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 Netherlands

 

Laboratory Project Identification

Project 503336

Substance 205003/A

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Castor Oil, Sulfated, Sodium Salt, 75% Project 503336

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1. CONTENTS

1. CONTENTS ................................................................................................................................ 2

2. STATEMENT OF GLP COMPLIANCE......................................................................................... 4

3. QUALITY ASSURANCE STATEMENT ........................................................................................ 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 ............................................................................................................................ 8

6. MATERIALS AND METHODS ..................................................................................................... 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 ................................................................................................. 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 concentrations ..................................................................................................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/range-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 ..........................................................................................................................16

Final Report
Castor Oil, Sulfated, Sodium Salt, 75% Project 503336

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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

APPENDICES 

APPENDIX 1 EC-VALUES.............................................................................................................17

APPENDIX 2 REFERENCE TEST ................................................................................................. 19

APPENDIX 3 CERTIFICATE OF ANALYSIS..................................................................................20

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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 440/2008, Part C.2, 2008 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 test 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 magnetically stirred 

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 group (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 nonspecific analytical confirmation of actual exposure concentrations were taken at the start and at the 

end of the test. 

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

Total 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. 54-78% 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-EC50 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). 

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Castor Oil, Sulfated, Sodium Salt, 75% Project 503336

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5. INTRODUCTION

5.1. Preface

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 December 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 EC50 at 24 and 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 guidelines: 

• OECD guidelines for Testing of Chemicals, guideline No. 202: "Daphnia sp., Acute Immobilisation

Test", Adopted April 13, 2004. 

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

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

• ISO International Standard 6341: "Water quality - Determination of the inhibition of the mobility of 

Daphnia 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 

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.

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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 EC50 is the concentration of test substance estimated to immobilise 50% of the daphnids after a 

defined period of exposure.

6. MATERIALS AND METHODS 

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 conditions 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)

Hygroscopic 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, 2nd to 5th 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.

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

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

6.3. Breeding

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

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

culture vessel.

Maximum age of the cultures 4 weeks

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

Temperature of medium 18-22°C

Feeding Daily, a suspension of fresh water algae.

Medium M7, as prescribed by Dr. Elendt-Schneider

(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An 

ultrastructural approach to antennal damage in Daphnia 

magna Straus. Protoplasma 154, 25-33).

Composition of medium M7: 

Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (ROwater, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):

Macro salts: CaCl2.2H2O 211.5 mg/l

 MgSO4.7H2O 88.8 mg/l 

 NaHCO3 46.7 mg/l 

 KCl 4.2 mg/l

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

concentrations: 

Trace elements: B 0.125 mg/l 

 Fe 0.05 mg/l

 Mn 0.025 mg/l

 Li, Rb and Sr 0.0125 mg/l

 Mo 0.0063 mg/l

Br 0.0025 mg/l

 Cu 0.0016 mg/l

 Zn 0.0063 mg/l

 Co and I 0.0025 mg/l 

Se 0.0010 mg/l

 V 0.0003 mg/l

 Na2EDTA.2H2O 2.5 mg/l 

 Macro nutrients: Na2SiO3. 9H2O 10.0 mg/l 

NaNO3 0.27 mg/l

 KH2PO4 0.14 mg/l

 K2HPO4 0.18 mg/l

 Vitamins: Thiamine 75.0 μg/l 

 B12 1.0 μg/l

 Biotin 0.75 μg/l

The hardness: 180 mg/l expressed as CaCO3 and the pH: 7.7 ± 0.3.

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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 liquid 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 magnetically stirred 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/range-finding test

The study started with a combined limit/range-finding 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 0.14, 1.4 and 14 mg/l; 

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

test concentration. 

6.6. Final 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 additives 

6.6.2. Test procedure and conditions

Test duration 48 hours

Test type Static

Test vessels 100 ml, all-glass 

Medium Adjusted ISO medium 

Number of daphnids 20 per concentration

Loading 5 per vessel containing 80 ml of test solution

Light 16 hours photoperiod daily

Feeding No feeding 

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Aeration No aeration of the test solutions.

Introduction of daphnids Within 45 minutes after preparation of the test solutions.

6.6.3. Sampling for analysis of test concentrations

Samples for Total Organic Carbon (TOC) analysis 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 analysis 

Apparatus TOC-VCPH 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 beginning 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;

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

USA): Temperature.

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6.8. Interpretation

6.8.1. Data handling

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-VCPH

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 concentrations

The average exposure concentrations were calculated as × CC tt == 480 , being the geometric means of 

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

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

Calculation of EC50

The 24 and 48h-EC50-value was calculated with the Weibull analysis from the probits of the 

percentages of affected daphnids and the logarithms of the corresponding average test substance 

concentrations using linear maximum likelihood 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 daphnids became immobilised.

2. The oxygen concentration 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 not affect the 

study outcome. 

The study integrity was not adversely affected by these 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. 

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7. RESULTS

7.1. Combined limit/range-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. The expected EC50 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 

concentration 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/range-finding test

Time (h) Replicate

Nominal concentration Castor Oil, Sulfated,

Sodium Salt, 75% (mg/l)

Control 0.14 1.4 14 141

0 A 5 5 5 5 5

B 5 5 5 5 5

C 5 5

D 5 5

Total introduced 20 10 10 10 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 70 100

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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 substance1

(mg/l)

Relative to 

nominal

(%)

Relative to 

initial

(%)

0 141 64.615 119 84

48 1.4

141

2.437

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. 54-78% 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. 

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 substance1

(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 Castor 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 EC50. 

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Table 4 Incidence of immobility 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

Concentration

Castor Oil, Sulfated, 

Sodium Salt, 75%

(mg/l)

Concentration

Active ingredient

(mg/l)

24h-EC50 19 (15 - 23) 14 (11 - 17)

48h-EC50 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 units; 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).

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Table 6 pH and oxygen concentrations during the final test

Nominal concentration

Castor Oil, Sulfated, 

Sodium Salt, 75%

(mg/l)

Start (t=0 h) End (t=48 h)

pH O2 pH O2

control 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

901 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-EC50 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). 

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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]

10 20 504030

% 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 EC50

Value (mg/l) 19.143

lower 95%-cl 14.827

upper 95%-cl 23.194

Final Report
Castor Oil, Sulfated, Sodium Salt, 75% Project 503336

- Page 18 - 

APPENDIX 1 EC-VALUES – 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: 2.0000

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]

10 3020

% 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 Oil, Sulfated, Sodium Salt, 75% at 48h

Table 10 Results of the probit analysis at 48h

Parameter EC50

Value [mg/L] 5.699

lower 95%-cl 4.713

upper 95%-cl 7.145

Final Report
Castor Oil, Sulfated, Sodium Salt, 75% 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 (K2Cr2O7) (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 K2Cr2O7 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 (K2Cr2O7, 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

K2Cr2O7

Number

Exposed

% immobile

24h 48h

Expected response (%)

After 48 hours 1

(mg/l) Minimal Maximal

control 20 0 0 0 102

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 K2Cr2O7 are within the ranges of the expected 

responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/l. Hence, the 

sensitivity of this batch of D. magna was in agreement with the historical data collected at WIL 

Research Europe.

The 24h-EC50 was estimated to be 0.98 mg/l. 

The 48h-EC50 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.

Final Report
Castor Oil, Sulfated, Sodium Salt, 75% Project 503336 

- Page 20 - 

APPENDIX 3 CERTIFICATE OF ANALYSIS