## FINAL REPORT

Study Title



## DETERMINATION OF 'READY' BIODEGRADABILITY: CARBON

DIOXIDE (CO 2 

## ) EVOLUTION TEST (MODIFIED STURM TEST) O F

1- PROPANESULFONIC ACID , 2

- HYDROXY -3-

(2-PROPEN-1-YLOXY)- , SODIUM SALT (1:1)

Author

M.J.E. Desmares

- Koopmans , Bachelor, ERT

Test Facility

WIL Research Europe B.V. Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 500675

Substance 204240/A

- Page 1 of 18
-1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 2 - 1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COM PLIANCE

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

3. QUALITY ASS URANCE STATEMENT

............................................................................................ 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

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

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. Test concentration and preparation of test solutions

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

6.2. Reference substance

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

6.2.1. Reference substance

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

6.2.2. Reference substance concentration and preparation of test solutions

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

6 .3. Test system

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

6.4. Test procedure and conditions

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

6.4.1. Preparation of bottles

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

6.4.2. Determination of CO 2

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

6.4.3. Measurements and recording

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

6.5. Electronic data capture

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

6.6. Interpretation

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

6.6.1. Data evaluation

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

6.6.2. Acceptabilit y of the test

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

6.7. List of deviations

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

6.7.1. List of protocol deviations

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

6.7.2. List of standard operating procedures deviations

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

7. RESULTS

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

7.1. Theoretical CO 2 production

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

7.2. Biodegradation

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

7.3. Monitoring of temperature and pH

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

8. CONCLUSION

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

TABLES

Table 1 Calculation of the Carbon content.

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

Table 2 pH values of different test media.

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

Table 3 HCl titrated in duplicate blank bottles

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

Table 4 HCl titrated in Ba(OH) 2 solution (background bottles)

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

Table 5 CO 2 production in the blank.

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

Table 6 CO 2 production and percentage biodegradation of the positive control substance.

..... 16

Table 7 CO 2 production and percentage biodegradation of the test substance (bottle A).

....... 16

Table 8 CO 2 production and percentage biodegradation of the test substance (bottle B).

....... 17

Table 9 CO 2 production and percentage biodegradation of the toxicity control.

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

Table 10 Comparison of biodegradation of the test substance in bottles A and B.

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

FIGURES Figure 1 Biodegradation of 1

- Propanesulfonic acid, 2

- hydroxy -3- (2 -p ropen -1- yloxy)

- , sodium salt (1:1) and sodium acetate in the modified Sturm test

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

APPENDICES

APPENDIX 1 TABLES

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

APPENDIX 2 CERTIFICATE OF ANALYSIS

...................................................................................... 181- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 5 - 4. SUMMARY

Determination of ‘ready’ biodegradability: carbon dioxide (CO 2 ) evolution test (modified Sturm test)

with 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

.

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In
addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142 , Part C.4

- C and ISO 9439, 1999.

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was a clear colourless liquid and consisted of 3 components. The purity and composition were used for the calculation of the carbon content (14%). In the test a concentration of 86 mg/l was tested (172 mg in 2 Litres final medium), corresponding with 12 mg TOC/l. The Theoretical CO 2 production (ThCO 2 ) of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was calculated to be 0.51 mg CO 2 /mg.

The study consisted of six bottles:

- 2 inoculum blanks (no test substance),

- 2 test bottles ( 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) ),

- 1 positive control (sodium acetate) and

- 1 toxicity control ( 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) plus sodium acetate).

Since 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , so dium salt (1:1) was easily soluble in water the test media were prepared using a stock solution of 1 g/l in Milli

- RO water. Aliquots of 172 ml

of the clear and colourless stock solution were added to the test substance bottles A and B and to the
toxicity c ontrol. These test bottles contained medium with microbial organisms (final volume: 2 litres).
The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 d ays (last CO 2 - measurement on the 29 th

day).

The relative biodegradation values calculated from the measurements performed during the test period revealed 8% and 21% biodegradation of 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10

- day window) was not met.
In the toxicity control, 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) wa s designated as not readily biodegradable.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- 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. Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director M.J.E. Desmares

- Koopmans, Bachelor, ERT

Study Plan Start : 23 January 2013 Completio n : 02 May 2013

5.2. Aim of the study

The purpose of the study was to evaluate a non

- volatile test substance for i ts ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with the supernatant of
activated sludge.

5.3. Guidelines

The study procedures described in this report were based on the Organization for Economic Co

oper ation and Development (OECD), OECD guidelines for Testing of Chemicals, Section 3, Degradation and Accumulation, guideline No. 301 B: "Ready Biodegradability: CO 2 Evolution Test" adopted July 17, 1992.

In addition, the procedures were designed to meet the test methods prescribed by the following
guidelines:

• Commission Regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.4. "Biodegradation: determination of the 'ready' biodegradability, C .4

- C: Carbon dioxide (CO 2 ) evolution test (Modified Sturm Test).

• ISO International Standard 9439 “Water Quality

- Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium

- carbon dioxide evolution test (1999).

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 Europe 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 sam ple until the expiry date, but no longer than

10 years after finalization of the report. After this period the sample will be destroyed.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 7 - 5.5. Definitions

Readily biodegradable are those test substances giving a result of at least 60% biodegradation

within 28 days. This pass level must be reached within the 10 days immediately following the attainment of 10% biodegradation (10

- day window).

Theoretical carbon dioxide (ThCO 2 ) is the quantity of carbon dioxide calculated (mg) to be produced from the known or measured carbon content of the test substance when fully mineralized; also
expressed as mg carbon dioxide evolved per mg test substance.

Total Organic Carbon (TOC) of a sample is the sum of the organic carbon in solution and in
suspension.

6. MATERIALS AND MET HODS

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 we ight 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 substance storage At room temperature in the dark Stability under storage conditions Stable Expiry date 13 August 2013

For the Certificate of Analysis see APPENDIX 2

.

6. 1.2. Study specific test substance information

Hygroscopic No

Volatile No Stability in water 1 year Solubility in water 1- 45%1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 8 - 6.1.3. Test concentration and preparation of test solutions

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was a c lear colourless liquid and consisted of 3 components. The purity and composition were used for the calculation of the carbon content (14%, see Table 1 )

. In the test a concentration of 86 mg/l was tested (172 mg in 2

L itres final medium), corresponding with 12 mg TOC/l.

Table 1 Calculation of the Carbon content.

Purity/Composition CAS Number Percent Molecular

formula Molecular weight % C

Sodium

- 1

- allyloxy

- 2

- hydroxypropane sulphonate 52556

- 42

- 0 38.64 C 6 H 11 NaO 5 S 218.22 33.0

3

- allyl

- 1,2

- propanediol 123

- 34

- 2 2.26 C 6 H 12 O 3 132.16 54.5

Water 7732

- 18

- 5 59.1 H 2 O 19.02 0

Total 100 14.0

Since 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was easily soluble in

wate r the test media were prepared using a stock solution of 1 g/l in Milli

- RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). A weighed amount of 1.0031 g of

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was dissolved in Milli

- RO
water and made up to 1000 ml. Aliquots of 172 ml of the clear and colourless stock solution were added to the test substance bottles A and B and to the toxicity control. These test bottles contained medium with microbial organisms (final volume: 2 litres). The test solutions were continuously stirred
during the test, to ensure optimal contact between the test substance and the test organisms.

6.2. Reference substance

6.2.1. Reference substance

Identification number RS186

Name S odium acetate Description White powder (determined at WIL Research Europe )

Molecular formula CH 3 COONa (taken from label) Molecular weight 82.03 (taken from label)

Batch number K34333668

Article number 1.06268.0250 Purity ≥ 99.0% Expiry Date 28 Februa ry 2015

Certified Yes Storage conditions At room temperature in the dark Supplier Merck, Darmstadt, Germany

6.2.2. Reference substance concentration and preparation of test solutions

A solution of sodium acetate was prepared by dissolving 400.8 mg in Milli -R O water and making this up to a total volume of 100 ml. Volumes of 20 ml from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final

concentration of 40 mg sodium acetate per litre (12 mg TOC/l).1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 9 - 6.3. Test system

Source The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant:
'Waterschap Aa en Maas ', Heeswijk

- Dinther, The Netherlands, receiving predominantly domestic s ewage.

Treatment The freshly obtained sludge was kept under continuous

ae ration until further treatment. The concent ration of
suspended solids was 3 g/l in the concentrated sludge (information obtained from the municipal sewage treatment
plant [3.0 g/l] a nd confirmed by the determination of suspended solids [3.1 g/l] ). Before use, the sludge was allowed to settle ( 34 minutes) and the supernatant liquid was

use d as inoculum at the amount of 1 5 ml/l 1 of mineral

medium.

Reason for selection The test has been accepted internationally for determining the 'ready' biodegradability of test substances under aerobic

conditions.

6.4. Test procedure and conditions

Test duration 28 days (last CO 2 - measurement on the 29 th day).
During the test period the test media were aerated and stirred continuously.

Test vessels 2 litre all

- glass brown coloured bottles.

Milli

- RO / Milli

- Q water Tap

- water purified by reverse osmosis (Milli

- RO) and
subsequently passed over activated carbon and ion

exchange cartridges (Milli

- Q) (Millipor e Corp., Bedford, Mass., USA).

Stock solutions of A) 8.50 g KH 2 PO 4 mineral components 21.75 g K 2 HPO 4 67.20 g Na 2 HPO 4 .12H 2 O
0.50 g NH 4 Cl

dissolved in Milli

- Q water and made up to 1 litre,
pH 7.4 ± 0.2 B) 22.50 g MgSO 4 .7H 2 O dissolved in Milli

- Q water
and made up to 1 litre. C) 36.40 g CaCl 2 .2H 2 O dissolved in Milli

- Q water and
made up to 1 litre. D) 0.25 g FeCl 3 .6H 2 O dissolved in Milli

- Q water and
made up to 1 litre.

Mineral medium 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli

- RO water.

Barium hydroxide 0.0125 M Ba(OH) 2 (Boom, The Netherlands), stored in a sealed vessel to prevent absorption of CO 2 from the air.

1 See also paragraph 6.7.1

.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 10
- Synthetic air (CO 2 < 1 ppm) A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was

pass ed through a bottle, containing 0.5
- 1 litre 0.0125 M Ba(OH) 2 solution to trap CO 2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1

- 2 bubbles per second (ca. 30

- 100 ml/min)

.

6.4.1. Preparation of bottles

Pre

- incubation medium The day before the start of the test (day

- 1) m ineral
components, Milli

- RO water (ca. 80% total volume) and inoculum (1 .5 % final volume) were added to each bottle.
This mixture was aerated with synthetic air overnight to purge the system of CO 2

.

Type and number of bottles Test suspension: containing test substance and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles) Positive control: containing reference substance and inoculum (1 bottle). Toxicity control: containing test substance, reference substance and inoculum (1 bottle).

Preparation At the start of the test (day 0) test and reference substance were added to the bottles containing the microbial orga nisms
and mineral componen ts

. The volumes of suspensions were made up to 2 litres with

Milli

- RO water, resulting in the mineral medium described

before.

Three CO 2 - absorbers (bottles filled with 100 ml 0.0125 M Ba(OH) 2 ) were connected in series to the exit air

line of each test bottle.

6.4.2. Determination of CO 2

Experimental CO 2 production The CO 2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO 2 produced was determined by titrating the remaining Ba(OH) 2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® amp o ul e ), Merck, Darmstadt, Germany).

Measurements Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the

28 th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.

Each time the CO 2 - absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A

new CO 2 - absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used
as pH

- indicator.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 11
- On the 28 th day, the pH of all test suspe nsions was measured and 1 ml of concentrated HCl (37%, Merck) was

added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off

CO 2 present in the test suspension. The final titration was made on day 29.

T heoretical CO 2 production The theoretical CO 2 production was calculated from the

molecular formula.

6.4.3. Measurements and recording

pH At the start of the test (day 0) and on the 28 th day.

Temperature of medium Continuously in a vessel with Milli

- RO water in the same

room.

6.5. Electronic data capture

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



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

6.6. Interpretation

6.6.1. Data evaluation

ThCO 2 , expressed as mg CO 2 /mg test substance, that can be generated by a test substance was

calculated as follows:

substancetweight tes Molecular 2 CO weight Molecular substance testinatoms carbonofNo.

2 ThCO ×

=

The first step in calculating the amount of CO 2 produced is to correct for background ( endogenous)

CO 2 production. Thus the amount of CO 2 produced by a test substance is determined by the difference (in ml of titrant) between the experimental and blank Ba(OH) 2 traps.

The amount of 0.05 N HCl titrated is converted into mg of CO 2 produced:

titrated HCl ml1.144 2 titrated HCl ml05.0 CO mg 2 ∆×=× ∆×

=

Relative biodegradation values were calculated from the cumulative CO 2 production relative to the total expected CO 2 production based on the total carbon content of the amount of test substance present in the test bottles. A figure of more than 10% biodegradation was considered significant.
The relative biodegradation values were plotted versus time together with the relative biodegradation of the positive control. If applicable, the number of days is calculated from the attainment of 10% biodegradation until 60% biodegradation. Should this period be ≤ 10 days (10

- day window), then the
test substance is designated as readily biodegradable.

Toxicity control: if less than 25% biodegradation (based on ThCO 2 ) occurred within 14 days, the test substance was assumed to be inhibitory.

The total CO 2 evolution in the inoculum blank was determined by the cumulative difference (in ml of titrant) between the blank Ba(OH) 2 traps and fresh Ba(OH) 2

.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 12
- 6.6.2. Acceptability of the test

1. The positive control su bstance was biodegraded by at least 60% (71%) within 14 days.

2. The difference of duplicate values for %

- degradation of the test substance was always less than

20.

3. The total CO 2 release in the blank at the end of th e test did not exceed 40 mg/l (62.7 mg CO 2 per 2 litres of medium, corresponding to 31.4 mg CO 2 /l).

4. The Inorganic Carbon content (IC) of the test substance (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test
medium was prepared in tap

- water purified by reverse osmosis (Milli

- RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test substance, 12 mg TOC/l).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

6.7. List of deviations

6.7.1. List of protocol deviations

1. The concentration of suspended solids of the sludge as obtained from the municipal sewage treatment plant was at the lower limit of the range as described by the protocol (3.0 g/l). Therefore, the sludge will be allowed to settle and the liquid will be decanted for use as inoculum

at the amount of 15 ml/l instead of 10 ml/l of mineral medium.

Evaluation: A logical correction on the amount of inoculum was made. Since all criteria for acceptability of the test were met, this deviation was considered to have no effect on the outcome of this study.

2. On day 1 temporary breakdown in the aeration (< 1 day) was noted in blank control A.

Evaluation: T his relat ive short breakdown was considered to have no effect on the outcome of this study (the criterion for Ready biodegradability).

The study integrity was not adversely affected by the deviations.

6.7.2. 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.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 13
- 7. RESULTS

7.1. Theoretical CO 2 production

The ThCO 2 of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was
calculated to be 0.51 mg CO 2 /mg.

The ThCO 2 of sodium acetate was calculated to be 1.07 mg CO 2 /mg.

The ThCO 2 per test bottle are given in Tables 5

- 8 ( APPENDIX 1 )

.

7.2. Biodegradation

All data are presented in APPENDIX 1

. The results of CO 2 production and biodegradation in blank bottles, background bottles and each test bottle are listed in Table 3 to 8. Table 10 contains the comparison of biodegradation of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in bottles A and B.

Figure 1 shows the curves for biodegradation of the two bottles with 1-P ropanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1), the positive control and the toxicity control.

Figure 1 Biodegradation of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy )- , sodium salt (1:1) and sodium acetate in the modified Sturm test

The relative biodegradation values calculated from the measurements performed during the test
period r evealed 8 % and 21 % biodegradation of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10

- day window) was not met.

In the toxicity control more than 25% biodegradation occurred within 14 days (3 3 %, based on ThCO 2 ). Therefore, the test substance was assumed not to inhibit microbial activity.

Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve (see also section 6.6.2 )

.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 14
- 7.3. Monitoring of temperature and pH

The temperature recorded in a vessel with water in the same room varied between 22 and 23°C.

The pH values of the different test media are presented in Table 2

.

Table 2 pH values of different test media.

Test medium: At the start of the test: On day 28:

Blank control (A) 7.6 7.5

Blank control (B) 7.6 7.5

Positive control 7.6 7.8

1- Propanesulfonic acid, 2

- hydroxy -3-(2- propen -1- yloxy)

- , sodium salt (1:1) (A) 7.6 7.5

1- Propanesulfonic acid, 2

- hydroxy -3-(2- propen -1- yloxy)

- , sodium salt (1:1) (B) 7.6 7.5

Toxicity control 7.6 7.8

8. CONCLUSION

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was not readily biodegradable under the conditions of the modified Sturm test presently performed.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 15
- APPENDIX 1 TABLES

Notes: Except for the percentages biodegradation, a ll calculations are performed without rounding off.

Produced CO 2 : negative values are expressed as 0.00 ml HCl.

Table 3 HCl titrated in duplicate blank bottles

Day HCl (0.05 N) titrated (ml)

Blank A Blank B Mean Value

2 47.16 47.11 47.14

5 45.28 46.20 45.74

7 46.04 46.53 4 6.29

9 47.23 46.93 47.08

14 46.33 46.20 46.27

19 45.91 46.00 45.96

23 45.02 45.40 45.21

27 45.29 45.05 45.17

29 44.80 44.90 44.85

29 49.11 49.58 49.35

29 50.16 50.10 50.13

Table 4 HCl titrated in Ba(OH) 2 solution ( backgro und bottles)

Day HCl (0.05 N) titrated (ml)

Bottle A Bottle B Mean value

2 52.16 51.62 51.89

5 50.91 52.13 51.52

7 50.40 50.33 50.37

9 50.68 51.55 51.12

14 51.50 52.56 52.03

19 53.01 52.95 52.98

23 52.48 52.71 52.60

27 52.37 52.21 52.29

29 51.6 5 51.56 51.61

29 51.93 51.92 51.93

29 51.66 52.05 51.861- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 16
- APPENDIX 1 TABLES
– CONTINUED

Table 5 CO 2 production in the blank.

Day HCl (0.05 N) titrated (ml) Produced

CO 2 Produced

CO 2 Cumulative

CO 2 Ba(OH) 2 1) Blank (mean) (ml HCl) (mg) (mg)

2 51.89 47.14 4.76 5.2 5.2

5 51.52 45.74 5.78 6.4 11.6

7 50.37 46.29 4.08 4.5 16.1

9 51.12 47.08 4.04 4.4 20.5

14 52.03 46.27 5.77 6.3 26.9

19 52.98 45.96 7.03 7.7 34.6

23 52.60 45.21 7.39 8.1 42.7

27 52.29 45.17 7.12 7.8 50.5

29 5 1.61 44.85 6.76 7.4 58.0

29 51.93 49.35 2.58 2.8 60.8

29 51.86 50.13 1.73 1.9 62.7

1) : "Strength" of untreated 0.0125 M Ba(OH) 2 solution

Table 6 CO 2 production and percentage biodegradation of the positive control substance.

Day HCl (0.05 N) titrated (ml) Produced

CO 2 (ml HCl) Produced

CO 2 (mg) Cumulative

CO 2 (mg) Biodegradation 1)

(%) Blank (mean) Positive

control

2 47.14 44.61 2.53 2.8 2.8 3

5 45.74 24.51 21.23 23.4 26.1 30

7 46.29 32.55 13.74 15.1 41.2 48

9 47.08 38.80 8.28 9.1 50.3 59

14 46.27 36.40 9.87 10.9 61.2 71

1) : Calculated as the ratio between CO 2 produced (cumulative) and the ThCO 2 of sodium acetate: 85.8 mg CO 2 /2l

Table 7 CO 2 production and percentage biodegradation of the test substance (bottle A).

Day HCl (0.05 N) titrated (ml) Produced

CO 2 Produced

CO 2 Cumulative

CO 2 Biodegradation 1)

(%) Blank (mean) Bottle A (ml HCl) (mg) (mg)

2 47.14 46.34 0.79 0.9 0.9 1

5 45.74 45.26 0.48 0.5 1.4 2

7 46.29 45.35 0.93 1.0 2.4 3

9 47.08 47.08 0.00 0.0 2.4 3

14 46.27 45.26 1.01 1.1 3.5 4

19 45.96 45.22 0.73 0.8 4.3 5

23 45.21 44.33 0.88 1.0 5.3 6

27 45.17 45.23 0.00 0.0 5.3 6

29 44.85 44.54 0.31 0.3 5.7 6

29 49.35 47.91 1.44 1.6 7.2 8

29 50.13 49.94 0.19 0.2 7.4 8

1) : Calcu lated as the ratio between CO 2 produced (cumulative) and the ThCO 2 of the test substance: 88.0 mg CO 2 /2l1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 17
- APPENDIX 1 TABLES
– CONTINUED

Table 8 CO 2 production and percentage biodegradation of the test substance (bottle B).

Day H Cl (0.05 N) titrated (ml) Produced

CO 2 (ml HCl) Produced

CO 2 (mg) Cumulative

CO 2 (mg) Biodegradation 1)

(%) Blank (mean) Bottle B

2 47.14 47.90 0.00 0.0 0.0 0

5 45.74 46.17 0.00 0.0 0.0 0

7 46.29 45.35 0.93 1.0 1.0 1

9 47.08 46.03 1.05 1.2 2.2 2

14 46.27 43.69 2.58 2.8 5.0 6

19 45.96 42.78 3.18 3.5 8.5 10

23 45.21 42.31 2.90 3.2 11.7 13

27 45.17 42.13 3.04 3.3 15.0 17

29 44.85 42.81 2.04 2.2 17.3 20

29 49.35 48.36 0.98 1.1 18.4 21

29 50.13 49.99 0.14 0.2 18.5 21

1) : Calculated as the ratio between CO 2 produced (cumulative) and the ThCO 2 of the test substance: 88.0 mg CO 2 /2l

Table 9 CO 2 production and percentage biodegradation of the toxicity control.

Day HCl (0.05 N) titrated (ml) Produced

CO 2 (ml HCl) Produced

CO 2 (mg) Cumulative

CO 2 (mg) Biodegradation 1)

(%) Blank (mean) Toxicity

control

2 47.14 45.61 1.53 1.7 1.7 1

5 45.74 28.06 17.68 19.4 21.1 12

7 46.29 32.96 13.33 14.7 35.8 21

9 47.08 38.51 8.57 9.4 45.2 26

14 46.27 34.93 11.34 12.5 57.7 33

1) : Calculated as the ratio between CO 2 produced (cumulative) and the sum of the ThCO 2 of the test substance and

positive control: 173.8 mg CO 2 /2l (ThCO 2 test substance: 88.0 mg CO 2 /2l + ThCO 2 sodium acetate:

85.8 mg CO 2 /2l)

Table 10 Comparison of biodegradation of the test substance in bottles A and B.

Day Biodegradation (%)

Bottle A Bottle B Mean A and B ∆ A

- B 1)

2 1 0 1 1

5 2 0 1 2

7 3 1 2 2

9 3 2 3 1

14 4 6 5 2

19 5 10 8 5

23 6 13 10 7

27 6 17 12 11

29 6 20 1 3 14

29 8 21 15 13

29 8 21 15 13

1) : Absolute difference in biodegradation between bottles A and B1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500675

- Page 18
- APPENDIX 2 CERTIFICATE OF ANALYSIS