## FINAL REPORT

Study Title



## 96

- HOUR ACUTE TOXICITY STUDY IN FATHEAD MINNOW 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 503337

Substance 205003/A

- Page 1 of 20

-Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- 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

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

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

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

6.2. Test System

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

6.3. Holding

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

6.4. Preparation of test solutions

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

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

6.6.1. TOC analysis

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

6.6.2. Measurements and recordings

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

6.7. Electro nic data capture

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

6.8. Interpretation

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

6.8.1. Data handling

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

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

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

7.1. Combined l imit/range

- finding test

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

7.2. Final test

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

7.2.1. Measured test substance concentrations

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

7.2.2. Mortality and other effects

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

7.2.3. Determination of effect concentrations

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

7.2.4. Exp erimental conditions

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

8. CONCLUSION

.......................................................................................................................... 16Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 3 - TABLES

Table 1 Incidence of mortality and total mortality during the combined limit/range

- finding

test

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

Table 2 Measured TOC contents during the combined limit/range

- finding test

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

Tab le 3 Measured TOC contents during the final test

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

Table 4 Incidence of mortality and total mortality during the final test

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

Table 5 Effect parameters

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

Table 6 pH

- values and dissolved oxygen concentrations (mg/l) during the final test

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

Table 7 Tempe ratures (°C) measured during the final test

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

Table 8 Parameters of the probit analysis at 24h

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

Table 9 Results of the probit analysis at 24h

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

Table 10 Parameters of the probit analysis at 48, 72 and 96h

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

Table 11 Results of the probit analysis at 48, 72 and 96h

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

FIGURES

Figure 1 Percentage of mortality of fish plotted against the test substance concentration

after 24 hours of exposure

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

Figure 2 Percentage of mortality of fish plotted against the test substance concentration

after 48, 72 and 96 hours of exposure

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

APPENDICES

APPENDIX 1 LC

- VALUES

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

APPENDIX 2 REFERENCE TEST

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

APPENDIX 3 CERTIFICATE OF ANALYSIS

.................................................................................. 20Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 6 - 4. SUMMARY

96

- Hour Acute Toxicity Study in Carp with Castor Oil, Sulfated, Sodium Salt, 75%

.

The study procedure described in this report was based on the OPPTS 850.1075, 1996

. In addition,

the procedures were designed to meet the test methods of the OECD guideline No. 203, 1992, the

Commission Regulation (EC) No 440/200 8

, Part C.1, 200 8 and the ISO International Standard 73 46

- 1:

Static method, 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 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

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

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

were clear and colourless up to nominal concentrations of 31 mg test substance per litre. Higher

concentrations became increasingly hazy and formed homogeneous dispersions.

Fourteen fish per group (two replicates, seven fish per replicate) were exposed to nominal test

substance concentrations of 6.5

, 14

, 31

, 65 and 141 mg / l, containing 4.9

, 10

, 23

, 49 and

105 mg a.i. per litre. Simultaneously, seven fish per group (one replicate) were exposed to a control.

The total exposure period was 96 hours and samples for non

- specific analytical confirmation of actual

expos ure concentrations were taken at the start, after 48 hours of exposure and at the end of the test.

In addition, samples were taken from the two highest test concentrations after 24 hours of exposure.

N on

- 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 mean initial concentrations in samples taken from nominal 6.5, 14, 31, 65 and 141 mg/l

were calculated to be 3.1, 8.8, 25, 54 and 119 mg/l, respectively, i.e. 45

- 85% of nominal. Most

concentrations decreased during the test period. Effect parameters were based on the average

exposure concentrations for the four lowest test concentrations and on the initial concentration for the

highest test concentration: 2. 6 , 5.1, 20, 45 and 119 mg test substance per litre, containing 1.9, 3.8, 15,

34 and 89 mg a.i. per litre.

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

The 96h

- LC 50 was 7.3 mg test substance per litre (95% confidence interval between 5.2 and 12 mg/l)

, containing 5.4 mg a.i. per litre (95% confidence interval between 3.9 and 9.0 mg/l ). This LC 50 value

was already reached after 48 hours of exposure.Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 7 - 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 : 20 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 in Pimeph ales promelas during an exposure period of 96 hours and, if possible, to determine

the LC 50 at all observation times.

5.3. Guidelines

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

Guidelines, OPPTS 850.1075, Fish Acute Toxicity Test, Freshwater and marine. ‘Public Draft’,

EPA 712 -C- 96

- 118, April 1996.

In addition, the procedure was designed to meet the test methods prescribed by the following

guidelines:

• OECD guidelines for Testing of Chemicals, guideline No. 203: "Fish Acute Toxicity Test", Adopted

17 July, 1992.

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

of ecotoxicity, Publication No. L142 , C. 1 . " Acute Toxicity for Fish ".

• ISO International Standard 7346

- 1: “Water qu ality

– Determination of the acute lethal toxicity of

substances to a freshwater fish [ Brachydanio rerio Hamilton

- Buchanan (Teleostei, Cyprinidae)]”

– Part 1: Static method, Second edition, 1996

- 06

- 15

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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 8 - 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 no longer than 10

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

5.5. Definitions

Fish were considered to be dead when no reaction was observed after touching the caudal peduncle

and visible breathing movements were absent. In addition, fish that were convulsing or showing other

severe forms of distress not considered transient in nature and likely to become more severe before

the exposure is terminated, will be sacrificed for humane reasons. These fish will be treated as havin g died in the test, based on OECD guideline on humane endpoint (ENV/JM/MONO/(2000)7).

The LC 50 is the concentration killing 50% of the fish after a defined period of exposure.

6. MATERIALS AND METHOD S

6.1. Test Substance

6.1.1. Test substance information

Identificatio n 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 (expir y 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 Gra vity / 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 a reference test with pentachlorophenol ( PCP) ( APPENDIX 2 )

.

6.2. Test System

Species Fa thead minnow ( Pimephales promelas , Teleostei,

Cyprinidae) Rafinesque

Source In house cultureCastor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 9 - Mean length 1 Combined limit/range

- finding test: 2.7 ± 0. 3 cm

Final test: 2.9 ± 0.3 cm

Mean weight 1 Combined limit/range

- finding test: 0.60 ± 0.22 g

Final t est : 0.66 ± 0.22 g

Reason for selection This system has been selected as an internationally

accepted species.

Total fish used 104

6.3. Holding

Medium Adjusted ISO medium, formulated using RO- water (tap

water purified by reverse osmosis; GEON

Waterbehandeling

, Berkel

- Enschot

, The Netherlands ) with

the following composition:

CaCl 2 .2H 2 O 2 11.5 mg/l

MgSO 4 .7H 2 O 88.8 mg/l

NaHCO 3 46.7 mg/l

KCl 4.2 mg/l

Measurements Conductivit y, pH, nitrate, nitrite and ammonia concentration:

once a week. Temperature: continuous. In addition, pH and

temperature were measured before transferring the fish to

the test system.

Water quality parameters Were kept within the optimum limits for the re spective fish

species.

Feeding Daily with pelleted fish food (Cyprico Crumble Excellent

(300

- 500 um), Coppens International bv, Helmond, The

Netherlands )

Validity of batch In the batch of fish used for the test, mortality during the

seven days prior to the start of the test was less than 5%.

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/compo sition 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

. Th is 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 concentration in test medium. The final test solutions

were clear and colourless up to nominal concentrations of 31 mg test substance per litre. Higher

concentrations became increasingly hazy and formed homogeneous dispersions.

1 Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 10

- 6.5. Combined limit/r ange

- finding test

The study started with a combined limit/range

- finding test. Fourteen fish (Two replicates, seven fish

per replicate) were exposed to a nominal test substance concentration of 141 mg/l and seven fish (one

replicate) were exposed to a control. Test procedure and conditions were similar to those applied in

the final test with the following exceptions:

• Three fish per concentration (one replicate) were exposed to nominal concentrations of 1.4

and 14 mg/l;

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

the highest test concentration with surviving fish;

• Aeration was introduced after 48 hours of exposure, as the oxygen concentration had

d ropped slightly below the optimum level for testing with Fathead minnow, i.e. below 5 mg/l.

6.6. Final test

6.6.1. Test concentrations

Castor Oil, Sulfated, 6.5, 14, 31, 65 and 141 mg test substance per litre,

Sodium Salt, 75% containing 4.9, 10, 23, 49 and 105 mg a .i. per litre

Control Test medium without test substance or other additives

6.6.2. Test procedure and conditions

Test duration 96 hours

Test type Static

Test vessels 9.8 litres, all

- gl ass, containing 9 litres of test solution

Test medium Adjusted ISO medium with a hardness of 180 mg CaCO 3 per

litre and a pH of 7. 7 ± 0. 3.

Number of fish 7 for the control and 14 for each test concentration

Number of replicates 1 for the control;

2 for the test concentrations, containing 7 fish each

Loading 0.51 g fish/litre, i.e. 7 fish per 9 litres of test medium

Illumination 16 hours photoperiod daily

Aeration The test media were not aerated during the test.

Feeding No feeding from 24 hours prio r to the test a nd during the

total test period

Introduction of fish Within 35 minutes after preparation of the test media from a

holding tank with comparable water quality parameters and

pH and temperature differences between test and holding

tank media of less than 0.5 unit and 0.5 °C

.

Euthanasia At the end of the test the surviving fish were rapidly killed by

exposing them to ca. 1.2% ethylene glycol monophenylether

in water.Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 11

- 6.6.3. Sampling for analysis of test concentrations

Samples for Total Organic Carbon (TOC) analysis were taken from the control and all test

concentrations with surviving fish according to the schedule below. In addition, samples were taken

from the two highest test concentrations after 24 hours of exposure.

Frequency at t=0 h, t= 48 h an d t= 96 h

Volume 40 ml

Storage Samples were stored in a refrigerator until analysis.

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.

6.6.1. TOC analysis

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

6.6.2. Measurements and recordings

Mortality and other effects At 5 , 24, 48, 72 and 96 hours following the start of exposure.

In addition, every afternoon from day 0 and every morning

from day 1 to observe for any dead or severely distressed

fish. Dead fish were removed when observed.

Dissolved oxygen content, Daily in all vessels with surviving fish , beginning at the

pH and temperature start of the test (day 0).

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;

Temperature during sample storage was monitored continuously using the following programme:

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

USA)

.

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 -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%.Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 12

- Determination of the average exposure concentrations

The average exposure concentrations for nominal 6.5 and 14 mg/l were calculated as:

2 CC CC t 48 t 48 t0t 96

== == ×+×

, being the mathematical means of the concentrations of Castor Oil,

Sulfated, Sodium Salt, 75% measured in the samples taken at the start (C t=0 ), after 48 hours (C t=48 )

and the end of the test (C t=96 ).

The average exposure concentrations for no minal 31 and 65 mg/l were calculated as: 240

==

× 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 after 24 hours of exposure (C t=24 ).

Calculatio n of LC 50

The LC 50 - value s w ere calculated with the Weibull analysis from the probits of the percentages of

affected fish 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. No mortality was observed in the control at the end of the test.

2. Test conditions were maintained constant throughout the test.

3. The dissolved oxygen concentration has been at least 60% of the air saturation value throughout

the test (> 5 mg/l at 22°C).

4. Since the measured concentrations deviated by more than 20% from nominal, results were based

on the average exposure concentration.

6.9. List of dev iations

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.

2. Combined li mit/range

- finding test: The oxygen concentration in the control has been slightly below

5.0 mg/l, i.e. 4.75 mg/l, after 48 hours of exposure.

Evaluation: Aeration was introduced and the oxygen concentration was kept above 5.0 mg/l during

the remainder of the test. No fish died or were observed to be otherwise affected.

3. Combined limit/range

- finding test: At the end of the first day of exposure fish were not observed

for mortality and sub

- lethal effects.

Evaluation: This does not affect the outcome o f the study.

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.

7. RESULTS

7.1. Combined limit/r ange

- finding test

The results of the combined limit/ range

- finding test are presented in Table 1

. Within 4 hours of

exposure all fish exposed to the highest test concentration died. No mortality or clinical effects were

observed at any of the lower test concentrations and the control during the test period. From 48 hoursCastor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 13

- onwards the nominal 14 mg/l concentration was observed to be slightly hazy. T he expected LC 50 was

between nominal test substance concentrations of 14 and 14 1 mg/l.

Samples taken from nominal 14 and 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 concentrations were 13 and 1 18/119 mg/l,

respectively, i.e. 93 and 84% of nominal. Both concentrations remained stable during the first 24 hours

of exposure ( 90

- 100 % of initial) . The highest test concentration remained also stable during the

remainder of the test (85/87% of initial) while nominal 14 mg/l slightly decreased to 73% of initial at the

end of the test.

Temperatu re and pH were maintained within the limits prescribed by the protocol. The oxygen

concentration in the control slightly decreased below the optimum range after 48 hours of exposure.

Therefore, aeration was introduced and maintained until the end of the te st, see also section 6.9.1.

Table 1 Incidence of mortality and total mortality during the combined limit/ range

finding test

Nominal c onc

. Test substance 1

(mg/l) Vessel

number Initial

number

of fish Cumulative mortality Total

Mor tality

(%) 4h 24h 48h 72h 96h

Control A 7 0 0 0 0 0 0

1.4 A 3 0 0 0 0 0 0

14 A 3 0 0 0 0 0 0

1 41 A

B 7

7 7

7 7

7 7

7 7

7 7

7 100

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

Table 2 Measured TOC c ontents 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 14

141A

141B 7.179

64.24

64. 99 13

118

119 93

84

84

48 14

141A

141B 7.005

58.10

58.37 13

107

107 93

76

76 100

91

90

96 14

141A

141B 5.182

56.07

55.23 9.5

103

101 68

73

72 73

87

85

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

7.2. Final test

7.2.1. Measured test substance conce ntrations

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 (when measured). The mean initial concentrations in samples taken from

nominal 6.5

, 14

, 31

, 65 and 141 mg/l were calculated to be 3.1

, 8.8

, 25

, 54 and 1 19 mg/l, respectively,

i.e. 45 -85 % of nominal. Most concentrations decreased during the test period. Effect parameters were

based on the average exposure concentrations for the four lowest test concentrations and on the i nitial

concentration for the highest test concentration : 2. 6

, 5.1

, 20

, 45 and 1 19 mg test substance per litre,

containing 1.9

, 3.8

, 15

, 34 and 89 mg a.i. per litre.Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 14

- Table 3 Measured TOC contents during the final test

Time of

sam pling

(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

6.5A

6.5B

14A

14B

31A

31B

65A

65B

141A

141B 0.3087

1.912

2.065

5.172

5.037

13.83

13.86

29.98

30.00

64.84

65.51 -

2.9

3.2

8.9

8.7

25

25

54

54

118

120 -

45

49

64

62

81

81

83

83

84

85

24 31A

31B

65A

65B 8.985

8.890

22.17

18.93 16

16

41

35 52

52

63

54 64

64

76

65

48 0

6.5A

6.5B

14A

14B 0.1397

1.918

1.652

3.065

2.452 -

3.3

2.8

5.4

4.2 -

51

43

39

30 -

114

88

61

48

96 0

6.5A

6.5B

14A

14B 0.1795

0.9477

0.9142

2.036

1.539 -

1.4

1.3

3.4

2.5 -

22

20

24

18 -
48

41

38

29

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

7.2.2. Mortality and other effects

Table 4 shows the mortality data recorded during the final test. Within 5 hours of exposure all fish

exposed to the highest test concentration died. Within 24 hours all fish exposed to nominal

concentrations of 31 and 65 mg/l died and 29% of the fish exposed to a nominal concentration of

14 mg/l died. No mortality or clinical effects were observed at the lowest test concentration and the

control during the test period. After 24 hours the nominal 31 mg/l concentration was observed to be

hazy. After 48 hours the nominal 14 mg/l concentration was observed to be slightly hazy. The

responses recorded in this test allowed for reliable determination of an LC 50

.

Table 4 Incidence of mortality and total mortality during the final test

Nominal c onc

. Test subs tance 1

(mg/l) Vessel

number Initial

number

of fish Cumulative mortality Total

Mortality

(%) 5h 24h 48h 72h 96h

Control A 7 0 0 0 0 0 0

6.5 A

B 7

7 0

0 0

0 0

0 0

0 0

0 0

14 A

B 7

7 0

0 0

1 2

2 2

2 2

2 29

31 A

B 7

7 0

0 7

7 7

7 7

7 7

7 100

65 A

B 7

7 2

0 7

7 7

7 7

7 7

7 100

1 41 A

B 7

7 7

7 7

7 7

7 7

7 7

7 100

1 Test substance was Castor Oil, Sulfated, Sodium Salt, 75%Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 15

- 7.2.3. Determination of effect concentrations

Table 5 shows the effect parameters based on average ex posure 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 oncentrati on

Active ingredient

(mg/l)

24h

- L C 50 9.7 ( 6.0

- 34 ) 7.2 ( 4.5

- 25 )

48h

- L C 50 7.3 ( 5.2

- 12 ) 5.4 ( 3.9

- 9.0 )

72h

- LC 50 7.3 (5.2

- 12) 5.4 (3.9

- 9.0)

96h

- LC 50 7.3 (5.2

- 12) 5.4 (3.9

- 9.0)

( ) 95% confidence interval

7.2.4. Experimental conditions

The results of measurement of pH and oxygen concentrations are presented in Table 6

. A eration was

introduced after 2 hours of exposure, in order to keep the oxygen concentration above 5 mg/l. The

temperatures measured during the study in the various test vessels are presented in Table 7

. All test

conditions remained within the ranges prescribed by the protocol (pH: 6.0

- 8.5, constant within 1 unit;

temperature 20

- 24 °C, constant within 2°C; oxygen > 60% of air saturati on).

Table 6 pH

- values and dissolved oxygen concentrations (mg/l) during the final test

Nominal c onc

. Test substance 1

(mg/l) Vessel

number Day 0 Day 1 Day 2 Day 3 Day 4

pH 2 O 2 pH O 2 pH O 2 pH O 2 pH O 2

C ontrol A 7.4 10.0 8.0 7.2 8.1 8.3 7.9 8.8 7.9 7.9

6.5 A

B 7.7

7.7 10.0

10.0 7.6

7.5 7.3

6.4 7.9

7.6 8.0

6.8 7.7

7.6 8.4

7.5 7.9

7.8 7.7

7.6

14 A

B 7.6

7.6 10.0

9.9 7.4

7.3 6.8

6.9 7.6

7.5 7.4

5.8 7.5

7.5 7.9

7.9 7.8

7.8 7.5

7.5

31 A

B 7.5

7.5 9.9

9.9 7.4

7.5 7.9

8.0

65 A

B 7.4

7.4 9.8

9.8 7.4

7.3 8.7

8.6

1 41 A

B 7.3

7.3 9.5

9.5

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

2 pH of culture medium was : 7.7Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 16

- Table 7 Temperatures (°C) meas ured during the final test

Nominal c onc

. Test substance 1

(mg/l) Vessel

number Day 0 2 Day 1 Day 2 Day 3 Day 4

C ontrol A 20.0 20.7 20.4 21.1 21.0

6.5 A

B 20.2

20.1 20.7

20.9 20.8

21.2 21.1

21.2 20.8

21.1

14 A

B 20.2

20.1 20.7

20.9 20.8

21.2 21.0

21.1 20.9

21.0

31 A

B 20.1

20.0 20.7

20.9

65 A

B 20.0

20.0 20.7

20.6

141 A

B 20.5

20.5

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

2 Temperature of culture medium was : 20.7 °C. Fish were acclimatized to 20.4°C before the start of the te st.

8. CONCLUSION

The 96h

- LC 50 was 7.3 mg test substance per litre (95% confidence interval between 5.2 and 12 mg/l)

, containing 5.4 mg a.i. per litre (95% confidence interval between 3.9 and 9.0 mg/l ). This LC 50 value

was already reached after 48 hours of exposure.Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 17

- APPENDIX 1 LC- VALUES

Table 8 Parameters of the probit analysis at 24h

Parameter Value

Computation runs: 8.0000

Slope b: 8.4763

Intercept a:

- 8.7212

Variance of b: 9.9591

Goodness of Fit

Chi2: 0.0885

Degrees of freedom: 3.0000

p(Chi2): 0.9932

Log EC50: 0.9857

SE Log EC50: 0.1256

g

- Criterion: 0.5325

F: 244.4990

p(F) (df: 1;3): 0.0010

Data

Function

95%-CL

Concentration [mg/l] 10 100% Mortality 100

90

80

70

60

50

40

30

20

10

0

Figure 1 Percentage of mortality of fish plotted against the test subst ance concentration

after 24 hours of exposure

Table 9 Results of the probit analysis at 24h

Parameter EC50

Value ( mg/l ) 9.675

lower 95%

- cl 5.985

upper 95%

- cl 33.705Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 18

- APPENDIX 1 LC- VALUES

– continued

–

Table 10 Parameters of the probit analysis at 48, 72 and 96h

Parameter Value

Computation runs: 6.0000

Slope b: 6.2544

Intercept a:

- 5.76311

Variance of b: 2.9736

Goodness of Fit

Chi2: 0.8480

Degrees of freedom: 3.0000

p(Chi2): 0.8380

Log EC50 : 0.8629

SE Log EC50: 0.0850

g

- Criterion: 0.2920

F: 46.5380

p(F) (df: 1;3): 0.0060

Data

Function

95%-CL

Concentration [mg/l] 1 10 100% Mortality 100

90

80

70

60

50

40

30

20

10

0

Figure 2 Percentage of mortality of fish plotted against the test substance concentration

after 48, 72 and 96 hours of exposure

Table 11 Results of the probit analysis at 48, 72 and 96h

Parameter EC50

Value ( mg/l ) 7.292

lower 95%

- cl 5.164

upper 95%

- cl 12.191Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 19

- APPENDIX 2 REFERENCE TEST

96

- hour acute toxicity study in the Fathead minnow with pentachlorophenol (PCP); WIL Research

Europe Project 504370 (Batch FM 13

- 04).

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In

addition, the procedures were designed to meet the test methods of the ISO International Standard

7346

- 1: Static method, 1996.

Start: 16 December 2013

End: 20 December 2013

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

Research Europe. The reference substance was pentachlorophenol (PCP, SIGMA, Art. P2604, Batch

06324ED

- 405).

Concentrations: 0.10, 0.22 and 0.46 mg/l in ISO

- medium.

Incidence of mortality observed in the reference study:

Concentration

PCP

(mg/l) Initial

number

of fish Cumulative mortality Total

Mortality

(%) 2h 24h 48h 72h 96h

0.10 5 0 1 2 2 2 40

0.22 5 0 5 5 5 5 100

0.46 5 0 5 5 5 5 100

During the test the pH, oxygen concentration and the temperature of the medium were within the

optimal ranges for fish.

Under the conditions of the present test with Fathead minnow exposed to PCP, the 96h

- LC 50 was

0.11 mg/l based on nominal concentrations. This effect was already reached within 48 hours of

exposure.

No historical data is available on the range of the 96h

- LC 50 for Fathead minnow. However, this test

sh ows that toxicity is similar for Fathead minnow and carp (Teleostei Cyprinidae). For Carp the

96h

- LC 50 is generally between 0.10 and 0.46 mg/l based on historical data of reference tests

performed approximately every 3 months from April 1988 until the end of 2000, and annually since

then.

The raw data and report from this study are kept in the WIL Research Europe archives. The test

described above was performed under GLP

- conditions with a QA

- check.Castor Oil, Sulfated, Sodium Salt, 75% Project 503337

- Page 20

- APPENDIX 3 CERTIFICATE OF ANALYSIS