## FINAL REPORT

Study Title



## ACUTE TOXICITY STUDY IN **DAPHNIA MAGNA** WITH

CASTOR OIL, SULFATED , SODIUM SALT, 75%

(STATIC)

Author

L.M. Bouwman, MSc.

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlan ds

Laboratory Project Identification

Project 503336

Substance 205003/A

- Page 1 of 20

-Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- 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. Definit ions

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

6. MATERIALS AND METHOD S

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

6.1. Test Substance

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

6.1.1. T est substance information

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

6.1.2. Study specific test substance information

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

6.1.3. Reference substance

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

6.2. Test system

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

6.3. Breeding

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

6.4. Preparation of test solutions

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

6.5. Combined limit/range

- finding test

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

6.6. Final test

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

6.6.1. Test co ncentrations

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

6.6.2. Test procedure and conditions

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

6.6.3. Sampling for analysis of test concentrations

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

6.6.4. TOC analysis

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

6.6.5. Measurements and recordings

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

6.7. Electronic data capture

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

6.8. Interpretation

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

6.8.1. Data handling

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

6.8.2

. Acceptability of the test

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

6.9. List of deviations

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

6.9.1. List of protocol deviations

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

6.9.2. List of standard operating procedures deviations

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

7. RESULTS

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

7.1. Combined limit/ran ge

- finding test

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

7.2. Final test

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

7.2.1. Measured concentrations

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

7.2.2. Immobility

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

7.2.3. Determination of effect concentrations

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

7.2.4. Experimental conditions

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

8. CONCLUSION

.......................................................................................................................... 16Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 3 - TABLES

Table 1 Incidence of immobility in the combined limit/range

- finding test

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

Table 2 Measured TOC contents during the combined limit/range

- finding test

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

Table 3 Measured TOC contents during the final test

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

Table 4 Incidence of immobility in the final test

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

Table 5 Effect parameters

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

Table 6 pH and oxygen concentrations during the final test

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

Table 7 Parameters of the probit analysis at 24h

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

Table 8 Results of the probit analysis at 24h

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

Table 9 Parameters of the probit analysis at 48h

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

Table 10 Results of the probit analysis at 48h

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

FIGURES

Figure 1 Percentage response (=immobility) of Daphnia magna as function of the log

concentration of Castor Oil, Sulfated, Sodium Salt, 75% at 24h

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

Figure 2 Percentage response (=immobility) of Daphnia magna as function of the log

concentration of Castor Oil, Sulfated, Sodium Salt, 75% at 48h

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

A PPENDICES

APPENDIX 1 EC

- VALUES

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

APPENDIX 2 REFERENCE TEST

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

APPE NDIX 3 CERTIFICATE OF ANALYSIS

.................................................................................. 20Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 6 - 4. SUMMARY

Acute Toxicity Study in Daphnia magna with Castor Oil, Sulfated, Sodium Salt, 75%

.

The study procedures described in this report were based on the OPPTS 850.1010

, 1996

. In addition,

the procedures were designed to meet the test methods of the OECD guideline No. 202

, 2004, the

Commission Regulation (EC) No 440/200 8

, Part C.2, 200 8 and the ISO International Standard 6341,

1996

.

The batch of Castor Oil, Sulfated, Sodium Salt, 75% tested was a clear amber liquid and completely

soluble or completely dispersible in t est medium at the concentrations tested.

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

- finding test.

Preparation of test solutions for the final test started with a nominal concentration of 141 mg/l

containing 105 mg of the active ingredient (a.i.) per litre. This concentration was magnetic ally stirr ed

for 15 minutes to accelerate the dissolving of the test substance in the test medium. The lower test

concentrations were prepared by subsequent dilutions of the 141 mg/l concentration in test medium.

The final test solutions were clear and colourless up to nominal concentrations of 45 mg test

substance per litre

. Higher concentrations became increasingly hazy and formed homogeneous

dispersions

.

Twenty daphnids per grou p ( four replicates

, five daphnids per replicate) were exposed to a control and

to nominal test substance concentrations of 1.4, 2.8, 5.6, 11, 23, 45 and 90 mg / l, containing 1.0, 2.1,

4.2, 8.2, 17, 34 and 67 mg a.i. per litre . The total exposure period was 48 hours and samples for non

specific analytical confirmation of actual exposure concentrations were taken at the start and at the

end of the test.

N on

- specific analysis was used for the analytical support of the aquatic ecotoxicological studies, i.e.

To tal Organic Carbon (TOC) analysis was performed. Subsequently, the concentration of test

substance in the aquatic samples was calculated using the TOC content of pure test substance (i.e.

54.48 %). The initial concentrations in samples taken from nominal 5.6, 11, 23 and 45 mg/l were

calculated to be 3.0, 5.9, 15 and 35 mg/l, i.e. 5 4-78 % of nominal, respectively. The three highest test

concentrations remained stable during the exposure period (80

- 91% of initial), while the lowest test

concentration decreased to 60% of initial. Given these results, effect parameters were based on the

average exposure concentrations: 2.3, 5.3, 13 and 33 mg test substance per litre, containing 1.7, 4.0,

9.7 and 25 mg a.i. per litre.

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

The 48h

- EC 50 was 5.7 mg test substance per litre (95% confidence interval between 4.7 and 7.1 mg/l)

, containing 4.3 mg a.i. per litre (95% confidence interval between 3.5 and 5.3 mg/l ).Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 7 - 5. INTRODUCTION

5.1. Pr eface

Sponsor Sulfated Castor Oil Consortium, Society of Chemical

Manufacturers & Affiliates

1850 M Street NW, Suite 700

Washington, DC 20036

USA

Study Monitor

(on behalf of the Sponsor) Rodney Boatman, Ph.D.

PCA Services

2704 Trail Wood Drive

Durham, NC 27705

USA

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD 's

- Hertogenbosch

The Netherlands

Study Director L.M. Bouwman, MSc.

Study Plan Start : 25 November 2013

Completion : 19 December 2013

TOC analysis : 04 and 23 Dece mber 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 an d 48 hours of exposure.

5.3. Guidelines

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

Guidelines, OPPTS 850.1010, Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids. ‘Public

Draft’, EPA 712 -C- 96

- 114, April 1996.

In addition, the procedures were designed to meet the test methods of the following guide lines :

• OECD guidelines for Testing of Chemicals, guideline No. 20 2 : " Daphnia sp.

, Acute Immobilisation

Test", Adopted April 13

, 2004

.

• Commission Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the d etermination

of ecotoxicity, Publication No. L142, C.2. "Daphnia Sp. Acute Immobilisation T est".

• ISO International Standard 6341: "Water quality

- Determination of the inhibition of the mobility of

Dap hnia magna Straus

- Acute toxicity test, Third edition, 1996

- 04

- 01.

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

Res earch 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 afte r

finalization of the report.Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 8 - 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.

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 Castor Oil, Sulfated, Sodium Salt, 75%

CAS Number 68187

- 76

- 8

Description Clear amber liquid

Batch R

- 10135

- 064

Purity/Composition 74.4%

Test substance storage At room temperature in the dark

Stable under storage c onditions until 18 July 2014 (expiry date)

See APPENDIX 3 : Certificate of analysis.

6.1.2. Study specific test substance information

Purity/composition correction factor

required Yes, correction factor is 1.34 (74.4% purity)

Hygros copic No

Volatile No

Specific Gravity / Density 1.049

Stability in water Yes, >24 hrs., 5%

Solubility in water Complete

6.1.3. Reference substance

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

( APPENDIX 2 )

.

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.

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.Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 9 - Characteristics For the test selection of young daphni ds with an age of < 24

hours, from parental daph nids of more than two weeks old.

6.3. Breeding

Start of each batc h 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

Renewal of the cultures After 7 days of cultivation half of the medium twice a we ek.

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 Osmosis (RO

water

, GEON Waterbehandeling

, Berkel

- Enschot

, The Netherlands ):

Macro s alts: 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

concentrat ions:

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.Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 10

- 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). A correction was made for the purity/composition of the test

substance. A correction factor of 1.41 was used. This factor was based on the information known

during testing. Afterwards, the information on purity/composition of the test substance was changed

, see also section 6.9.1

. As a consequence slightly higher concentrations than necessary were tested.

The batch of Castor Oil, Sulfated, Sodium Salt, 75% tested was a clear amber liqu id and completely

soluble or completely dispersible in test medium at the concentrations tested.

Preparation of test solutions started with a nominal concentration of 141 mg/l containing 105 mg of the

active ingredient (a.i.) per litre. This concentration was magnetic ally stirr ed for 15 minutes to

accelerate the dissolving of the test substance in the test medium. The lower test concentrations were

prepared by subsequent dilutions of the 141 mg/l concentration in test medium. The final test solutions

were clear and colourless up to nominal concentrations of 45 mg test substance per litre

. Higher

concentrations became increasingly hazy and formed homogeneous dispersions.

6.5. Combined limit/r ange

- finding test

The study started with a combined limit/range

- findi ng test. Twenty daphnids per concentration (four

replicates, 5 daphnids per vessel) were exposed to a control and a nominal test substance

concentration of 141 mg/l. Test procedure and conditions were similar to those applied in the final test

with the following exceptions:

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

concentrations of 0.1 4 , 1. 4 and 1 4 mg/l ;

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

test concentration

.

6.6. Fi nal test

6.6.1. Test concentrations

Castor Oil, Sulfated, 1.4, 2.8, 5.6, 11, 23, 45 and 90 mg test substance per litre,

Sodium Salt, 75% containing 1.0, 2.1, 4.2, 8.2, 17, 34 and 67 mg a.i. per litre

Control Test medium without test substance or other additive s

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 feedingCastor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 11

- Aeration No aeration of the test solutions.

Introduction of daphni ds Within 45 minutes after preparation of the test solutions.

6.6.3. Sampling for analysis of test concentrations

S amples for Total Organic Carbon (TOC) analys is were taken from all test concentrations and the

control according to the schedule below.

Frequency at t=0 h and t= 48 h

Volume 40 ml

Storage Samples were stored in a refrigerator until analysis.

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

.

Additionally, reserve samples of 40 ml were taken for possible analysis. If not used, these samples

were stored in a refrigerator 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. TOC analys is

Apparatus TOC -V CPH total organic carbon analyser combined with a

Shimadzu ASI

- V autosampler.

Calibration solution TC standard: Potassium hydrogen phtalate solution

Parameters TOC = NPOC (Non

- Purgeable Organic Carbon)

Analysis set

- up No. of repeats: at least 3

6.6.5. Measurements and recordings

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

pH At the begi nning and at the end of the test, for all

concentrations and the control.

Dissolved oxygen At the beginning and at the end of the test, for all

concentrations and the control. In addition after 24 hours,

immediately after counting the immobilized daphnids oxygen

levels were measured in the highest test concentration

.

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

the start of the test.

6.7. Electronic data capture

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

programme(s):

- Shimadzu TOC

- Control V version 2.00 (Shimadzu, Kyoto, Japan): TOC analysis;

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

USA) : Temperature.Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 12

- 6.8. Interpretation

6.8.1. Data han dling

Test substance concentrations

The amount of test substance present in test medium was calculated from the measured amount of

TOC in the test medium and the TOC content of the pure test substance.

A sample of the pure test substance was taken for determination of the Total Organic Carbon (TOC)

content ( WIL Research Project 503334 ). TOC analysis was performed using a Shimadzu TOC -V CPH total organic carbon analyzer combined with a Shimadzu SSM

- 5000A (Solid Sample module for Total

Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan). Calibration standards were Glucose as total

carbon (TC) standard and Sodium carbonate as inorganic carbon (IC) standard. The TOC content of

the test substance was determined to be 54.48 %.

Determination of the average exposure c oncentrations

The average exposure concentrations were calculated as 480

== × tt CC , being the geometric means of

the concentrations of Castor Oil, Sulfated, Sodium Salt, 75% measured in the samples taken at the

start (C t=0 ) and the end of the test (C t=48 ).

Calculation of EC 50

The 24 and 48h

- EC 50 - value was calculated with the Weibull analysis from the probits of the

percentages of affected daphni ds and the logarithms of the corresponding average test substance

concentrations using linear maximum li kelihood regression

.

ToxRat Professional v 2.10.05 (ToxRat Solutions ® GmbH, Germany) was used to perform the

calculations

.

6.8.2. Acceptability of the test

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

2. The oxygen concentrati on at the end of the test was ≥ 3 mg/l in control and test vessels.

6.9. List of deviations

6.9.1. List of protocol deviations

1. A correction factor of 1.41 was used for the purity/composition of the test substance.

Evaluation: Slightly higher concentrations than necessary were tested, but this did n ot affect the

study outcome.

The study integrity was not adversely affected by the se 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.Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 13

- 7. RESULTS

7.1. Combined limit/r ange

- finding test

Table 1 shows the responses recorded during the combined limit/ range

- finding test. After 24 hours of

exposure all daphnids exposed to the highest test concentration were immobilised. The presence of

undissolved test substance was not the cause of the immobility, this was checked under a microscope.

After 48 hours of exposure 70% of the daphnids exposed to nominal 14 mg test substance per litre

were immobilised. Again, the presence of undissolved test substance was not the cause of the

immobility. No immobility was observed at any of the lower test concentrations during the test period.

Note that a maximum of 10% immobilisation is acceptable for the control. T he expected EC 50 was

between nominal test substance concentrations of 1.4 and 14 mg/l.

Samples taken from nominal 141 mg test substance per litre were analysed for TOC. The

concentration of test substance in the aquatic samples was calculated using the TOC content of pure

test substance ( fourth column of Table 2 ) . The initial concentration was 119 mg/l , i.e. 84% of nominal

and this concentration remained stable during the test period ( 82 % of initial at the end of the test). In

addition the sample taken at the end of the test from nominal 1.4 mg/l was analysed and the measured

concentration was calculated to be 4.5 mg/l, which was above nominal

. This relatively high

concent ration can be related to the fact that measurements could not be corrected for the control.

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

Table 1 Incidence of immobility in the combined limit/ r ange

- finding test

Time (h) Replicate Nominal concentration Castor Oil, Sulfated,

Sodium Salt, 75% (mg/l)

Control 0.1 4 1. 4 1 4 1 41

0 A 5 5 5 5 5

B 5 5 5 5 5

C 5 5

D 5 5

Total introduced 20 1 0 1 0 1 0 20

24 A 0 0 0 1 5

B 0 0 0 0 5

C 0 5

D 0 5

Total immobilised 0 0 0 1 20

Effect % 0 0 0 10 100

48 A 0 0 0 3 5

B 1 0 0 4 5

C 1 5

D 0 5

Total immobilised 2 0 0 7 20

Effect % 10 0 0 70 100Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 14

- Table 2 Measured TOC contents during the combined limit/range

- finding test

Time of

sampling

(hours) Nominal conc.

(mg/l) Measured

organic carbon

conc.

(mg/l) Calculated

conc.

Test substance 1

(mg/l) Relative to

nominal

(%) Relative to

initial

(%)

0 141 64.615 119 84

48 1.4

141 2.43 7

52.85 4.5

97 321

69 -

82

- Sample was not analysed at the start of the test

2 Test substance was Castor Oil, Sulfated, Sodium Salt, 75%

7.2. Final test

7.2.1. Measured concentrations

The results of TOC analysis of the samples taken during the final test are described in Table 3

. Based

on the TOC content of the pure test substance (i.e. 54.48 %) the concentration of test substance in the

aquatic samples was calculated ( fourth column of Table 3 ) . A correction was made for the measured

amount of TOC in the control. The initial concentrations in samples taken from nominal 5.6, 11, 23 and

45 mg/l were calculated to be 3. 0

, 5.9, 15 and 35 mg/l, i.e. 5 4-78 % of nominal, respectively. The t hree

highest test concentrations re mained stable during the exposure period ( 80-91 % of initial) , while the

lowest test concentration decreased to 60 % of initial . Given these results, effect parameters were

based on the average exposure concentrations: 2.3, 5.3, 13 and 33 mg test substance per litre,

containing 1.7, 4.0, 9.7 and 25 mg a.i. per litre.

Table 3 Measured TOC contents during the final test

Time of

sampling

(hours) Nominal conc.

(mg/l) Measured

organic carbon

conc.

(mg/l) Calculated

conc.

Test substance 1

(mg/l) Relative to

nominal

(%) Relative to

initial

(%)

0 0

5.6

11

23

45 0.2239

1.837

3.460

8.206

19.16 -

3.0

5.9

15

35 -

54

54

65

78

48 0

5.6

11

23

45 0.2050

1.191

2.750

6.821

17.71 -

1.8

4.7

12

32 -

32

43

52

71 -

60

80

80

91

1 Test substance was Cas tor Oil, Sulfated, Sodium Salt, 75%

7.2.2. Immobility

Table 4 shows the responses recorded during the final test. After 48 hours of exposure all daphnids

exposed to the three highest test concentrations and 45% of the dapnids exposed to nominal 11 mg/l

were immobilised. No immobility was observed at any of the lower concentrations during the test

period. The responses recorded in this test allowed for reliable determination of an EC 50

.Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 15

- Table 4 Incidence of imm obility in the final test

Time (h) Replicate Nominal concentration Castor Oil, Sulfated, Sodium Salt, 75% (mg/l)

Control 1.4 2.8 5.6 11 23 45 90

0 A 5 5 5 5 5 5 5 5

B 5 5 5 5 5 5 5 5

C 5 5 5 5 5 5 5 5

D 5 5 5 5 5 5 5 5

Total introduced 20 20 20 20 20 20 20 20

24 A 0 0 0 0 0 2 4 5

B 0 0 0 0 0 1 5 5

C 0 0 0 0 0 1 5 5

D 0 0 0 0 0 1 5 5

Total immobilised 0 0 0 0 0 5 19 20

Effect % 0 0 0 0 0 25 95 100

48 A 0 0 0 0 3 5 5 5

B 0 0 0 0 1 5 5 5

C 0 0 0 0 3 5 5 5

D 0 0 0 0 2 5 5 5

Total immobilised 0 0 0 0 9 20 20 20

Effect % 0 0 0 0 45 100 100 100

7.2.3. Determination of effect concentrations

Table 5 shows the effect parameters based on average exposure concentrations, which were

calculated from non

- specific TOC

- analysis , see also APPENDIX 1

.

Table 5 Effect parameters

Parameter C oncentration

Castor Oil, Sulfated,

Sodium Salt, 75%

(mg/l) C oncentration

Active ingredient

(mg/l)

24h

- EC 50 1 9 (1 5

- 2 3 ) 1 4 (1 1

- 1 7 )

48h

- EC 50 5. 7 ( 4.7

- 7.1 ) 4. 3 ( 3.5

- 5.3 )

( ) 95% confidence interval

7.2.4. Experimental conditions

The results of measurement of pH and oxygen concentrations (mg/l) are presented in Table 6

. These

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

- 8.5, not varying by more

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

The temperature continuously measured in a temperature control vessel varied between 18.8 and

19.3 °C during the test, and complied with the requirements as laid down in the protocol (18

- 22°C,

constant within 2°C).Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 16

- Table 6 pH and oxygen concentrations during the final test

Nominal c oncentration

Castor Oil, Sulfated,

Sodium Salt, 75%

(mg/l) St art (t=0 h) End (t=48 h)

pH O 2 pH O 2

c ontrol 7.8 9.7 8.0 9.7

1.4 7.8 9.6 7.9 9.5

2.8 7.7 9.5 7.9 9.5

5.6 7.7 9.5 7.9 9.5

11 7.7 9.5 7.8 9.5

23 7.7 9.4 7.8 9.5

45 7.6 9.5 7.8 9.5

90 1 7.6 9.4 7.7 9.5

1 The oxygen concentration after 24 hours of exposure was 9.1 mg/l

8. CONCLUSION

The 48h

- EC 50 was 5.7 mg test substance per litre (95% confidence interval between 4.7 and 7.1 mg/l)

, containing 4.3 mg a.i. per litre (95% confidence interval between 3.5 and 5.3 mg/l ).Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 17

- APPENDIX 1 EC

- VALUES

Table 7 Parameters of the probit analysis at 24h

Parameter Value

Computation runs: 7.0000

Slope b: 6.3920

Intercept a:

- 8.56111

Variance of b: 1.4864

Goodness of Fit

Chi2: 0.6423

Degrees of freedom: 2.0000

p(Chi2): 0.7253

Log EC50: 1.2820

SE Log EC50: 0.0429

g

- Criterion: 0.1398

F: 85.5900

p(F) (df: 1;2): 0.0110

Data

Function

95%-CL

Concentration [mg/L] 504030 20 10% Immobility 100

90

80

70

60

50

40

30

20

10

0

Figure 1 Percentage response (=immobility) of Daphnia magna as function of the log

concentration of Castor Oil, Sulfated, Sodium Salt, 75% at 24h

Table 8 Results of the probit analysis at 24h

Parameter EC 50

Value ( mg/ l) 19.143

lower 95%

- cl 14.827

upper 95%

- cl 23.194Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 18

- APPENDIX 1 EC

- VAL UES

– continued

–

Table 9 Parameters of the probit analysis at 48h

Parameter Value

Computation runs: 5.0000

Slope b: 8.2378

Intercept a:

- 6.59282

Variance of b: 4.1093

Goodness of Fit

Chi2: 0.6124

Degrees of freedom: 2.000 0

p(Chi2): 0.7362

Log EC50: 0.7558

SE Log EC50: 0.0433

g

- Criterion: 0.2326

F: 53.9330

p(F) (df: 1;2): 0.0180

Data

Function

95%-CL

Concentration [mg/L] 3020 10% Immobility 100

90

80

70

60

50

40

30

20

10

0

Figure 2 Percentage response (=immobility) of Daphnia magna as function of the log

concentration of Castor Oi l, Sulfated, Sodium Salt, 75% at 48h

Table 10 Results of the probit analysis at 48h

Parameter EC 50

Value [mg/L] 5.699

lower 95%

- cl 4.713

upper 95%

- cl 7.145Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 19

- APPENDIX 2 REFERENCE TEST

Start: 21 October 2013

End : 23 October 2013

48

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

Europe Project 504049).

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 daphnids 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:

Concentration

K 2 Cr 2 O 7 Number

Exposed % immobile

24h 48h Expected response (%)

After 48 hours 1

(mg/l) Minimal Maximal

control 20 0 0 0 10 2

0.10 20 0 0 0 10

0.18 20 0 0 0 10

0.32 20 0 0 0 30

0.56 21 0 48 0 100

1.0 20 60 100 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

responses at the different concentrations, i.e. the 48h

- EC 50 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 estimated to be 0.98 mg/l.

The 48h

- EC 50 was estimated to be 0.56 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.Castor Oil, Sulfated, Sodium Salt, 7 5% Project 503336

- Page 20

- APPENDIX 3 CERTIFICATE OF ANALYSIS