## FINAL REPORT

Study Title



## ACUTE TOXICITY STUDY IN **DAPHNIA MAGNA** 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. Hambakenwetering 7

52 31 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 500676

Substance 204240/A

- Page 1 of 23 -1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 2 - 1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COM PLIANCE

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

3. QUALITY ASSURANCE ST ATEMENT

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

4. SUMMARY

................................................................................................ ........................................ 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. Breeding

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

6.4. Preparation of test solutions

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

6.5. Range

- finding test

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

6.6. Limit test

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

6.6.1. Test concentration s

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

6.6.2. Test procedure and conditions

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

6.6.3. Sampling for analysis of test concentrations

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

6.6.4. Measurements and recordings

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

6.7. Electronic data capture

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

6.8. Interpretation

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

6.8.1. Data handling

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

6.8.2. Acceptability of the test

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

6.9. List of deviations

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

6.9.1. List of protocol deviations

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

6.9.2. List of standard operating procedures deviations

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

7. RESULTS

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

7.1. Range

- finding test

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

7.2. Limit test

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

7.2.1. Measured concentrations

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

7.2.2. Immobility

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

7.2.3. Determination of effect concentrations

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

7.2.4. Experimental conditions

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

8. CONCLUSION

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

TABLES

Table 1 Incidence of immobility in the range

- finding test

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

Table 2 Acute immobilisation of daphnids after 24 and 48 hours in the limit test

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

Table 3 Effect parameters

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

Table 4 pH and oxygen concentrations during the limit test

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

APPENDICES

APPENDIX 1 REFERENCE TEST

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

APPENDIX 2 CERTIFICATE OF ANALYSIS

...................................................................................... 15

APPENDIX 3 ANALYTICAL REPORT

................................................................................................ 161- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 5 - 4. SUMMARY

Acute Toxicity Study in Daphnia magna with 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

.

The study procedure described in this report was based on the OPPTS 850.1010

, 1996

. In addi tion, the procedures were designed to meet the test methods of the OECD guideline No. 202, 2004

.

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.

A limit test was performed based on the results of a preceding range

- finding test. 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. Approximately 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 colourless.

Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to a nominal concentration of 1000 mg/l and a control. The total test period was 48 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the

test.

Analysis of the sample taken at the start of the test showed that the actual test concentration was in
agreement with nominal (100%). This concentration remained stable during the test period (102% of

initial at the end of the te st). Given these results, the effect parameters were based on the nominal
exposure concentration.

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

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) did not induce acute

immobilisation of Daphnia magna at an analytically confirmed nominal concentration of 1000 mg/l.
This concentration contained 386 mg of the active ingredient per litre.

Hence, t he 48h

- EC 50 was >1000 mg test substance /l , c orresponding to >386 mg a.i./l

.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 6 - 5. INTRODUCTION

5.1. Preface

Sponsor Society of Chemical Manufacturers and Affiliates
1850 M Street, NW Suite 700
WASHINGTON, DC 20036

USA

Study Monitor Dr. C. T. Helmes

Test Facility WIL Research Europe B.V.

Hambakenweterin g 7

5231 DD 's

- Hertogenbosch

The Netherlands

Study Director L.M. Bouwman, MSc.

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

Study Plan Start : 09 April 2013 Completion : 15 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 on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to

determine the EC 50 at 24 and 48 hours of exposure.

5.3. Guidelines

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

Guidelines, OPPTS 850.10 10

, Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids. ‘Public

Draft’, EPA 712 -C- 96

- 11 4 , 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. 20 2 : " Daphnia sp.

, Acute Immobilisation
Test", Adopted April 13

, 2004

.

5.4. Storage 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 final report are retained in the WIL Research
Europe archives for a period of at least 2 years after finalization 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 Europ e for as long as the quality of the specimens permits evaluation but no longer than three months after finalization of the report.

WIL Research Europe will retain a test substance sample until the expiry date, but no longer than 10 years after finalization of the report. After this period the sample will be destroyed.

5.5. Definitions

Immobile are those animals not able to swim within 15 seconds after gentle agitation of the test

vessel.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 7 - The EC 50 is the concentration of test substance estimated to immobilise 50% of the daphni ds after a
defined period of exposure.

6. MATERIALS AND METHOD S

6.1. Test Substance

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

W ater CAS Number Percent

52556

- 42

- 0 38.64

123

- 34

- 2 2.26

7732

- 18

- 5 59.1 Test substance storage At room temperature in the dark Stability under storage conditions Stable Expiry date 13 August 2013

See APPENDIX 2 : Certifi cate of analysis.

6.1.2. Study specific test substance information

Purity/composition correction 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 the most recent reference test with potassium dichromate ( APPENDIX 1 )

.

6.2. Test system

Species Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at
least third generation, obtained by acyclical parthenogenesis
under specified breeding conditions.

Source In

- house laboratory culture with a known history.

Reason for selection This system has been selected as an internationally
accepted invertebrate species.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 8 - Validity of batch Daphnids originated from a healthy stock, 2 nd to 5 th brood,
showing no signs of stress such as mortality >20%,
presence of males, ephippia or discoloured animals and
there was no delay in the production of the first brood.

Characteristics For the test selection of young daphni ds with an age of < 24

hours, from parental daphnids of more than two weeks old.

6.3. Breeding

Start of each batch With newborn daphnids, i.e. less than 3 days old, by placing

about 250 of them into 5 litres of medium in an all

- glass

culture vessel.

Maximum age of the cultures 4 weeks

R enewal of the cultures After 7 days of cultivation half of the medium twice a week.

Temperature of medium 18

- 22°C

Feeding Daily, a suspension of fresh water algae.

Medium M7, as prescribed by Dr. Elendt

- Schneider
(Elendt, B.

- P., 1990: Selenium deficiency in Crustacea. An
ultrastructural approach to antennal damage in Daphnia

magna Straus. Protoplasma 154, 25

- 33).

Composition of medium M7 :

Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmos is (RO

water

, GEON Waterbehandeling

, Berkel

- Enschot

, The Netherlands ):

Macro salts: CaCl 2 .2H 2 O 211.5 mg/l

MgSO 4 .7H 2 O 88.8 mg/l

NaHCO 3 46.7 mg/l

KCl 4.2 mg/l

Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO medium to reach the following

concentrations:

Trace elements: B 0.125 mg/l

Fe 0.05 mg/l

Mn 0.025 mg/l

Li, Rb and Sr 0.0125 mg/l

Mo 0.0063 mg/l

Br 0.0025 mg/l

Cu 0.0016 mg/l

Zn 0.0063 mg/l

Co and I 0.0025 mg/l

Se 0.0010 mg/l

V 0.0003 mg/l

Na 2 EDTA.2H 2 O 2.5 mg/l

Macro nutrients: Na 2 SiO 3

. 9H 2 O 10.0 mg/l

NaNO 3 0.27 mg/l

KH 2 PO 4 0.14 mg/l

K 2 HPO 4 0.18 mg/l

Vitamins: Thiamine 75.0 μg/l

B 12 1.0 μg/l

Biotin 0.75 μg/l

The hardness: 180 mg/l expressed as CaCO 3 and the pH: 7.7 ± 0. 3.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 9 - 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 ac id, 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. Approximately fifteen

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

medium. The lower test concentrations for the r ange

- finding test were prepared by subsequent dilutions of the 1000 mg/l concentration in test medium. The final test solutions were all clear and

colourless.

6.5. Range

- finding test

A range

- finding test was performed to provide information about the range of concentrations to be
used in the final test. Test procedure and conditions were similar to those applied in the limit test with
the following exceptions:

• Ten daphni ds per concentration (in duplicate, 5 per vessel) were exposed to nominal

concentrations of 0.10, 1.0, 10, 100 and 1000 mg/l, containing 0.039, 0.39, 3.9, 39 and

386 mg a.i. per litre, respectively;

• Dissolved oxygen concentrations and pH were only measured in the control and the highest

test concentration ;

• No sampling for determination of actual test concentrations was performed.

6.6. Limit test

6.6.1. 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

6.6.2. Test procedure and conditions

Test duration 48 hours

Test type Static

Test vessels 100 ml, all

- glass

Medium Adjusted ISO medium

Number of daphni ds 20 per concentration

Loading 5 per vessel containing 80 ml of test solution

Light 16 hours photoperiod daily

Feeding No feeding1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 10 - Aeration No aeration of the test solutions.

Introduction of daphni ds Within 20 minutes after preparation of the test solutions.

6.6.3. Sampling for analysis of test concentrations

S amples for possible analysis were taken from bot h the control and 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 and t= 48 h

Volume 1.5 ml from the approximate centre of the test vessels
Storage Samples were stored in a freezer until analysis.

At the end of the exposure period , the replicates were pooled at each concentration before sampling

.

Additionally, reserve samples of 1.5 ml were taken for possible analysis. If not 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.

6.6.4. Measurements and recordings

Immobility (including mortality) At 24 hours and at 48 hours.

pH and dissolved oxygen At the beginning and at the end of the test, for both the

control and the limit concentration.

Temperature of medium Continuously in a temperature control vessel, beginning at

the start of the test.

6.7. Elect ronic data capture

Observations/measurements in the study were recorded electronically using the following programme(s):

- R EES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA) : Temperature.

6.8. Interpretation

6.8.1. Data han dling

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

- toxic (EC 50 > maximum

concentration tested ).

6.8.2. Acceptability of the test

1. In the control, no daphni ds became immobilised.

2. The oxygen concentration at the end of the te st was ≥3 mg/l in control and test vessels.

6.9. List of deviations

6.9.1. List of protocol deviations

There were no deviations from the protocol.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 11 - 6.9.2. 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. Range

- finding test

Table 1 shows the responses recorded during the range

- finding test. No i mmobility was observed at any of the test concentrations during the test period. Therefore, the expected EC 50 was above a

nominal concentration of 10 00 mg/l, containing 386 mg a.i. per litre

.

All test conditions were maintained within the limits prescribed by the protocol.

Table 1 Incidence of immobility in the range

- finding test

Nominal c onc

. Test substance 1

(mg/l) Vessel

number Number

Daphnia

exposed Response at 24 h Response at 48 h

number Total

% number Total

%

control A

B 5

5 0

0 0 0

0 0

0.1 0 A

B 5

5 0

0 0 0

0 0

1.0 A

B 5

5 0

0 0 0

0 0

10 A

B 5

5 0

0 0 0

0 0

100 A

B 5

5 0

0 0 0

0 0

1000 A

B 5

5 0

0 0 0

0 0

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

7.2. Limit test

7.2.1. Me asured 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 sample taken at the start of the test showed that the actual test concentration was in

agr eement with nominal (100%). This concentration remained stable during the test period (102% of
initial at the end of the test). Given these results, the effect parameters were based on the nominal
exposure concentration.

7.2.2. Immobility

Table 2 shows the responses recorded during the limit test. No immobility was observed in the limit

concentration during the test period. The responses recorded in this test allowed for reliable

determination of an EC 50

.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 12 - Table 2 Acute immobilisation of daphni ds after 24 and 48 hours in the limit test

Concentration

Test substance 1

(mg/l) Vessel

number Number

Daphnia

exposed Response at 24 h Response at 48 h

number Total

% number Total

%

control A

B

C

D 5

5

5

5 0

0

0

0 0 0

0

0

0 0

1000 A

B

C

D 5

5

5

5 0

0

0

0 0 0

0

0

0 0

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

7.2.3. Determination of effect concentrations

Table 3 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 3 Effect parameters

Parameter Concentration

Test substance 1

(mg/l) Concentration

Acti ve ingredient 2

(mg/l)

24 & 48 h

- EC 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- hydroxypropane sulphonate

7.2.4. Experimental conditions

The results of measurement of pH and oxygen concentrations (mg/l) are presented in Table 4

. These
test conditions remained within the limits prescribed by the protocol (pH: 6.0

- 8.5, not varying by more

than 1 .5 unit; oxygen: ≥ 3 mg/l at the end of the test).

The temperature of the test medium ranged between 20.3 and 20.5°C at the start of the test. The
temperature continuously measured in a temperature control vessel varied between 19.4 and 20.3°C
during the test, and complied with the requirements as laid down in the protocol (18

- 22°C, constant
within 2°C).

Table 4 pH and oxygen concentrations during the limit test

Concentration

Test substance 1

(mg/l) Start (t=0 h) End (t=48 h)

pH O 2 pH O 2

c ontrol 7.8 9.2 7.9 8.8

10 00 7.6 9.2 7.8 8.8

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 13 - 8. CONCLUSION

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

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) did not induce acute immobilisation of Daphnia magna at an analytically confirmed

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

litre.

Hence, t he 48h

- EC 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 500676

- Page 14 - APPENDIX 1 REFERENCE TEST

Start: 04 March 2013

End : 06 March 2013

48

- hour Acute Toxicity Study in Daphnia magna with potassium dichromate (K 2 Cr 2 O 7 ) (WIL Research
Europe Project 502370).

The study procedures described in this report were based on the OECD guideline No. 202: "Daphnia
sp., Acute Immobilisation Test", Adopted April 13, 2004 and the ISO International Standard 6341.

The reference test was carried out to check the sensitivity of the test system as used by WIL Research
Europe. Daphnia were exposed for a maximum of 48 hours to K 2 Cr 2 O 7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/l and to a control. Twenty daphnia were exposed per concentration.

The reference substance, potassium dichromate (K 2 Cr 2 O 7 , art. 1.04864, batch no. K34869764 607)
was obtained from Merck, Darmstadt, Germany.

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium

dichromate:

K 2 Cr 2 O 7
Nominal concentration

(mg/l) Number

Exposed % immobile Expected response (%)

After 48 hours 1

24h 48h Minimal Maximal

control 20 0 0 0 10 2

0.10 20 0 5 0 10

0.18 20 0 0 0 10

0.32 20 0 0 0 30

0.56 20 15 55 0 100

1.0 20 70 100 40 100

1.8 20 100 100 100 100

1: Based on historical data of the previous years (n>60). 2: A maximum response of 10% does not invalidate the results of the test.

The actual responses in this reference test with K 2 Cr 2 O 7 are within the ranges of the expected

re sponses at the different concentrations, i.e. the 48h

- EC50 was between 0.3 and 1.0 mg/l. Hence, the
sensitivity of this batch of D. magna was in agreement with the historical data collected at WIL
Research Europe.

The 24h

- EC 50 was 0.78 mg/l with a 95% confidence interval between 0.69 and 0.93 mg/l.

The 48h

- EC 50 was 0.53 mg/l with a 95% confidence interval between 0.49 and 0.62 mg/l.

The raw data from this study are kept in the WIL Research Europe archives. The test described above
was performed under GLP with a QA

- check.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 15 - APPENDIX 2 CERTIFICATE OF ANALYSIS1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 16 - APPENDIX 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 500676

- Page 17 - 1. CONTENTS

1. CONTENTS

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

2. REPORT APPROVAL

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

3. INTRODUCTION

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

3.1. Preface

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

3.2. Aim of the study

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

4. MATERIALS AND METHOD S

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

4.1. Reagents

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

4.2. Samples

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

4.3. Analytical method

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

4.3.1. Analytical conditions

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

4.3.2. Preparation of solutions

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

4.3.3. Sample injections

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

4.4. Electronic data capture

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

4.5. Formulas

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

5. RESULTS

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

5.1. Calibration curves

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

5.2. Samples

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

5.2.1. Procedural recovery samples

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

5.2.2. Test samples

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

TABLES

Table 1 Procedural recovery samples

........................................................................................... 23

Table 2 Concentra tions of the test substance in test medium

....................................................... 231- propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500676

- Page 19 - 3. INTRODUCTION

3.1. Preface

Study plan analytical phase Start : 21 M ay 201 3 Completion : 22 M ay 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

syste m (Millipore, Bedford, MA, USA)

Di

- Sodium hydrogenphosphate dihydrate Merck, Darmstadt, Germany ( Na 2 HPO 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 500676

- Page 20 - 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 solutions
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 sampl es 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 500676

- Page 21 - 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 500676

- Page 22 - 5. RESULTS

5.1. Calibration curves

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

were used. Linear r egression 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 500676

- Page 23 - 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 1000 100

48 15

- May

- 2013 21

- May

- 2013 0 n.d. n.a.

1000 1022 102 102

1 Samples were stored in the freezer ( ≤

- 15°C ) until the day of analysis.

n.d. Not detected.

n.a. Not applicable.