reference study:

Concentration

PCP (mg/l)

Nominal Initial

Number

Of fish Cumulative number of dead fish recorded at

various time points after start of exposure Total

Mortality

(%) 1 3⁄4 h 24h 48h 72h 96h

0.10 5 0 0 0 0 0 0

0.22 5 0 3 3 3 3 60

0.46 5 0 4 4 5 5 100

During the test the pH, oxygen concentration and the temperature of the medium were within the

optimal ranges for fish.

Under the conditions of the present test with Fathead minnow exposed to PCP, the 96h

- LC 50 was

0.20 mg/l based on nominal concentrations, with a 95% confidence interval between 0.16 and

0.32 mg/l. This effect was already reached within 72 hours of exposure.

No historical data is available on the range of the 96h

- LC 50 for Fathead minnow. However, this test

shows that toxicity is similar for Fathead minnow and carp (Teleostei Cyprinidae). For Carp the

96h

- LC 50 is generally between 0.10 and 0.46 mg/l based on historical data of reference tests

performed approximately every 3 months from April 1988 until the end of 2000, and annually since

then.

The raw data and report from this study are kept in the WIL Research Europe archives. The test

described above was performed under GLP

- conditions with a QA

- check.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 14

- APPENDIX 2 CERTIFICATE OF ANALYSIS1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 15

- APPEND IX 3 ANALYTICAL REPORT



# DETERMINATION OF THE

CONCENTRATIONS

Author

M.J.C. Brekelmans , MSc.1- propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 16

- 1. CONTENTS

1. CONTENTS

................................................................................................ ................................... 16

2. REPORT APPROVAL

.................................................................................................................... 17

3. INTRODUCTION

........................................................................................................................... 18

3.1. Preface

................................................................................................ ................................... 18

3.2. Aim of the study

..................................................................................................................... 18

4. MATERIALS AND METHOD S

....................................................................................................... 18

4.1. Reagents

................................................................................................ ................................ 18

4.2. Samples

................................................................................................ ................................ . 18

4.3. Analytical method

................................................................................................................... 19

4.3.1. Analytical conditions

.................................................................................................... 19

4.3.2. Preparation of solutions

............................................................................................... 19

4.3.3. Sample injections

......................................................................................................... 19

4.4. Electronic data capture

.......................................................................................................... 19

4.5. Formulas

................................................................................................ ................................ 20

5. RESULTS

................................................................................................ ...................................... 21

5.1. Calibration curves

.................................................................................................................. 21

5.2. Samples

................................................................................................ ................................ . 21

5.2.1. Procedural recovery samples

...................................................................................... 21

5.2.2. Test samples

................................................................................................................ 21

TABLES

Table 1 Procedural recovery samples

........................................................................................... 22

Table 2 Concentra tions of the test substance in test medium

....................................................... 221- propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 18

- 3. INTRODUCTION

3.1. Preface

Study plan analytical phase Start : 21 M ay 201 3

Completion : 22 May 201 3

3.2. Aim of the study

The purpose of the analytical phase was to determine the actual concentrations in samples taken from

the test solutions used during the ecotoxicity test.

4. MATERIALS AND METHOD S

4.1. Reagents

Water Tap water purified by a Milli

- Q water purification

system (Millipore, Bedford, MA, USA)

Di

- Sodium hydrogenphosphate dihydrate Merck, Darmstadt, Germany

( Na 2 H PO 4 · 2H 2 O)

Sodium dihydrogenphosphate dihydrate Merck

(NaH 2 PO 4 · 2H 2 O)

Sodium nitrate Merck

(NaNO 3 )

ISO

- medium see main report

All reagents were of analytical grade, unless specified otherwise.

4.2. Samples

The samples were stored in the freezer ( ≤

- 15°C ) . Storage stability of samples under these conditions

was de monstrated in project 500674

.

On the day of analysis, the samples were defrosted at room temperature and analysed.1- propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 19

- 4.3. Analytical method

4.3.1. Analytical conditions

Quantitative analysis was based on the analytical method validated for the test substance in project

500674

.

Instrument Alliance Separation Module 2695 (Waters, Milford, MA, USA)

Detector Refractive Index Detector 2414 (Waters)

Column

Stationary phase Synergi Hydro

- RP 80A

Dimensions 250 mm × 4.6 mm i.d., dp =4 μm

Brand Phenomenex, Torrance, CA , USA

Column temperature 40°C ± 1°C

Injection volume 50 μl

Mobile phase water containing 0.05M Na 2 HPO 4 , 0.05M NaH 2 PO 4 and 0.05M

NaNO 3

Flow 1 ml/min

RI detection

Temperature 30°C

Sensitivity 4

Time constant 1 seconds

4.3.2. Preparation of solutions

Stock and spiking solutions

Stock solutions of the test substance were prepared in water at concentrations of 4935 and

5945 mg/l..

Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of

the spiking solutions was water

.

Calibration so lutions

Calibration solutions in the concentration range of 80

– 1200 mg/l were prepared from two stock

solutions. The end solution of the calibration solutions was mobile phase

.

Procedural recovery samples

1.6 ml ISO

- medium was spiked with the test substance at a target concentration of 100 or 1000 mg/l.

The accuracy samples were analysed without further treatment.

4.3.3. Sample injections

Calibration solutions were injected in duplicate. Test samples and procedural recovery samp les were

analysed by single injection.

4.4. Electronic data capture

System control, data acquisition and data processing were performed using the following programme:

- Empower version 7.00 (Waters, Milford, MA, USA).

Temperature, relative humidity and/or atmospheric pressure during sample storage and/or

performance of the studies was monitored continuously using the following programme:

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ,

USA)

.1- propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 20

- 4.5. Formulas

Response (R ) Peak area test substance [units]

Calibration curve b C a R N +=

where:

C N = nominal concentration [mg/l]

a = slope [units × l/mg]

b = intercept [units]

Analysed concentration (C A ) [mg/l] d

a b) (R C A × −

=

where:

d = dilution factor

Recovery [%] 100

C C

N

A

×

Relative to nominal concentration [%] 100

C C

N

A

×

Relative to initial concentration [%] 100 hours) 0 (t C hours)x (t C

A A

×

= =1- propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 21

- 5. RESULTS

5.1. Calibration curves

A calibration curve was constructed using five concentrations. For each concentration, two responses

were used. Linear regression analysis was performed using the least squares method with a

1/concentration 2 weighting factor. The coefficient of correlation (r) was > 0.99.

5.2. Samples

5.2.1. Procedural recovery samples

The results for the procedural recovery samples are given in Table 1

.

The mean recoveries of the procedural recovery samples fell within the criterion of 70

- 110%. It

demonstrated that the analytical method was adequate for the determination of the test substance in

the test samples.

5.2.2. Test samples

The results for the test samples are given in Table 2

.1- propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500677

- Page 22

- TABLES

Table 1 Procedural recovery samples

Date of

preparation Date of

analysis Target

concentration

[mg/l] Nominal

concentration

[mg/l] Analysed

concentration

[mg/l] Recovery

[%] Mean

recovery

[%]

21

- May

- 2013 21

- May

- 2013 100 103 106 103 103

103 107 104

21

- May

- 2013 21

- May

- 2013 1000 1032 1048 102 102

1032 1061 103

Table 2 Concentrations of the test substance in test medium

Time of

sampling

[ hours ] Date of

sampling Date of

analysis 1 Concentration Relative to

nominal

[%] Relative to

initial

[%] Nominal

[mg/l] Analysed

[mg/l]

0 13

- May

- 2013 21

- May

- 2013 0 n.d. n.a.

1000 1034 2 103

1000 1024 3 102

48 15

- May

- 2013 21

- May

- 2013 0 n.d. n.a.

1000 1011 2 101 98

1000 1006 3 101 98

96 17

- May

- 2013 21

- May

- 2013 0 n.d. n.a.

1000 1025 2 103 99

1000 1037 3 104 101

1 Samples were stored in the freezer ( ≤

- 15°C ) until the day of analysis.

2 Sample A

3 Sample B

n.d. Not detected.

n.a. Not applicable.