## FINAL REPORT

Study Title



## FRESH WATER ALGAL GROWTH INHIBITION TEST

WITH

1- PROPANESULFONIC ACID , 2

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

YLOXY)- , SODIUM SALT (1:1)

Author

L.M. Bouwman, MSc.

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 500678

Substance 204240/A

- Page 1 of 29

-1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 2 - 1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COM PLIANCE

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

3. QUALITY ASSURANCE ST ATEMENT

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

4. SUMMARY

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

5. INTRODUCTION

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

5.1. Preface

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

5.2. Aim of the study

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

5.3. Guidelines

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

5.4. Storage and retention of records and materials

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

5.5. Definitions

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

6. MATERIALS AND METHOD S

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

6.1. Test Substance

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

6.1.1. Test substance information

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

6.1.2. Study specific test substance information

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

6.1.3. Reference substance

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

6.2. Test System

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

6.3. Fresh water algae culture

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

6.4. Preparation of test solut ions

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

6.5. Range

- finding tests

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

6.6. Limit test

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

6.6.1. Te st concentrations

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

6.6.2. Test procedures and conditions

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

6.6.3. Sampling for analysis of test concentrations

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

6.6.4. Measurements

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

6.6.5. Recording of cell densities

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

6.7. Electroni c data capture

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

6.8. Interpretation

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

6.8.1. Data handling

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

6.8.2. Acceptability of the limit test

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

6.9. List of deviations

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

6.9.1. List of protocol deviations

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

6.9.2. List of standard operating procedures deviations

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

7. RESULTS

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

7.1. First range

- finding test

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

7.1.1. Mean cell densities, reduction of growth rate and inhibition of yield

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

7.2. Second range

- finding test

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

7.2.1. Mean cell densities, reduction of growth rate and inhibition of yield

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

7.3. Limit test

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

7.3.1. Measured test substance concentrations

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

7.3.2. Mean cell densities

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

7.3.3. Reduction of growth rate and inhibition of yield

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

7.3.4. Determination of effect concentrations

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

7.3.5. Experimental condit ions

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

8. CONCLUSION

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

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 3 - TABLES

Table 1 Mean cell densities (x10 4 cells/ml) during the first range

- fi nding test

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

Table 2 Percentage reduction of growth rate, inhibition of yield and inhibition of cell

growth during the first range

- finding test

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

Table 3 Mean cell densities (x10 4 cells/ml) during the second range

- finding test

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

Table 4 Percentage reduction of growth rate, inhibition of yield and inhibition of cell

growth during the second range

- finding test

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

Table 5 Mean cell densities (x 10 4 cells/ml) during the limit test

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

Table 6 Per centage reduction of growth rate, inhibition of yield and inhibition of cell

growth during the limit test

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

Table 7 Effect parameters

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

Table 8 pH levels recorded during the limit test

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

Table 9 Individual cell densities (x10 4 cells/ml)

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

Table 10 Calc ulation of growth rate, yield and cell growth

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

FIGURES

Figure 1 Growth curves at different concentrations of 1

- Propanesulfonic acid, 2

- hydroxy -3-

(2

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

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

APPENDICES

APPENDIX 1 WORKSHEET DATA

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

APPENDIX 2 REFERENCE TEST

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

APPENDIX 3 CERTIFICATE OF ANALYSIS

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

APPENDIX 4 ANALYTICAL REPORT

................................................................................................ 221- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 6 - 4. SUMMARY

Pseudokirchneriella subcapitata , Fresh Water Algal Growth Inhibition Test 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 OCSPP 850.4500, 2012

. In addition,

the procedures were designed to meet the test methods of the OECD guideline No. 201, 2006 ; Annex

5 corrected 28 July 2011

.

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 tes ted.

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

- finding tests. 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- hydroxypropa ne 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.

Six replicates of exponentially growing algal cultures were exposed to a nominal concentration of

1000 mg/l and a control. The initial cell density was 10 4 cells/ml and the total test period was 96 hours

. S amples for analytical confirmation of actual exposure concentrations were taken at the start, after 2 4

hours of exposure and at 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 (101%). This concentration remained stable during the test period (102% of

initia l at the end of the test). 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.

N o reduction of growth rate, inhibition of yield or inhibition of cell growth was recorded at an

analytically confirmed nominal concentration of 1000 mg/l. This concentration contained 386 mg of the

active ingredient per litre.

The IC 50 for growth rate reduction ( 96h -I R C 50 ) , yield inhibition (9 6h -I Y C 50 ) and cell growth inhibition

(96h -I B C 50 ) was > 1000 mg test substance /l , corresponding to >386 mg a.i./l

.

The 96h

- NOEC for growth rate reduction, yield inhibition and cell growth inhibition was 1000 mg/l.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 7 - 5. INTRODUCTION

5.1. Preface

Sponsor Society of Ch emical 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 L.M. Bouwma n, MSc.

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

Study Plan Start : 08 April 2013

Completion : 17 May 2013

5.2. Aim of the study

The purpose of the study is to evaluate the test substance for its ability to generate toxic effects in

Pseudokirchne riella subcapitata during an exposure period of 96 hours and, if possible, to determine

the IC 50 for reduction of growth rate, inhibition of yield and area under the growth curve (biomass). In

addition, the NOEC or IC 05 for these measures will be determine d.

5.3. Guidelines

The study procedures described in this report were based on the EPA Ecological Effects Test

Guidelines, OCSPP 850.4500, Algal Toxicity, EPA 712 -C- 006

, January 2012

.

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

guideline:

• OECD guideline s for Testing of Chemicals, guideline No. 201: "Freshwater Alga and

Cyanobacteria , Growth Inhibition Test", Adopted March 23, 2006; Annex 5 corrected 28 July 2011

.

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 sample until the expiry date, but n o longer than 10

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

5.5. Definitions

Cell density is the number of cells per millilitre.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 8 - Growth is the increase of cell density over the test period. The I B C 50 is the concentration of test

substance that results in a 50% inhibition of cell growth relative to the control.

Growth rate is the increase in cell density per unit time. It is derived from the slope of the growth

curve in a logarithmic plot. Following from the mathematical nature of exponential growth, the measure

of the specific growth rate is preferable over cell density or yield. The I R C 50 is the concentration of test

substance that results in a 50% reduction in growth rate relative to the control.

Yield is define d as the cell density at the end of the exposure period minus the cell density at the start

of the exposure period. The I

y C 50 is the concentration of test substance that results in a 50% inhibition

of yield relative to the control.

No Observed Effect Concentration (NOEC) is the highest tested concentration at which the

measured parameter(s) show(s) no significant effect on algal growth relative to control values.

6. MATERIALS AND METHOD S

6.1. Test Substance

6.1.1. Test substance information

Identification 1

- Propanesul fonic 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/Co mposition

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

See APPENDIX 3 : Certificate of analysis.

6.1.2. Study specific test substance information

Purity/composition correction factor

required No

Hygroscopic No

Volatile No

Specifi c Gravity / Density 1.17

Stability at higher temperatures Yes, maximum temperature: 80 ° C,

maximum duration: 1 hour

Stability in water 1 year1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 9 - 6.1.3. Reference substance

This report includes the results of the most recent reference test with potassium dichrom ate

( APPENDIX 2 )

.

6.2. Test System

Species Pseudokirchneriella subcapitata , strain: NIVA CHL 1

Source In

- house laboratory culture.

Reason for selection This system is an unicellular algal species sensitive to toxic

substances in the aquatic ecosystem and has been selected

as an internationally accepted species.

6.3. Fresh water algae culture

Stock culture Algae stock cultures were started by inoculating growth

medium with algal cells from a pure culture on agar. The

suspensions were cont inuously aerated and exposed to light

in a climate room at a temperature of 2 1- 24 °C.

Light intensity 60 to 120 μE/m 2 /s when measured in the photosynthetically

effective wavelength range of 400 to 700 nm.

Stock culture medium M1; according to the NPR 650 5 (“ Nederlandse Praktijk

Richtlijn no. 6505 ”) formulated using Milli

- RO water (tap

water purified by reverse osmosis; Millipore Corp., Bedford,

Mass., USA) with the following composition:

NaNO 3 500 mg/l

K 2 HPO 4 .3H 2 O 52 mg/l

MgSO 4 .7H 2 O 75 mg/l

Na 2 CO 3 .10H 2 O 54 mg/l

C 6 H 8 O 7 .H 2 O 6 mg/l

NH 4 NO 3 330 mg/l

CaCl 2 .2H 2 O 35 mg/l

C 6 H 5 FeO 7 .xH 2 O 6 mg/l

H 3 BO 3 2.9 mg/l

MnCl 2 .4H 2 O 1.81 mg/l

ZnCl 2 0.11 mg/l

CuSO 4 .5H 2 O 0.08 mg/l

(NH 4 ) 6 Mo 7 O 24 .4H 2 O 0.018 mg/l

Pre

- culture 3 to 4 days before the start of the test, cells from the algal

stock culture were inoculated in culture medium at a cell

density of 1 x 10 4 cells/ml. The pre

- culture was maintained

under the same conditions as used in the test. The cell

density was measured immediately before use.

Pre

- culture medium M2 ; according to the OECD 201 Guideline, formulated using

Milli

- RO water and with the following composition:

NH 4 Cl 15 mg/l

MgCl 2 .6H 2 O 12 mg/l

CaCl 2 .2H 2 O 18 mg/l

MgSO 4 .7H 2 O 15 mg/l

KH 2 PO 4 1.6 mg/l

FeCl 3 .6H 2 O 64 μg/l

Na 2 EDTA.2H 2 O 100 μg/l

H 3 BO 3 185 μg/l

MnCl 2 .4H 2 O 415 μg/l

ZnCl 2 3 μg/l

CoCl 2 .6H 2 O 1.5 μg/l

CuCl 2 .2H 2 O 0.01 μg/l

Na 2 MoO 4 .2H 2 O 7 μg/l

NaHCO 3 50 mg/l

Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO 3 /l)1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 10

- pH 8. 1 ± 0.2

6.4. Preparation of test solutions

The standard test procedures required generation of test solutions, which contained completely

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

testing of concentrations that would disturb the test system was prevented as much as possible (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 acid, 2

- hy droxy -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

th e 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 range

- fin ding test s were prepared by subsequent

dilutions of the 1000 mg/l concentration in test medium. The final test solutions were all clear and

colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration.

Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of

10 4 cells/ml.

6.5. Range

- finding test s

Two range

- finding test s w ere 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:

• Exponentially growing algal cultures 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;

• Three replicates were tested per concentration and three replicates in the control group ;

• During the second range

- finding test pH was only measured in the control and the highest test

concentration ;

• At the end of the second range

- finding test algae were not observed to verify a normal and

healthy appearance ;

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

6.6. L imit 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

Replicates 6 replicates of both the control and the test concentration;

1 extra replicate of both the control and the test

concentration for sampling purposes;

2 replicate s of the test concentration without algae.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 11

- 6.6.2. Test procedures and conditions

Test duration 96 hours

Test type Static

Test vessels 1 25 ml, all

- glass, containing 50 ml of test solution

Medium M2

Cell de nsity An initial cell density of 1 x 10 4 cells/ml.

Introduction of algal cells Immediately after preparation of the test solutions.

Illumination Continuously using TLD

- lamps of the type ‘Cool

- white’ of

30 Watt, with a light intensity within the range of 59 to

66 μ E.m -2 .s -1

.

Incubation Capped v essels were distributed at random in the incubator

and as such were daily repositioned . During incubation the

algal cells were kept in suspension by continuous shaking.

6.6.3. Sampling for analysis of test concentrations

S amples for possible analysis were taken from both the control and the test concentration according to

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

( APPENDIX 4 )

.

Frequency at t=0 h, t= 24 h and t= 96 h

Volume 1.5 ml

Storage Samples were stored in a freezer until analysis.

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

sampling

.

Compliance with the Quality criteria regarding maintenance of actual concentrations was

demonstrated by running a test vessel at the highest substance concentration but without algae and

samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test

period.

Addition ally, 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.

6.6.4. Measurements

pH At the beginning and at the end of the test.

The pH of the solutions should preferably not deviate by

more than 1.5 units during the test.

Temperature of medium Continuously in a temperature control vessel.

Appea rance of the cells At the end of the first range

- finding test and the limit test

microscopic observations were performed on the limit

concentration to observe for any abnormal appearance of

the algae.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 12

- 6.6.5. Recording of cell densities

At the beginning of the test, cells were counted using a microscope and a counting chamber.

Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm

using a spectrophotometer with imme rsion probe (pathlength =20 mm)

. Algal medium was used as

blank. One extra test vessel per concentration without algae was used as background for the

determination of the algal cell density at each time interval.

6.7. Electronic data capture

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

programme(s):

- Shimadzu Spectrophotometer UV

- 1800 including UVProbe 2.33 software (Shimadzu, Kyoto,

Japan): Algal cell density.

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

USA) : Temperature.

6.8. I nterpretation

6.8.1. Data handling

Calibration curve

Quantification of cell densities was based on a calibration curve. Cell density was plotted versus

extinction using spectrophotometric measurements of a minimum of six dilutions of an algal

suspension with different cell densities. The calibration curve was composed using linear regression.

The software automatically calculates the cell densities based on this curve for the spectrophotometric

measurements at the various points in time during the test period.

Co mparison of average growth rates

The average specific growth rate for a specific period is calculated as the logarithmic increase in the

biomass from the equation for each single vessel of controls and treatments:

)day( lnln

1

−

−

− −

=

ij ij

ji tt XX

μ

Where: μ i-j = t he average specific growth rate from time i to j

X

i = the biomass at time i

X

j = the biomass at time j

The average growth rate at each test substance concentration is then compared with the control value

and the percentage reduction in growth rate is ca lculated :

100 % × −

=

C TC

r μ μμ I

Where: % I

r = percent inhibition in average specific growth rate

μ C = mean value for average specific growth rate in the control group

μ T = average specific growth rate for the treatment replicate

Yield

The percent inhibition in yield is calculated for each treatment replicate as follows:

100 % × −

=

C TC

y Y YY I

Where: % I

y = percent inhibition of yield

Y

C = mean value for yield in the control group1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 13

- Y

T = value for yield for the treatment replicate

Comparison of areas under the growth curves

The area below the growth curve is calculated using the formula:

b 1

- b 0 b 1 + b 2 - 2 * b 0 b n-1 + b

n - 2 * b 0 A = _____ * t 1 + _____________ * (t 2 -t 1 ) + ______________ * (t

n -t n-1 )

2 2 2

Where: A = area

b 0 = biomass (cell density) at the start of the test

b 1 = biomass (cell density) at t 1

b

n = biomass (cell density) at t

n

t 1 = time of first measurements after beginning of the test

t

n = time of n th measurement after beginning of the test

The percentage inhibition of cell growth at each test concentration (I T ) is calculated using the following

formula:

A C -A T

I T = _____ _ * 100

A C

Where: A C = area below the growth curve obtained in the control

A T = area below the growth c urve at each test substance concentration

Doubling time of controls

The doubling time (T d ) of cell density in controls at test termination is calculated using the following

formula:

c d

μ T )2ln(

=

Where: μ C = average specific growth rate in the control group

Determination of the NOEC and calculation of the IC 50 Statistical analysis of the data was not needed as the effects recorded were not significant (<10%).

No IC 50 - values could be calculated because the test substance proved to be non

- toxic ( IC 50 >

maximum concentration tested ).

6.8.2. Acceptability of the limit test

1. In the controls, cell density increased by an average factor of > 100 within 3 days.

2. The coefficient of variation of average specific growth rates during the whole test period in

replicate control cultures did not exceed 15%.

3. The coefficient of variation of average yield during the whole test period in replicate control

cultures did not exceed 15 %.

4. Test conditions and test set

- up were according to the guideline, with the exception of the pH at

the end of the test which slightly exceeded the upper limit of the optimum temperature range. This

however did not affect test validity, see section 6.9.1

.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 14

- 6.9. List of deviations

6.9.1. List of protocol deviations

1. First range

- finding test: The M2 medium used for the test contained 2.6 μg ZnCl 2 per litre, 2 μg

CoCl 2 * 6H 2 O per litre, 0.017 μg CuCl 2 * 2H 2 O per litre and 12.6 μg Na 2 MoO 4 * 2H 2 O per litre.

Evaluation: The deviations of the concentrations in the medium were only minimally and did not

affe ct the validity criteria of the study.

2. First range

- finding test: Microscopic observations were performed on the highest test

concentration to observe for any abnormal appearance of the algae at the end of the test period.

Evaluation: This is not required but does not affect the study integrity.

3. Second range

- finding test: Temperature varied by more than 2°C during the test period.

Evaluation: Temperature was kept within the optimum range of 22

- 26°C and algal growth was not

affected.

4. Final test: p H at the end of the test was lightly above the optimum range of 6.0

- 9.0 units.

Moreover, pH varied by 1.5

- 1.6 unit during the test period.

Evaluation: pH was similar for the control and the test concentration and algal growth was not

affected.

The study integrity was not adversely affected by the deviations.

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. First r ange

- finding test

7.1.1. Mean cell densities, reduction of growth rate and inhibition of yield

The mean cell densities measured during the first range

- finding test are presented in Table 1

. Table 2

presents the percentages growth rate reduction

, yield inhibition and cell growth inhibition per

concentration. Although the test proved to be valid a second range

- finding test was performed

because of the large variation in cell density between the replicate vessels of the test concentrations.

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

Table 1 Mean cell densities (x10 4 cells/ml) during the first range

- finding test

Nominal c onc

. Exposure time (hours)

Test substance 1

(mg/l) 0 24 48 72 96

control 1.0 5.4 26.9 111.1 270.9

0.10 1.0 4.8 26.2 113.9 269.0

1.0 1.0 5.0 18.7 70.5 186.6

10 1.0 5.1 22.6 90.3 236.3

100 1.0 5.1 20.6 71.6 1 88.5

1000 1.0 4.1 16.2 68.9 187.8

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 500678

- Page 15

- Table 2 Percentage reduction of growth rate

, inhibition of yield and inhibition of cell

growth duri ng the first range

- finding test

Nominal c onc

. Mean g rowth rate Yield (0

- 96 h) Cell growth (0

- 96 h)

Test substance 1

(mg/l) μ (0

- 96 h) Reduction (%) x10 4 cells/ml Inhibition (%) Area (A) Inhibition (%)

control 0.05835 269.89 6608.05

0.1 0. 05828 0.1 268.05 0.7 6623.37

- 0.2

1.0 0.05400 7.5 185.65 31.2 4415.94 33.2

10 0.05681 2.6 235.29 12.8 5583.48 15.5

100 0.05427 7.0 187.49 30.5 4512.95 31.7

1000 0.05401 7.4 186.78 30.8 4310.42 34.8

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

7.2. Second r ange

- finding test

7.2.1. Mean cell densities, reduction of growth rate and inhibition of yield

The mean cell densities measured during the second range

- finding test are presented in Table 3

. Table 4 presents the percentages growth rate reduction

, yield inhibition and cell growth inhibition per

concentration. T he expected IC 50 for growth rate reduction , yield inhibition and cell growth inhibition

was above a nomin al concentration of 1000 mg/l, containing 386 mg a.i. per litre.

pH was maintained within the limits prescribed by the protocol. Temperature varied by more than 2°C

during the test period, see section 6.9.1.

Table 3 Mean cell densities (x10 4 cells/ml) during the second range

- finding test

Nominal c onc

. Exposure time (hours)

Test substance 1

( mg/l ) 0 24 48 72 96

control 1.0 8.7 38.0 215.0 457.3

0.10 1.0 8.1 38.1 216.9 453.8

1.0 1.0 8.1 36.2 206.3 458.9

10 1.0 8.1 39.4 203.5 456.9

100 1.0 7.7 36.6 211.6 450.3

1000 1.0 6.4 26.3 184.8 453.1

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

Table 4 Percentage reduction of growth rate

, inhibition of yie ld and inhibition of cell

growth during the second range

- finding test

Nominal c onc

. Mean growth rate Yield (0

- 96 h) Cell growth (0

- 96 h)

Test substance 1

( mg/l ) μ (0

- 96 h) Reduction (%) x10 4 cells/ml Inhibition (%) Area (A) Inhibition (%)

con trol 0.06380 456.30 11685.45

0.10 0.06371 0.1 452.82 0.8 11677.31 0.1

1.0 0.06383

- 0.1 457.90

- 0.4 11437.54 2.1

10 0.06379 0.0 455.88 0.1 11421.45 2.3

100 0.06364 0.2 449.32 1.5 11460.02 1.9

1000 0.06370 0.1 452.07 0.9 10573.18 9.5

1 Test substanc e 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 500678

- Page 16

- 7.3. Limit test

7.3.1. Measured test substance concentrations

The results of analysis of the samples taken during the limit tes t are described in Tabl e 2 of the

appende d Analytical Repor t.

Anal ysis of the sample taken at the start of the test showed that the actual test concentration was in

agreement with nominal (101%). 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.

7.3.2. Mean cell densities

Table 5 shows mean cell densities measured at 24

- hour intervals at the different concentrations of 1-

Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1). The respective growth

curves are shown in Figure 1 (see APPENDIX 1 for the cell densities per replicate).

Table 5 Mean cell densities (x 1 0 4 cells/ml) during the limit test

Concentration Exposure time (hours)

Test substance 1

(mg/l) 0 24 48 72 96

control 1.0 5.2 30.4 112.3 220.5

1000 1.0 4.5 25.0 106.8 207.4

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yl oxy)

- , sodium salt (1:1)

Figure 1 Growth curves at different concentrations of 1- Propanesulfonic acid, 2

- hydroxy

3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

7.3.3. Reduction of growth rate and inhibition of yield

Table 6 shows the calculation of the percentages of growth rate reduction

, yield inhibition and cell

growth inhibition (see APPENDIX 1 for the values of growth rate

, yield and cell growth per replicate).

No biologically signif icant differences were recorded between the values for growth rate

, yield or cell

growth at the test concentration when compared to the control group

.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 17

- Microscopic observations at the end of the test revealed a normal and healthy appearance of the

exposed cells when compared to the control.

Table 6 Percentage reduction of growth rate

, inhibition of yield and inhibition of cell

growth during the limit test

Concentration Mean growth rate Yield (0

- 96 h) Cell growth (0

- 96 h)

Test substance 1

(mg/l) μ (0

- 96 h) Reduction (%) x10 4 cells/ml Inhibition (%) Area (A) Inhibition (%)

control 0.05620 219.53 6110.45

1000 0.05557 1.1 206.43 6.0 5678.08 7.1

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

7.3.4. Determination of effect concentrations

Table 7 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 7 Effect parameters

Parameter Concentration

Test substance 1

(mg/l) Concentration

Active ingredient 2

(mg/l)

96 h

- NOE R C 1000 386

96 h

- I R C 50 >1000 >386

96h

- NOE Y C 1000 386

96 h-I Y C 50 >1000 >386

96h

- NOE B C 1 000 386

96 h

- I B C 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

7.3.5. Experimental conditions

Table 8 sh ows the pH recorded at the beginning and the end of the test. The pH was within the limits

prescribed by the protocol (6.0

- 9.0) at the start of the test

. However, the pH of both the control and the

test concentration slightly exceeded the upper limit at the end of the test and therefore var ied by 1.5

-

1.6 unit , see also section 6.9.1

. The temperature of the test medium was 23.5 °C at the start of the test.

During the exposure period the temperature measured in the incubator was maintained between 24.2

and 24. 9 °C. Temperature remained within the limits prescribed by the protocol (2 2-26 °C , constant

within 2 °C ).

Table 8 pH levels recorded during the limit test

Concentration Exposure time (hours)

Test substance 1

(mg/l) 0 96

control 8.0 9.6

1000 8.0 9.5

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 500678

- Page 18

- 8. CONCLUSION

Under the conditions of the present study with Pseudokirchneriella subcapitata , no reduction of growth

rate

, inhibition of yield or inhibition of cell growth was recorded at an analytically confirmed nominal

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

The IC 50 for growth rate reduction ( 96 h-I R C 50 )

, yield inhibition ( 96 h-I Y C 50 ) and cell growth inhibition

(96h -I B C 50 ) was > 1000 mg test substance /l , corresponding to >386 mg a.i./l

.

The 96 h- NOEC for growth rate reduction

, yield inhibition and cell growth inhibition was 1000 mg/l.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 19

- APPENDIX 1 WORKSH EET DATA

Table 9 Individual cell densities (x10 4 cells/ml)

Concentration Vessel Exposure time (hours)

Test substance 1 number

(mg/l) 0 24 48 72 96

control 1 1.00 4.88 34.60 115.65 220.73

2 1.00 5.58 27.80 96.25 206. 67

3 1.00 5.28 26.76 111.56 221.14

4 1.00 5.19 31.76 109.43 218.59

5 1.00 4.96 31.47 125.22 239.89

6 1.00 5.09 29.71 115.84 216.15

1000 1 1.00 4.26 20.15 101.78 202.05

2 1.00 4.59 32.42 116.19 211.38

3 1.00 5.65 24.15 98.70 203.52

4 1.00 4.4 2 26.86 102.78 200.59

5 1.00 4.00 20.78 111.75 220.40

6 1.00 4.28 25.63 109.85 206.66

1 Test substance is 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

Table 10 Calculation of growth rate

, yield an d cell growth

Concentration Vessel Growth rate Yield Area (A) Growth rate red. Yield inhib. Growth inhib.

Test substance 1 number (μ) (x10 4 cells/ml) (%) (%) (%)

(mg/l) 0

- 96 h 0

- 96 h 0

- 96 h 0

- 96 h 0

- 96 h 0

- 96 h

control 1 0.05622 219.73 6287.60

2 0.05553 205.67 5507.32

3 0.05624 220.14 6016.19

4 0.05612 217.59 6052.48

5 0.05709 238.89 6674.04

6 0.05600 215.15 6125.06

mean 0.05620 220 6110

CV 1% 5% 6%

1000 1 0.05530 201.05 5369.06 2 8 12

2 0.05577 210.38 6129.22 1 4 0

3 0.05537 202.52 5442.22 1 8 11

4 0.05522 199.59 5540.27 2 9 9

5 0.05620 219.40 5837.58 0 0 4

6 0.05553 205.66 5750.12 1 6 6

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 500678

- Page 20

- APPENDIX 2 REFERENCE TEST

Pseudokirchneriella subcapitata , strain: NIVA CHL

- 1. Fresh water algal growth inhibition test with

potassium dichromate (Project 502369).

Start of first exposure: 05 March 2013

Completion last exposure: 08 March 2013

T he study procedures described in this report were based on the OECD guideline No. 201, Adopted

March 23, 2006 and ISO Standard 8692, Second edition, 01 October 2004.

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

Europe to Potassium dichromate (Merck, Art. 1.04864, Batch K34869764 607).

Algae were exposed for a period of 72 hours to K 2 Cr 2 O 7 (Potassium dichromate) concentrations of

0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a control. The initial cell density was 1.0 x 10 4 cells/ml.

Results:

Overview of % reduction of growth rate and % inhibition of yield in the reference test:

K 2 Cr 2 O 7

Nominal concentration

(mg/l) Mean growth rate Yield (0

- 72 h)

μ (0

- 72 h) Reduction

(%) x10 4 cells/ml Inhibition (%)

control 0.06848 137.53

0.18 0.06215 9.2 87.62 36.3

0.32 0.05582 18.5 56.23 59.1

0.56 0.04090 40.3 18.05 86.9

1.0 0.02982 56.5 7.58 94.5

1.8 0.02352 65.6 4.44 96.8

3.2 0.01787 73.9 2.62 98.1

Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations

of 0.18 mg/l and higher.

The EC 50 for growth rate reduction (E R C 50 : 0

- 72h) was 0.97 mg/l with a 95% confidence interval

ranging from 0.62 to 1.5 mg/l. The historical ranges for growth rate reduction lie between 0.82 and

2.3 mg/l. Hence, the E R C 50 : 0

- 72h for the algal culture tested corresponds with this range.

The EC 50 for yield inhibition (E Y C 50 : 0

- 72h) was 0.20 mg/l with a 95% confidence interval ranging from

0.05 to 0.72 mg/l. The historical ranges of the 72h EC 50 for yield inhibition lie between 0.43 and

1.1 mg/l. Hence, the E Y C 50 : 0

- 72h for the algal culture tested was lower than this range. However, this

does not invalidates the results as EC 50 for yield is not a validity criterion for this test.

The protocol, raw data and report of 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 500678

- Page 21

- APPENDIX 3 CERTIFICATE OF ANALYSIS1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 22

- APPE NDIX 4 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 500678

- Page 23

- 1. CONTENTS

1. CONTENTS

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

2. REPORT APPROVAL

.................................................................................................................... 24

3. INTRODUCTION

........................................................................................................................... 25

3.1. Preface

................................................................................................ ................................... 25

3.2. Aim of the study

..................................................................................................................... 25

4. MATERIALS AND METHOD S

....................................................................................................... 25

4.1. Reagents

................................................................................................ ................................ 25

4.2. Samples

................................................................................................ ................................ . 25

4.3. Analytical method

................................................................................................................... 26

4.3.1. Analytical conditions

.................................................................................................... 26

4.3.2. Preparation of solutions

............................................................................................... 26

4.3.3. Sample injections

......................................................................................................... 26

4.4. Electronic data capture

.......................................................................................................... 26

4.5. Formulas

................................................................................................ ................................ 27

5. RESULTS

................................................................................................ ...................................... 28

5.1. Calibration curves

.................................................................................................................. 28

5.2. Samples

................................................................................................ ................................ . 28

5.2.1. Procedural recovery samples

...................................................................................... 28

5.2.2. Test samples

................................................................................................................ 28

TABLES

Table 1 Procedural recovery samples

........................................................................................... 29

Table 2 Concentra tions of the test substance in test medium

....................................................... 291- propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500678

- Page 25

- 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

system (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 )

M2

- 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 500678

- Page 26

- 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 M2

- 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 500678

- Page 27

- 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 500678

- Page 28

- 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 500678

- Page 29

- 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

103 106 103

21

- May

- 2013 21

- May

- 2013 1000 1032 1055 102 103

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 1013 101

1000 1015 2 102

24 14

- May

- 2013 21

- May

- 2013 0 n.d. n.a.

1000 1009 101 100

1000 1068 2 107 105

96 17

- May

- 2013 21

- May

- 2013 0 n.d. n.a.

1000 1029 103 102

1000 915 2 92 90

1 Samples were stored in the freezer ( ≤

- 15°C ) until the day of analysis.

2 Without algae.

n.d. Not detected.

n.a. Not applicable.