# FINAL REPORT

Study Title



# FRESH WATER ALGAL GROWTH INHIBITION TEST

WITH

CASTOR OIL, SULFATED , SODIUM SALT, 75%

Author

L.M. Bouwman, MSc.

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 503338

Substance 205003/A

- Page 1 of 28

-

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- 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

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

6.2. Test System

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

6.3. Fresh water algae culture

..................................................................................................... 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 procedures and conditions

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

6.6.3. Sampling for analysis of test concentrations

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

6.6.4. TOC analysis

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

6.6.5. Measurements and recordings

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

6.6.6. Re cording of cell densities

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

6.7. Electronic data capture

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

6.8. Interpretation

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

6.8.1. Data handling

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

6.8.2. Acceptability of the test

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

6.9. List of deviations

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

6.9.1. List of protocol deviations

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

6.9.2. List of standard operating procedures deviations

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

7. RESULTS

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

7.1. Combined limit/range

- finding test

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

7.2. Final test

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

7.2.1. Measured test substance concentrations

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

7.2.2. Mean cell densities

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

7.2.3. Inhibition of growth rate and inhibition of yield

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

7.2.4. Determination of effect concentrations

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

7.2.5. Experimental conditions

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

7.2.6. Algistatic/algicidal effects

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

8. CONCLUSION

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

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 3 - TABLES

Table 1 Mean cell densities (x10 4 

### cells/ml) during the combined limit/range

- finding test

......... 15

Table 2 Percentage inhibition of growth rate, yield and cell growth during the combin ed

limit/range

- finding test

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

Table 3 Measured TOC contents during the combined limit/range

- finding test

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

Table 4 Measured TOC contents during the final test

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

Table 5 Percentage inhibition of growth rate, yield and cell growth during the final test

.......... 17

Table 6 Effect parameters

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

Table 7 pH levels recorded during the final test

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

Table 8 Mean cell densities (x 10 4 

### cells/ml) d uring the recovery period in the final test

.......... 18

Table 9 Individual cell densities (x10 4 

### cells/ml)

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

Table 10 Calculation of growth rate, yield and cell growth

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

Table 11 IC- values for growth rate inhibition

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

Table 12 IC- values for yield inhibition

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

Table 13 IC- values for cell growth inhibition

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

FIGURES

Figure 1 Growth curves at different concentrations of Castor Oil, Sulfated, Sodium Salt,

75%

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

Figure 2 Percentage inhibition of growth rate as function of the log concentration (mg/l) of

Castor Oil, Sulfated, Sodium Salt, 75%.

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

Figure 3 Percentage inhibition of yield as function of the log concentration (mg/l) of Castor

Oil, Sulfated, Sodium Salt, 75%.

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

Fig ure 4 Percentage inhibition of cell growth as function of the log concentration (mg/l) of

Castor Oil, Sulfated, Sodium Salt, 75%.

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

APPENDICES

APPENDIX 1 WORKSH EET DATA

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

APPENDIX 2 STATISTICS: GROWTH RATE

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

APPENDIX 3 STATISTICS: YIELD

................................................................................................ 22

APPENDIX 4 STATISTICS: CELL GROWTH

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

APPENDIX 5 IC

- VALUES

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

APPENDIX 6 REFERENCE TEST

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

APPENDIX 7 CERTIFICATE OF ANALYSIS

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

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 6 - 4. SUMMARY

Pseudokirchneriella subcapitata , Fresh Water Algal Growth Inhibition Test with Castor Oil, Sulfated,

Sodium Salt, 75%

.

The study procedure described in this report was based on the OCSPP 850.4500, 2012

. In ad dition,

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

5 corrected 28 July 2011

.

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

soluble in test medium at the concentrations tested.

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

- finding test.

P reparation of test solutions started with a nominal concentration of 212 mg/l containing 158 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 212 mg/l concentration in test medium. The final test solutions

were clear and colourless

.

Four replicates of exponentially growing algal cultures were exposed to a control and to nominal test

substance concentrations of 1.4, 4.9, 17, 61 and 212 mg / l, containing 1.0, 3.7, 13, 46 and 158 mg a.i

. per litre. The initial cell density was 10 4 

### cells/ml and the total exposure period was 96 hours . S amples

for non

- specific analytical confirmation of actual exposure concentrations were taken at the start, after

24 and after 96 hours of exposure. Subsequently, subsamples of the highest test concentration were

incubated in M2 medium without test substance in order to determine if the effects on algal growth

were algistatic or algicidal.

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 initial concentrations in samples taken from nominal 1.4, 4.9, 17, 61 and 212 mg/l were

calculated to be 0.30, 3.6, 15, 48 and 132 mg/l, i.e. 21

- 88% of nominal, respectively. The three highest

test concentrations remained stable during the exposure period (100

- 113% of initial at the end of the

test), while the lower test concentrations decreased to below the Limit Of Detection (L OD =0.01 mg/l)

for the lowest test concentration and to 72% of initial for nominal 4.9 mg/l. Given these results, effect

parameters were based on the average exposure concentrations: 0.089, 2.4, 13, 51 and 140 mg test

substance per litre, containing 0.066, 1.8, 9.9, 38 and 104 mg a.i. per litre.

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

Castor Oi l, Sulfated, Sodium Salt, 75% inhibited growth rate , yield and cell growth of this fresh water

algae species significantly at an average exposure concentrations 51 mg/l and higher. A test

substance concentration of 51 mg/l contained 38 mg of the active ingredient per litre.

The IC 50 

### for growth rate inhibition ( 96h -I R 

### C 50 

### ) was 62 mg/l ( 95% confidence interval between 23 and

170 mg/l ), containing 46 mg a.i. per litre (95% confidence interval between 17 and 130 mg/l ).

The IC 50 

### for yield inhibition ( 96h -I Y 

### C 50 

### ) was 40 mg/l ( 95% confidence interval between 19 and 83 mg/l ),

containing 30 mg a.i. per litre (95% confidence interval between 14 and 62 mg/l ).

The IC 50 

### for cell growth inhibition ( 96h -I B 

### C 50 

### ) was 39 mg/l ( 95% confidence interval between 22 and

68 mg/l ), containing 29 mg a.i. per litre (95% confidence interval between 16 and 51 mg/l ).

The 96h

- NOEC for inhibition of growth rate, yield and cell growth was 13 mg/l , containing 9.7 mg a.i.

per litre

.

Incubation of subsamples taken from the highest test concentration in M2 medium without test

substance showed that the inhibitory effect of Castor Oil, Sulfated, Sodium Salt, 75% was algistatic.

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- 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 : 25 December 2013

TOC analysis : 04 December 2013 and 08 January 2014

5.2. Aim of the study

The purpose of the study was to evaluate the test substan ce for its ability to generate toxic effects in

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

the IC 50 

### for inhibition of growth rate, yield and area under the growth curve (biomass). In addition, the

N OEC or IC 05 

### for these measures were determined.

5.3. Guidelines

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

Guidelines, OCSPP 850.4500, Algal Toxicity, EPA 712 -C- 006, January 2012.

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

guideline:

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

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

.

5.4. S torage and retention of records and materials

Records and materials pertaining to the study including protocol, raw data, specimens (except

specimens requiring refrigeration or freezing) and the final report are retained in the WIL Research

Europe archives for a period of at least 2 years after finalization of the report. After this period, the

sponsor will be contacted to determine how the records and materials should be handled. WIL

Research Europe will retain information concerning decisions made.

Those specimens requiring refrigeration or freezing will be retained by WIL Research Europe for as

long as the quality of the specimens permits evaluation but no longer than three months after

finalization of the report.

WIL Research Europe will retain a test substance sample until the expiry date, but no longer than 10

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

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 8 - 5.5. Definitions

Cell density is the number of cells per millilitre.

Growth is the increase of cell density over the test period. The I B 

### C 50 

### is the concentration of test

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

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

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

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

### C 50 

### is the concentration of test

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

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

of the exposure period. The I

y 

### C 50 

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

of yield rel ative to the control.

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

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

Algicidal refers to having the property of killing algae.

Algicidal concentration is the lowest concentration tested which allows no net growth of algal cells

during either exposure to the test substance or during the recovery period in the absence of test

substance.

Algistatic refers to having the property of inhibiting algal growth.

Algistatic concentration is the highest concentration tested which allows no net growth of algal cells

during exposure to the test substance but permits regrowth when the algal cells are placed in test

substance

- free m edium.

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 r oom temperature in the dark

Stable under storage conditions until 18 July 2014 (expiry date)

See APPENDIX 7 : Certificate of analysis.

6.1.2. Study specific test substance information

Purity/composition correction factor

required Ye s, 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

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 9 - 6.1.3. Reference substance

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

( APPENDIX 6 )

.

6.2. Test System

Species Pseudokirchneriella subcapitata , strain: NIVA CHL 1

Source In

- house laboratory culture.

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

substances in the aquatic ecosystem and has been selected

as an internationally accepted species.

6.3. Fresh water algae culture

Stock culture Algae stock cultures were started by inoculating growth

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

suspensions were continuously aerated and exposed to light

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

Light intensity 60 to 120 μE/m 2 

### /s when measured in the photosynthetically

effective wavelength range of 400 to 700 nm.

Stock culture mediu m M1; according to the NPR 6505 (“Nederlandse Praktijk

Richtlijn no. 6505 ”) formulated using Milli

- RO water (tap

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

Mass., USA) with the following composition:

NaNO 3 500 mg/l

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

MgSO 4 .7H 2 O 75 mg/l

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

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

NH 4 NO 3 330 mg/l

CaCl 2 .2H 2 O 35 mg/l

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

H 3 BO 3 2.9 mg/l

MnCl 2 .4H 2 O 1.81 mg/l

ZnCl 2 0.11 mg/l

CuSO 4 .5H 2 O 0.08 mg/l

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



### Pre

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

culture were inoculated in culture medium at a cell density of

1 x 10 4 

### cells/ml. The pre

- culture was maintained under the

same conditions as used in the test. The cell density was

measured immediately before use.

Pre

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

Milli

- RO water and with the following composition:

NH 4 Cl 15 mg/l

MgCl 2 .6H 2 O 12 mg/l

CaCl 2 .2H 2 O 18 mg/l

MgSO 4 .7H 2 O 15 mg/l

KH 2 PO 4 1.6 mg/l

FeCl 3 .6H 2 O 64 μg/ l

Na 2 EDTA.2H 2 O 100 μg/l

H 3 BO 3 185 μg/l

MnCl 2 .4H 2 O 415 μg/l

ZnCl 2 3 μg/l

CoCl 2 .6H 2 O 1.5 μg/l

CuCl 2 .2H 2 O 0.01 μg/l

Na 2 MoO 4 .2H 2 O 7 μg/l

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 10

- NaHCO 3 50 mg/l

Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO 3 /l)

pH 8. 1 ± 0.2



### 6.4. Preparation of test solutions

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

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

testing of concentrations that would disturb the test system was prevented as much as possible (e.g.

film of the test substance on the water surface). A correction was made for the purity/composition of

the test substance. A correction fact or 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 in test medium at the concentrations tested.

P reparation of test solutions started with a nominal concentration of 141 mg/l containin g 105 mg of the

active ingredient (a.i.) per litre for the combined limit/range

- finding test and with a nominal

concentration of 212 mg/l containing 1 58 mg of the active ingredient (a.i.) per litre for the final test

. Th ese concentration s w ere 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 or 212 mg/l concentration s in test medium. The final test solutions were clear and colourless

.

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

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

10 4 

### cells/ml.

6.5. Combined limit/r ange

- find ing test

The study started with a combined limit/range

- finding test. Six replicates of exponentially growing

algae 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:

• Three replicates per concentration were exposed to nominal concentrations of 0.1 4 , 1. 4 and

14 mg/l ;

• After 24 hours of exposure cell densities were determined by spectrophotometric measurement of

samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal

medium was used as blank and one extra test vessel per concentration without algae was used as

background

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

plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of

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

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

spectrophotometric measurements at the various points in time during the test period ;

• pH was only measured in the control and the highest test concentration ;

• Algistatic/algicidal effects were not determ ined

.

6.6. Final test

6.6.1. Test concentrations

Castor Oil, Sulfated, 1.4, 4.9, 17, 61 and 212 mg test substance per litre,

Sodium Salt, 75% containing 1.0, 3.7

, 13

, 46 and 158 mg a.i. per litre

Control Test medium without test substance or other additives

Replicates 4 replicates of the control and each test concentration ;

4 replicates of the control and each test concentration

without algae.

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 11

- 6.6.2. Test procedures and conditions

Test duration 96 hours

Test type Static

Test vessels 1 25 ml, all

- glass, containing 50 ml of test solution

Medium M2

Cell density An initial cell density of 1 x 10 4 

### cells/ml.

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

Illumination Continuously using TLD

- lamps , with a light intensity within

the range of 60 to 62 μ E.m -2 

### .s -1



### .

Incubation Capped vessels were distributed at random in the incubator

and as such were daily repositioned. During incubation the

algal cells were kept in suspension by continuous shaking.

6.6.3. Sampling for analysis of test concentrations

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

concentrations without algae according to the schedule below.

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

Volume 40 ml

Storage Samples were stored in a refrigerator u ntil analysis.

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

before sampling

.

Additionally, reserve samples of 40 ml were taken from all test solutions for possible analysis. If not

already 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

Shi madzu 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

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

The pH of the solutions should preferably not deviate by

more than 1.5 units during the test.

Temperature of medium Continuously in a temperature control vessel.

Appearan ce of the cells At the end of the 96h exposure period microscopic

observations were performed on all test concentration s to

observe for any abnormal appearance of the algae.

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 12

- Algistatic/algicidal effects At the end of the 96h exposure period a subsample wa s

removed from each replicate of the highest test

concentration. These subsamples were cultured in M2

medium without test substance for a period of up to 9 days.

6.6.6. Recording of cell densities

C ells were counted using a microscope and a counting chamber.

6.7. El ectronic data capture

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

programme(s):

- Shimadzu Spectrophotometer UV

- 1800 including UVProbe 2.33 software (Shimadzu, Kyoto,

Japan): Algal cell density (Only at 24 hours in the combined limit/range

- finding test)

.

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

USA) : Temperature.

6.8. Interpretation

6.8.1. Data handling

Comparison of average growth rates

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

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



### )day( lnln

1

−

−



### − −

=

ij ij

ji 

### tt XX

μ

Where: μ i-j 

### = the average specific growth rate from time i to j

X

i 

### = the biomass at time i

X

j 

### = the biomass at time j

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

and the percentage inhibition in growth rate is calculated :



### 100 % × −

=

C TC

r 

### μ μμ I

Where: % I

r 

### = percent inhibition in average specific growth rate

μ C 

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

μ T 

### = average specific growth rate for the treatment replicate

Yield

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



### 100 % × −

=

C TC

y 

### Y YY I

Where: % I

y 

### = percent inhibition of yield

Y

C 

### = mean value for yield in the control group

Y

T 

### = value for yield for the treatment replicate

Comparison of areas under the growth curves

The area below the growth curve is calcu lated using the formula:

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 13

- b 1

- 

### b 0 

### b 1 

### + b 2 

### - 2 * b 0 

### b n-1 

### + b

n 

### - 2 * b 0 

### A = _____ * t 1 

### + _____________ * (t 2 

### -t 1 

### ) + ______________ * (t

n 

### -t n-1 

### )

2 2 2

Where: A = area

b 0 

### = biomass (cell density) at the start of t he test

b 1 

### = biomass (cell density) at t 1



### b

n 

### = biomass (cell density) at t

n



### t 1 

### = time of first measurements after beginning of the test

t

n 

### = time of n th 

### measurement after beginning of the test

The percentage inhibition of cell growth at each test concentration (I T 

### ) is calculated using the following

formula:

A C 

### -A T



### I T 

### = ______ * 100

A C



### Where: A C 

### = area below the growth curve obtained in the control

A T 

### = area below the growth curve at each test substance concentration

Doubling time of controls

The doubling time (T d 

### ) of cell density in controls at test termination is calculated using the following

formula:

c d

μ T )2ln(

=



### Where: μ C 

### = average specific growth rate in the control group

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 wer e 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:

96 CC CC 24 t 24 t 24 t0t 96 72

== == ××+××



### , being

the Time Weight ed Average (TWA) of the concentrations of Castor Oil, Sulfated, Sodium Salt, 75%

measured in the samples taken at the start (C t=0 

### ), after 24 hours (C t=24 

### ) and the end of the test (C

t= 96 

### ).

In c ase concentrations measured were below the limit of detection or the limit of quantification, the

final exposure concentration(s) w ere taken as a factor of 2 below th is limit. This procedure is based on

the OECD “Guidance document on the use of the harmoni sed system for the classification of

chemicals which are hazardous for the aquatic environment”.

Determination of the NOEC and calculation of the IC 50 

### For determination of the NOEC and the IC 50 

### the approaches recommended in the OECD guideline 201

were used. An effect was considered to be significant if statistical analysis of the data obtained for the

test concentrations compared with those obtained in the negative control revealed significant inhibition

of growth rate, yield or cell growth (ANOVA, Bonfer roni t -T est, TOXSTAT Release 3.5, 1996, D.D.

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 14

- Gulley, A.M. Boelter, H.L. Bergman). Additionally, the IC 05 

### was determined.

Calculation of the IC 50 

### and IC 05 

### values was based on log

- linear regression analysis of the percentages

inhibition of growth rate, yield and cell growth versus the logarithms of the corresponding average

exposure concentrations of the test substance.

6.8.2. Acceptability of the test

1. In the controls, cell density increased by an average factor of > 100 within 96 hours.

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

replicate control cultures did not exceed 15%.

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

cultures did not exceed 15%.

4. Test conditions and test set

- up were according to the guideline.

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.

2. Combined limit/range

- finding test: The vessels for the pre

- culture have not been kept shaking

during the first 2 days of incubation.

Evaluation: Algal growth in the pre

- culture proved to be sufficient.

3. Final test: The test vessels of the highest test concentration were not pooled before the start of

the recovery phase. Instead, all vessels have been tested individually.

Evaluation: This did not have an effect on the outcome of the recovery phase.

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 operatin g procedures that affected the integrity of the study.

7. RESULTS

7.1. Combined limit/r ange

- finding test

The mean cell densities measured during the combined limit/ range

- finding test are presented in

Table 1

. Table 2 presents the percentages for inhibition of growth rate, yield and cell growth per

concentration. After 48 hours of exposure test concentrations of 1.41 mg/l and higher became

increasingly hazy with increasing concentration. After 72 and 96 hours of exposure no haziness was

observed at 1.41 and 14 mg/l but haziness, test substance particles and a precipitate were observed

at the highest test concentration , indicating that part of the test substance came out of solution at this

concentration. T he expected IC 50 

### for inhibition of growth rate, yield and cell growth was between

nominal test substance concentrations of 14 and 141 mg/l.

Samples taken from nominal 1.4 and 141 mg test substance per litre without algal cells 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 3 ) . The initial concentration s w ere 7.1 and 101

mg/l, respectively, i.e. 507 and 7 2% of nominal

. Both concentrations remained stable during the first

24 hours of exposure ( 83

- 94 % of initial) but decreased to 44

- 70% of initial at the end of the test

. The

relatively high concentration at nominal 1.4 mg/l can be related to the fact that me asurements could

not be corrected for the control.

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

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 15

- Table 1 Mean cell densities (x10 4 

### cells/ml) during the combined limit/ range

- finding test

Nomina l c onc

. Exposure time (hours)

Test substance 1

mg/l 0 24 48 72 96

control 1.0 6.0 17.9 85.5 244.3

0.14 1.0 6.3 18.1 88.7 246.9

1.4 1.0 7.4 19.2 85.3 257.8

14 1.0 7.7 17.6 66.0 188.3

141 1.0 6.0 7.3 6.1 10.4

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



### Table 2 Percentage inhibition of growth rate , yield and cell growth during the combined

limit/ range

- finding test

Nominal c onc

. Mean growth rate Yield (0

- 96 h) Cell growth (0

- 96 h)

Test substance 1

mg/l μ (0

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

control 0.05726 243.31 5473.29

0.14 0.05738

- 0.2 245.94

- 1.1 5594.07

- 2.2

1. 4 0.05783

- 1.0 256.81

- 5.5 5695.03

- 4.1

14 0.05456 4.7 187.25 23.0 4366.02 20.2

1 41 0.02442 57.4 9.44 96.1 505.18 90.8

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



### Table 3 Measured TOC contents during the combined limit/range

- finding test

Time of

sampling

(hours) Nominal conc.

(mg/l) Measured

or ganic carbon

conc.

(mg/l) Calculated

conc.

Test substance 1

(mg/l) Relative to

nominal

(%) Relative to

initial

(%)

0 1.4

141 3.876

54.87 7.1

1 01 507

72

24 1.4

141 3.231

51.83 5.9

95 421

67 83

94

96 1.4

141 1.668

38.54 3.1

71 221

50 44

70

1 Test substa nce was Castor Oil, Sulfated, Sodium Salt, 75%



### 7.2. Final test

7.2.1. Measured test substance concentrations

The results of TOC analysis of the samples taken during the final test are described in Table 4

. 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 4 ) . A correction was made for the measured

amount of TOC in the control. The initial concentrations in samples taken from nominal 1.4, 4.9 , 1 7

, 61

and 212 mg/l were calculated to be 0.30, 3. 6

, 15

, 48 and 132 mg/l, respectively, i.e. 21 -8 8% of

nominal. The three highest test concentrations remained stable during the exposure period ( 100

- 113 %

of initial at the end of the test ) which was not expected based on the results of the range

- finding test.

However, no explanation could be given for this contradiction. T he lowe r test concentration s

decreased to below the LOD for the lowest test concentration and to 72 % of initial for nominal 4.9

mg/l . Given these results, effect parameters were based on the average exposure concentrations:

0.089, 2.4, 13, 51 and 140 mg test substance per litre, containing 0.066

, 1.8 , 9. 9, 38 and 104 mg a.i.

per litre.

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 16

- Table 4 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

1.4

4.9

17

61

21 2 <LOD

0.1111

1.900

8.255

26.16

71.87 -

0. 30

3. 6

15

48

132 -

21

73

88

79

62

24 0

1.4

4.9

17

61

212 0.6626

0.7846

1.835

7.610

27.21

77.77 -

0.22

2.1

13

49

142 -

16

43

76

80

67 -

73

58

87

102

108

96 0

1.4

4.9

17

61

212 0.7760

0.6785

2.189

7.684

29.96

76.6 7 -

< LOD

2.6

13

54

139 -

-

53

76

89

66 -

-

72

100

113

105

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

LOD=0.01 mg/l



### 7.2.2. Mean cell densities

Figure 1 shows growth curves at different concentrations of Castor Oil, Sulfated, Sodium Salt, 75%

. The individual and group mean cell densities measured at 24h intervals are given in Table 9

, APPENDIX 1

. After 72 hours of exposure the two highest test concentrations became incr easingly

hazy with increasing concentration. This haziness was not observed after 96 hours of exposure.

Figure 1 Growth curves at different concentrations of Castor Oil, Sulfated, Sodium Salt,

75%

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 17

- 7.2.3. Inhibition of growth rate and inhibition of yield

Table 5 shows the calculation of the percentages of inhibition for growth rate, yield and cell growth

(see APPENDIX 1 for the individual values). Statistical analysis of the data is s hown in APPENDIX 2

, APPENDIX 3 and APPENDIX 4

.

Growth rate, yield and cell growth were in the range of the controls at average exposure

concentrations up to and including 13 mg/l during the 96

- hour test period, whereas the growth rate

, yield and cell growth of algae exposed to average exposure concentrations of 51 mg/l and higher were

increasingly inhibited . Statistically significant inhibition was found at test concentrations of 51 mg/l and

hig her

.

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

exposed cells when compared to the control.

Table 5 Percentage inhibition of growth rate , yield and cell growth during the final test

TWA conc. Mean growth rate Yield (0

- 96 h) Cell growth (0

- 96 h)

Test substance 1

(mg/l) μ (0

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

control 0.05767 252.88 5332.50

0.089 0.05722 0.8 244.25 3.4 5259.00 1.4

2.4 0.05917

- 2.6 293.00

- 15.9 6045.00

- 13.4

13 0.05803

- 0.6 264.31

- 4.5 5358.75

- 0.5

51 0.04386 23.9 * 68.63 72.9 * 1486.50 72.1 *

140 0.00519 91.0 * 0.88 99.7 * 120.00 97.7 *

1 Test substance was Castor Oi l, Sulfated, Sodium Salt, 75%

*

- effect was statistically significant



### 7.2.4. Determination of effect concentrations

Table 6 shows the effect parameters based on average exposure concentrations, which were

calculated from non

- specific T OC

- analysis

, see also APPENDIX 5

.

Table 6 Effect parameters

Parameter Concentration

Castor Oil, Sulfated,

Sodium Salt, 75%

(mg/l) Concentration

Active ingredient

(mg/l)

96h

- NOE R C 13 9.7

96 h

- I R C 05 19 (6. 8

- 51) 14 (5.1

- 38)

96 h

- I R C 50 62 (23

- 170) 46 (17

- 130)

96h

- NOE Y C 13 9.7

96 h-I Y C 05 14 (6.6

- 31) 10 (4.9

- 23)

96 h

- I Y C 50 40 (19

- 83) 30 (14

- 62)

96h

- NOE B C 13 9.7

96 h

- I B C 05 13 (7.3

- 24) 9.7 (5.4

- 18)

96 h-I B C 50 39 (22

- 68) 29 (16

- 51)

( ) 95% confidence interval



### 7.2.5. Experimental conditions

Table 7 shows the pH recorded at the beginning and the end of the test. The pH was within the limits

prescribed by the protocol (6.0

- 9.0, preferably not varying by more than 1.5 unit). The temperature of

the test medium was 23.1 °C at the start of the test. During the exposure period the temperature

measured in the incubator was maintained between 24.0 and 24.8°C. Temperature remained within

the limits prescribed by the protocol (2 2-26 °C , constant within 2 °C ).

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 18

- Table 7 pH levels recorded during the final test

TWA conc. Exposure time (hours)

Test substance 1

(mg/l) 0 96

control 8.3 8.6

0.089 8.3 8.6

2.4 8.3 8.5

13 8.2 8.5

51 8.1 8.3

140 7.8 7.8

1 Test su bstance was Castor Oil, Sulfated, Sodium Salt, 75%



### 7.2.6. Algistatic/algicidal effects

Table 8 shows mean cell densities directly after incubation of the subsamples in M2 medium without

test substance and after 72 , 96 and 120 hours of i ncubation

. The subculture test was discontinued

after 120 hours of exposure but cell density had already increased by an average factor of >16 after

96 hours of exposure

. This result showed that the inhibitory effect of Castor Oil, Sulfated, Sodium Salt,

7 5% was algistatic.

Table 8 Mean cell densities (x 10 4 

### cells/ml) during the recovery period in the final test

TWA c onc

. Replicate Exposure time (hours)

Test substance 1 Vessel

(mg/l) 0 72 96 120

140 A 0.5 0 2.5 0 22.50 126.50

B 0.50 1.50 15.25 101.25

C 0.75 2.00 26.00 111.00

D 0.25 2.50 27.00 105.25

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



### 8. CONCLUSION

Under the conditions of the present study with Pseudokirchneriella subcapitata

, Castor Oil, Sulfated,

Sodium Salt, 75% inhibited growth rate, yield and cell growth of this fresh water algae species

significantly at an average exposure concentrations 51 mg/l and higher. A test substance

concentration of 51 mg/l contained 38 mg of the active ingredient per litre.

The IC 50 

### for growth rate inhibition ( 96 h-I R 

### C 50 

### ) was 62 mg/l ( 95% confidence interval between 23 and

170 mg/l ), containing 46 mg a.i. per litre (95% confidence interval between 17 and 130 mg/l ).

The IC 50 

### for yield inhibition ( 96 h-I Y 

### C 50 

### ) was 40 mg/ l ( 95% confidence interval between 19 and 83 mg/l ),

containing 30 mg a.i. per litre (95% confidence interval between 14 and 62 mg/l ).

The IC 50 

### for cell growth inhibition ( 96h -I B 

### C 50 

### ) was 39 mg/l ( 95% confidence interval between 22 and

68 mg/l ), containing 29 mg a.i. per litre (95% confidence interval between 16 and 51 mg/l ).

The 96 h- NOEC for inhibition of growth rate , yield and cell growth was 13 mg/l , containing 9.7 mg a.i.

per litre

.

Incubation of subsamples taken from the highest test concentration in M2 medium without test

substance showed that the inhibitory effect of Castor Oil, Sulfated, Sodium Salt, 75% was algistatic.

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 19

- APPENDIX 1 WORKSHEET DATA

Table 9 Individual cell densities (x10 4 

### cells /m l)

TWA conc. Vessel Exposure time (hours)

Test substance 1 number

(mg/l) 0 24 48 72 96

control 1 1.00 4.25 18.00 79.75 260.25

2 1.00 4.00 18.75 71.00 249.00

3 1.00 3.25 17.00 73.75 239.25

4 1.00 3.50 19.50 82.25 267.00

0.089 1 1.00 4.5 0 19.50 92.75 288.75

2 1.00 4.00 17.50 64.00 210.75

3 1.00 4.00 18.50 73.00 267.00

4 1.00 4.50 20.75 77.00 214.50

2.4 1 1.00 4.50 18.50 88.25 280.50

2 1.00 3.25 16.75 85.75 324.75

3 1.00 4.00 18.75 86.50 307.50

4 1.00 3.50 17.50 86.25 263.25

13 1 1.00 4.25 12.75 75.00 333.00

2 1.00 3.50 16.75 73.75 252.75

3 1.00 4.00 15.00 76.00 238.50

4 1.00 3.50 15.50 76.50 237.00

51 1 1.00 3.75 7.25 19.50 101.00

2 1.00 3.00 10.25 17.50 66.75

3 1.00 2.75 8.00 21.50 53.00

4 1.00 2.50 9.00 17.50 5 7.75

140 1 1.00 2.75 4.75 1.25 3.75

2 1.00 1.75 3.00 1.50 1.25

3 1.00 2.75 3.75 1.00 1.25

4 1.00 2.00 4.00 1.75 1.25

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

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 20

- APPENDIX 1 WORKSHEET DATA

– continued

–

Table 10 Calculation of growth rate, yield and cell growth

TWA conc. Vessel Growth rate Yield Area (A) Growth rate red. Yield inhib. Growth inhib.

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

(mg/l) 0

- 96 h 0

- 96 h 0

- 96 h 0

- 96 h 0

- 96 h 0

- 96 h

c ontrol 1 0.05793 259.25 5487

2 0.05747 248.00 5154

3 0.05706 238.25 5043

4 0.05820 266.00 5646

mean 0.05767 253 5333

CV 1% 5% 5%

0.089 1 0.05902 287.75 6183

- 2

- 14

- 16

2 0.05574 209.75 4497 3 17 16

3 0.05820 266.00 5412

- 1

- 5

- 1

4 0.05592 213.50 4944 3 16 7

2.4 1 0.05871 279.50 5952

- 2

- 11

- 12

2 0.06024 323.75 6351

- 4

- 28

- 19

3 0.05967 306.50 6228

- 3

- 21

- 17

4 0.05805 262.25 5649

- 1

- 4

- 6

13 1 0.06050 332.00 6120

- 5

- 31

- 15

2 0.05763 251.75 5205 0 0 2

3 0.05702 237.50 5058 1 6 5

4 0.05696 236.00 5052 1 7 5

51 1 0.04807 100.00 1860 17 60 65

2 0.04376 65.75 1455 24 74 73

3 0.04136 52.00 1326 28 79 75

4 0.04225 56.75 1305 27 78 76

140 1 0.01377 2.75 171 76 99 97

2 0.00232 0.25 81 96 100 98

3 0.00232 0 .25 111 96 100 98

4 0.00232 0.25 117 96 100 98

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

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 21

- APPENDIX 2 STATISTICS: GROWTH RATE

**Chi**

**- Square Test for Normality** 

---------------------------------------------------------------------------- Actual and Expected Frequencies

INTERVAL <

- 1.5

- 1.5 to <

- 0.5

- 0.5 to 0.5 >0.5 to 1.5 >1.5

----- ------------- ----------- ----------- -----

EXPECTED 1.6080 5.8080 9.1680 5.8080 1.6080

OBSERVED 0 8 7 9 0

----------------------------------------------------------------------------

Chi

- Square = 6.3102 (p

- value = 0.1771)

Critical Chi

- Square = 13.277 (alpha = 0.01 , df = 4)

= 9.488 (alpha = 0.05 , df = 4)

---------------------------------------------------------------------------- Data PASS normality test (alpha = 0.01). Continue analysis.

**Levene's Test for Homogeneity of Variance**

ANOVA Table

----------------------------------------------------------------------------

SO URCE DF SS MS F

----------------------------------------------------------------------------

Between 5 0.0000 0.0000 0.4969

Within (Error) 18 0.0001 0.0000

----------------------------------------------------------------------------

Total 23 0.0001

----------------------------------------------------------------------------

(p

- value = 0.7745)

Critical F = 4.2479 (alpha = 0.01, df = 5,18)

= 2.7729 (alpha = 0.05, df = 5,18)

Since F < Critical F FAIL TO REJECT Ho: All equal (alpha = 0.01)

**ANOVA Table**

----------------------------------------------------------------------------

SOURCE DF SS MS F

----------------------------------------------------------------------------

Between 5 0.0090 0.0018 223.0309

Within (Error) 18 0.0001 0.0000

----------------------------------------------------------------------------

Total 23 0.0091

----------------------------------------------------------------------------

(p

- value = 0.0000)

Critical F = 4.2479 (alpha = 0.01, df = 5,18)

= 2.7729 (alpha = 0.05, df = 5,18)

Since F > Critical F REJECT Ho: All equal (alpha = 0.05)

**Bonferroni t**

**- Test** 

**- TABLE 1 OF 2**  Ho: Control=Treatment

----------------------------------------------------------------------------

TRANSFORMED MEAN CALCULATED IN SIG

GROUP IDENTIFICATION MEAN ORIGINAL UNITS t STAT 0.05

----- -------------------- ----------- ------------------ ------ ---

1 control 0.0577 0.0577

2 0.089 0.0572 0.0572 0.2217

3 2.4 0.0592 0.0592 0.7486

4 13 0.0580 0.0580 0.1806

5 51 0.0439 0.0439 6.8779 *

6 140 0.0052 0.0052 26.1478 *

---------------------------------------------------------------------------- Bonferroni t critical value = 2.8784 (2 Tailed, alpha = 0.05, df = 5,18)

**Bonferroni t**

**- Test** 

**- TABLE 2 OF 2**  Ho: Control=Treatment

----------------------------------------------------------------------------

NUM OF MIN SIG DIFF % OF DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

----- -------------------- ------- ---------------- ------- ------------

1 control 4

2 0.089 4 0.0058 10.0 0.0004

3 2.4 4 0.0058 10.0 0.0015

4 13 4 0.0058 10.0 0.0004

5 51 4 0.0058 10.0 0.0138

6 140 4 0.0058 10.0 0.0525

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 22

- APPENDIX 3 STATISTICS: YIELD

**Chi**

**- Square Test for Normality** 

---------------------------------------------------------------------------- Actual and Expected Frequencies

INTERVAL <

- 1.5

- 1.5 to <

- 0.5

- 0.5 to 0.5 >0.5 to 1.5 >1.5

----- ------------- ----------- ----------- -----

EXPECTE D 1.6080 5.8080 9.1680 5.8080 1.6080

OBSERVED 0 8 8 8 0

----------------------------------------------------------------------------

Chi

- Square = 5.0194 (p

- value = 0.2853)

Critical Chi

- Square = 13.277 (alpha = 0.01 , df = 4)

= 9.488 (alpha = 0.05 , df = 4)

---------------------------------------------------------------------------- Data PASS normality test (alpha = 0.01). Continue analysis.

**Levene's Test for Homogeneity of Variance**

ANOVA Table

----------------------------------------------------------------------------

SOURCE DF SS MS F

----------------------------------------------------------------------------

Between 5 2824.7005 564.9401 1.6176

Within (Error) 18 6286.2969 349.2387

----------------------------------------------------------------------------

Total 23 9110.9974

----------------------------------------------------------------------------

(p

- value = 0.2060)

Critical F = 4.2479 (alpha = 0.01, df = 5,18)

= 2.7729 (alpha = 0.05, df = 5,18)

Since F < Critical F FAIL TO REJECT Ho: All equal (alpha = 0.01)

**ANOVA T able** 

----------------------------------------------------------------------------

SOURCE DF SS MS F

----------------------------------------------------------------------------

Between 5 293939.3255 58787.8651 71.1049

Within (Error) 18 14881.9844 826.7769

----------------------------------------------------------------------------

Total 23 308821.3099

----------------------------------------------------------------------------

(p

- value = 0.0000)

Critical F = 4.2479 (alpha = 0.01, df = 5,18)

= 2.7729 (alpha = 0.05, df = 5,18)

Since F > Critical F REJECT Ho: All equal (alpha = 0.05)

**Bonferroni t**

**- Test** 

**- TABLE 1 OF 2**  Ho: Control=Treatment

----------------------------------------------------------------------------

TR ANSFORMED MEAN CALCULATED IN SIG

GROUP IDENTIFICATION MEAN ORIGINAL UNITS t STAT 0.05

----- -------------------- ----------- ------------------ ------ ---

1 control 252.8750 2 52.8750

2 0.089 244.2500 244.2500 0.4242

3 2.4 293.0000 293.0000 1.9735

4 13 264.3125 264.3125 0.5625

5 51 68.6250 68.6250 9.0621 *

6 140 0.8750 0.8750 12.3943 *

---------------------------------------------------------------------------- Bonferroni t critical value = 2.8784 (2 Tailed, alpha = 0.05, df = 5,18)

**Bonferroni t**

**- Test** 

**- TABLE 2 OF 2**  Ho: Control=Treatment

----------------------------------------------------------------------------

NUM OF MIN SIG DIFF % OF DI FFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

----- -------------------- ------- ---------------- ------- ------------

1 control 4

2 0.089 4 58.5243 23.1 8.6250

3 2.4 4 58.5243 23.1 40.1250

4 13 4 58.5243 23.1 11.4375

5 51 4 58.5243 23.1 184.2500

6 140 4 58.5243 23.1 252.0000

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 23

- APPENDIX 4 STATISTICS: CELL GROWTH

**Chi**

**- Square Test for Normality** 

---------------------------------------------------------------------------- Actual and Expected Frequencies

INTERVAL <

- 1.5

- 1.5 to <

- 0.5

- 0.5 to 0.5 >0.5 to 1.5 >1.5

----- ------------- ----------- ----------- -----

EXPECTED 1.6080 5.8080 9.1680 5.8080 1.6080

OBSERVED 0 9 7 8 0

----------------------------------------------------------------------------

Chi

- Square = 6.3102 (p

- value = 0.1771)

Critical Chi

- Square = 13.277 (alpha = 0.01 , df = 4)

= 9.488 (alpha = 0.05 , df = 4)

---------------------------------------------------------------------------- Data PASS normality test (alpha = 0.01). Continue analysis.

**Levene's Test for Homogeneity of Variance**

ANOVA Table

----------------------------------------------------------------------------

SOURCE DF SS MS F

----------------------------------------------------------------------------

Between 5 574732.8750 114946.5750 1.6544

Within (Error) 18 1250664.7500 69481.3750

----------------------------------------------------------------------------

Total 23 1825397.6250

----------------------------------------------------------------------------

(p

- value = 0.1967)

Critical F = 4.2479 (alpha = 0.01, df = 5,18)

= 2.7729 (alpha = 0.05, df = 5,18)

Since F < Critical F FAIL TO REJECT Ho: All equal (alpha = 0.01)

**ANOVA Table**

----------------------------------------------------------------------------

SOURCE DF SS MS F

----------------------------------------------------------------------------

Between 5 122938048.8750 24587609.7750 143.7608

Within (Error) 18 3078564.7500 171031.3750

----------------------------------------------------------------------------

Total 23 126016613.6250

----------------------------------------------------------------------------

(p

- value = 0.0000)

Critical F = 4.2479 (alpha = 0.01, df = 5,18)

= 2.7729 (alpha = 0.05, df = 5,18)

Since F > Critical F REJECT Ho: All equal (alpha = 0.05)

**Bonferroni t**

**- Test** 

**- TABLE 1 OF 2**  Ho: Control=Treatment

----------------------------------------------------------------------------

TRANSFORMED MEAN CALCULATED IN SIG

GROUP IDENTIFICATION MEAN ORIGINAL UNITS t STAT 0.05

----- -------------------- ----------- ------------------ ------ ---

1 control 5332.5000 5332.5000

2 0.089 5259.0000 5259.0000 0.2513

3 2.4 6045.0000 6045.0000 2.4365

4 13 5358.7500 5358.7500 0.0898

5 51 1486.5000 1486.5000 13.1518 *

6 140 120.0000 120.0000 17.8247 *

---------------------------------------------------------------------------- Bonferroni t critical value = 2.8784 (2 Tailed, alpha = 0.05, df = 5,18)

**Bonferroni t**

**- Test** 

**- TABLE 2 OF 2**  Ho: Control=Treatment

----------------------------------------------------------------------------

NUM OF MIN SIG DIFF % OF DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

----- -------------------- ------- ---------------- ------- ------------

1 control 4

2 0.089 4 841.7442 15.8 73 .5000

3 2.4 4 841.7442 15.8 712.5000

4 13 4 841.7442 15.8 26.2500

5 51 4 841.7442 15.8 3846.0000

6 140 4 841.7442 15.8 5212.5000

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 24

- APPENDIX 5 IC- VALUES

Table 11 IC- values for growth rate inhibition

Concentration X Y Slope: 86.0695

(mg/l) Log conc. (mg/l) Inhibition (%) I ntercept:

- 104.4140

0.089 *

- 2.34 Multiple R: 0.9297

0.089 * 3.35 n = number of observations: 12

0.089 *

- 0.93

0.089 * 3.03 Regression line: Y= 86.07X

- 104.41

2.4 *

- 1.82

2.4 *

- 4.46 Prediction of X values based on known Y values

2.4 *

- 3.48

2.4 *

- 0.67 Known Y 

### 10 Xreg 

### 10 X95%

- 

### 10 X95%+

13 1.11

- 4.92 Inhibition (%) (mg/l) (mg/l) (mg/l)

13 1.11 0.06 5 18.67 6.80 51.31

13 1.11 1.11 50 62.24 23.26 166.52

13 1.11 1.23

51 1.71 16.63

51 1.71 24.12

51 1.71 28.28

51 1.71 26.73

140 2.15 76.12

140 2.15 95.97

140 2.15 95.97

140 2.15 95.97

* Not included in IC

- calculations.



### Figure 2 Percentage inhibition of growth rate as function of the log concentration (mg/l)

of Castor Oil, Sulfated, Sodium Salt, 75%

.

Dashed curves represent the 95% confidence limits.

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 25

- APPENDIX 5 IC- VALUES

– continued

–

Table 12 IC- values for yield inhibition

Concentration X Y Slope: 102.6112

(mg/ l) Log conc. (mg/l) Inhibition (%) Intercept:

- 113.9142

0.089 *

- 13.8 Multiple R: 0.9583

0.089 * 17.1 n = number of observations: 12

0.089 *

- 5.2

0.089 * 15.6 Regression line: Y= 102.61X

- 113.91

2.4 *

- 10.5

2.4 *

- 28.0 Predictio n of X values based on known Y values

2.4 *

- 21.2

2.4 *

- 3.7 Known Y 

### 10 Xreg 

### 10 X95%

- 

### 10 X95%+

13 1.114

- 31.3 Inhibition (%) (mg/l) (mg/l) (mg/l)

13 1.114 0.4 5 14.42 6.64 31.31

13 1.114 6.1 50 39.58 18.92 82.78

13 1.114 6.7

51 1.708 60 .5

51 1.708 74.0

51 1.708 79.4

51 1.708 77.6

140 2.146 98.9

140 2.146 99.9

140 2.146 99.9

140 2.146 99.9

* Not included in IC

- calculations.



### Figure 3 Percentage inhibition of yield as function of the log concentration (mg/l) of

Castor Oil, Sulfated, Sodium Salt, 75%

.

Dashed curves represent the 95% confidence limits.

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 26

- APPENDIX 5 IC- VALUES

– continued

–

Table 13 IC- values for cell growth inhibition

Concent ration X Y Slope: 96.7307

(mg/l) Log conc. (mg/l) Inhibition (%) Intercept:

- 103.7140

0.089 *

- 15.9 Multiple R: 0.9752

0.089 * 15.7 n = number of observations: 12

0.089 *

- 1.5

0.089 * 7.3 Regression line: Y= 96.73X

- 103.71

2.4 *

- 11.6

2.4 *

- 19.1 Prediction of X values based on known Y values

2.4 *

- 16.8

2.4 *

- 5.9 Known Y 

### 10 Xreg 

### 10 X95%

- 

### 10 X95%+

13 1.114

- 14.8 Inhibition (%) (mg/l) (mg/l) (mg/l)

13 1.114 2.4 5 13.30 7.336 24.12

13 1.114 5.1 50 38.82 22.12 68. 13

13 1.114 5.3

51 1.708 65.1

51 1.708 72.7

51 1.708 75.1

51 1.708 75.5

140 2.146 96.8

140 2.146 98.5

140 2.146 97.9

140 2.146 97.8

* Not included in IC

- calculations.



### Figure 4 Percentage inhibition of cell growth as function of the log concentration (mg/l)

of Castor Oil, Sulfated, Sodium Salt, 75%

.

Dashed curves represent the 95% confidence limits.

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 27

- APPENDIX 6 REFERENCE TEST

Pseudokirchneriella s ubcapitata , strain: NIVA CHL

- 1. Fresh water algal growth inhibition test with

potassium dichromate (Project 504048).

Start of first exposure: 21 October 2013

Completion last exposure: 24 October 2013

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

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

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

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

Algae were exposed for a period of 72 hours to K 2 

### Cr 2 

### O 7 

### (Potassium dichromate) concentrations of

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

### cells/ml.

Overview of % inhibition of growth rate in the reference test:

Nominal conc.

K 2 Cr 2 O 7 (mg/l) Mean Std. Dev. n %Inhibition

c ontrol 1.491 0.0439 3 0.0

0.18 1.446 0.0460 3 3.0

0.32 1.390 0.0389 3 6.8

0.56 1.165 0.0064 3 21.9

1.0 0.797 0.0385 3 46.6

1.8 0.491 0.0076 3 67. 1

3.2 0.421 0.0131 3 71.7



### Overview of % inhibition of yield in the reference test:

No minal conc.

K 2 Cr 2 O 7 ( mg/l ) Mean Std. Dev. n % Inhibition

c ontrol 87.218 11.4001 3 0.0

0.18 76.057 10.7056 3 12.8

0.32 64.098 7.7032 3 26.5

0.56 31.927 0 .6330 3 63.4

1.0 9.962 1.2942 3 88.6

1.8 3.358 0.0995 3 96.1

3.2 2.542 0.1387 3 97.1



### Potassium dichromate inhibited growth rate of this fresh water algae species at nominal

concentrations of 0.18 mg/l and higher.

The EC 50 

### for growth rate inhibition (72h -E R 

### C 50 

### ) wa s 1.3 mg/l with a 95% confidence interval ranging

from 1.1 to 1.4 mg/l. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/l.

Hence, the 72h -E R 

### C 50 

### for the algal culture tested corresponds with this range.

The EC 50 

### for yield inhibition (72h -E Y 

### C 50 

### ) was 0.46 mg/l with a 95% confidence interval ranging from

0.42 to 0.50 mg/l. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the

72h -E Y 

### C 50 

### for the algal culture tested corresponds with this range.

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

described above was performed under GLP conditions with a QA

- check.

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503338

- Page 28

- APPENDIX 7 CERTIFICATE OF ANALYSIS