Final Research Report 

ECRA F07012 T 05012 ODA April19, 2007 

M.G.J. Geurts, J. van Dam and L. Lofgren 

ACUTE TOXICITY OF 

WATER FLEAS, DAPHNIA MAGNA 

ICS-103 CONFIDENTIAL 

Project identification 

Client 

Subclient 

Client No 

Subclient No 

Technology 

& Engineer ing 

Page 1 of 26 

Project No 

Task No 859.024 

Drafted by department Environmental Chemistry and Regulatory Affairs 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

Apri119, 2007 

ABSTRACT 

The acute toxicity of 

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to Daphnia magna (water flea) was tested under static 

conditions for 48 hours in accordance with OECD and EEC guidelines for testing chemicals. The test 

was performed in compliance with Good Laboratory Practice (GLP). 

The 48 h EC50 was calculated to be 1.21 mg/L with 95% confidence limits of 1.06 and 1.38 mg/L. The 

highest test concentration causing no immobility (no observed effect concentration, NOEC) after 48 

hours was observed at 0.8 mg/L. These results are calculated based on the test substance as received. 

According to the CoA the test substance contains 82% trimethylalkyl amine ammonium salt. After 

correction to 100% trimethylalkyl amine ammonium salt (active ingredient) the following effect data are 

obtained: 48 h EC50 = 1 mg/L and a NOEC of 0.66 mg/L. 

Sub-lethal effects, such as floating at the surface, were not observed in the test concentrations where 

no immobility was observed. 

The results of the chemical analyses show that the initial concentrations in the test solutions were 

close to the nominal (expected) concentrations. A decrease of< 6% of test substance concentration 

during the 48 hours of testing was observed. The effects observed are therefore expressed based on 

their nominal concentrations. 
;;; 

;,. 

ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19, 2007 

ACUTE TOXICITY OF 

Sponsors 

Study monitor 

STUDY ORGANIZATION 

Location 

Study director 

Quality Assurance Unit 

Management 

Experimental initiation date 

Experimental completion date 

ARCHIVING AND STORAGE 

DAPHNIA MAGNA 

Akzo Nobel Surface Chemistry AB 

Se 44485 Stenungsund 

Sweden 

-

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& Engineering 

Environmental Chemistry and Regulatory Affairs Dept. 

lng. H. van Daalen 

lr. A.R. Luttmer 

29-01-2007 

23-02-2007 

The project file including the final report, amendments to the final report, the study plan, amendments 

to the study plan, records of quality assurance inspections, all letters, memos and notes and raw data 

pertaining to the study will be retained in the archives of··············· 

- for a period of ten years. Other records including master schedule sheet, laboratory 

notebooks, logbooks, records of the maintenance and calibration of equipment, summary of training, 

curricula vitae and job descriptions of the personnel involved in the study, records related to location 

and storage of the test substance will also be kept in the 

••• archives for a period of ten years. Test material will be stored deep-frozen under the sample 

code T 05012 for ten years or only as long as the quality of the test substance permits evaluation. 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19, 2007 

GLP COMPLIANCE STATEMENT 

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The study was conducted in compliance with the following Good Laboratory Practice regulations: 

• OECD Principles on Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98) 17. 

• Commission Directive 1999/11/EC of 8 March 1999 adapting to technical progress the Principles 

of Good Laboratory Practice as specified in Council Directive 87/18/EEC on the harmonisation of 

laws. regulations and administrative provisions relating to the application of the Principles of 

Good Laboratory Practice and the verification of their applications for tests on chemical 

substances (O.J. No. L 77 of 23.3.1999). 

• Staatstoezicht op de Volksgezondheid. Voedsel en Waren Autoriteit. GLP Monitoring Unit. 

The report contains an accurate description of the results. The test substance data were submitted by 

the sponsor (unless stated otherwise), who accepted full responsibility for the validity of these data. The 

preliminary test was performed under identical conditions and in the same laboratory as the definitive 

test but was not performed under GLP. The chemical analyses were due to the complexity of the 

analytical method performed outside the GLP facility and therefore no GLP is claimed for this part of the 

results. This has however no impact on the quality of the chemical analyses and the final outcome of the 

test. 

Study director 

lng···· 

Management 

lr. A.R. Luttmer 

date 2~ ' 0{' - 2eD 7 

signature ~--

date 

signature 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19, 2007 

QUALITY ASSURANCE STATEMENT 

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This report was audited by the Quality Assurance Unit contracted by 

••••••• It is considered to be an accurate presentation of the methods and procedures 

applied in the course of the study and an accurate reproduction of the data recorded. 

Listed below are the dates of inspection of this study by the Quality Assurance Unit and the dates on 

which its findings were reported to Study Director and Management. 

Phase ofthe study 

Study plan 

Test (preparation of the test substance stock solution) 

Report 

Quality Assurance Unit 

lng. H. van Oaalen 

Dates of inspection 

30-01-2007 

30-01-2007 

04-04-2007 

Dates of reporting 

30-01-2007 

30-01-2007 

04-04-2007 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19, 2007 

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CONTENTS 

1. INTRODUCTION 7 

2. TEST GUIDELINES, MODIFICATIONS AND DEVIATIONS 7 

3. MATERIALS 8 

3.1 Test substance 8 

3.2 Chemicals 8 

3.3 De-ionized water 8 

3.4 Test vessels 8 

3.5 Test room and light regime 9 

3.6 Test animals 9 

4. METHODS 9 

4.1 Test principle and procedures 9 

4.2 Prepara1tion of the test medium 9 

4.3 Preparation of the stock solution 10 

4.4 Preparation of the test solutions 10 

4.5 Chemical analysis 10 

4.6 Determination of dissolved oxygen, pH, conductivity, water hardness and temperature in 

the test solutions 10 

4. 7 Evaluation of data 11 

5. RESULTS 11 

5.1 Toxicity 11 

5.2 Chemical analysis 11 

5.3 Oxygen, pH, conductivity, water hardness and temperature 12 

6. QUALITY CRITERIA 12 

7. AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN 

7.1 Amendments to the study plan 

7.2 Deviations form the study plan 

8. REFERENCES 

ANNEX 1 

ANNEX 2 

ANNEX3 

12 

12 

12 

13 

17 

18 

24 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19. 2007 

1. INTRODUCTION 

The purpose of this study was to determine the acute toxicity of 

magna (water flea) under continuous exposure during 48 hours in a static test. 

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to Daphnia 

Acute toxicity is the discernible adverse effect induced in an organism within a short time of exposure 

to a substance. 

The acute toxicity of a test substance is expressed as the median effective concentration (EC50), 

which is the concentration in water, which immobilizes 50% of the test animals within a period of 

continuous exposure. Those animals, which were not able to swim for 15 seconds after gentle 

agitation of the test vessel, were considered to be immobile .. 

Other endpoints that. if possible. were derived from the results are the highest tested concentration 

causing no immobilization (NOEC), the lowest tested concentration with significant immobility as 

compared to the control (LOEC) and the lowest concentration causing 100% (EC100) immobility. The 

swimming ability and other sub-lethal effects, such as being trapped at the surface, were recorded 

after 24 and 48 hours, if observed. 

2. TEST GUIDELINES, MODIFICATIONS AND DEVIATIONS 

The study was carried out in accordance with OECD (8.1) and EEC (8.2) guidelines for testing of 

chemicals with one modification of the test guideline: 

o The chemical analyses is not performed under GLP conditions 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19, 2007 

3. MATERIALS 

3.1 Test substance 

A sample 

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(project sample code T 05012) was received in June 2005. The 

following data on the test substance were submitted by the sponsor, who accepted full responsibility 

for the validity thereof. 

Test substance name 

Batch/Lot no. 

Purity 

Appearance 

Water solubility 

Octanol-water partition coefficient 

Vapour pressure 

Stability 

Storage until required 

6506001437 

82% 

Brown viscous liquid 

Soluble 

Not available 

A 

has a very low vapour pressure 

Assumed to be stable under test conditions 

At room temperature in the dark 

For additional information on the compound see Annex 1. 

3.2 Chemicals 

and 

All reagents used were of reagent grade quality and obtained from J.T. Baker Chemicals BV, 

Deventer, the Netherlands, ACROS chemicals (formally known as Janssen Chimica), Geel, Belgium 

and Fluka Chemie, Buchs, Switzerland. 

3.3 De-ionized water 

The de-ionized water used contained not more than 0.01 mg/L of copper, had a Non purgable organic 

carbon (NPOC) -content of not more than 2.0 mg/L and a conductivity of less than 5 JlS/cm. This water 

was produced from tap water in a water purification system according to Standard Operation 

Procedure K 10 (8.8). 

3.4 Test vessels 

As test vessels 50 ml glass beakers were used, containing 50 ml of test solution. They were covered by 

glass plates during the test. 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19, 2007 

3.5 Test room and light regime 

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The test was carried out in a temperature-controlled room. The test temperature was between 18 and 

22oc and the actual temperature was kept constant within ± 1 oc. The light regime was 16 h of ambient 

light per day, provided by fluorescent tubes. 

3.6 Test animals 

The test was performed with Daphnia magna Clone 5 (water fleas), which was obtained from NOTOX 

at 's Hertogenbosch according to Standard Operation Procedure E 1 (8.3). The animals used in the 

test were less than 24 hours old at the beginning of the test and were obtained from parent animals 

having an age of 2-4 weeks. 

4. METHODS 

4.1 Test principle and procedures 

The test was performed as a static test The total duration was 48 hours. 20 daphnids divided into 4 

groups of 5 animals were used at the test concentration and control. 

The daphnids were exposed to the chosen concentrations of the test substance as described below 

and immobility and sub-lethal effects were recorded at approximately 24 and 48 hours. The daphnids 

were considered immobile when they were not able to swim for 15 seconds after gentle agitation of 

the test vessel. In addition to immobility, sub-lethal effects such as floating at the surface were 

recorded. 

The daphnids were randomly placed in the test solutions and the test vessels were positioned in a 

random manner. During the test the vessels are covered with glass plates. No aeration of the test 

substance took place during the test period. The animals were not fed during the test. 

4.2 Preparation of the test medium 

The test medium, so called Dutch Standard Water (DSW), was used, having a pH of approximately 8.2, 

and a conductivity between 550 and 650 1-JS/cm, containing per liter of de-ionized water: 200 mg of 

CaCI2·2H20, 180 mg of MgS04·7H20, 100 mg of NaHC03 and 20 mg of KHC03 (Standard Operation 

Procedure01 (8.12)). 

The test medium was aerated before being used in the test. The air was water-saturated and purified by 

an active coal and cotton filter according to Standard Operating Procedure K 12 (8.9). Before the start 

of the test the pH of the DSW was measured to be 8.6 and adjusted to 8.2 using hydrochloric acid. 
JCS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19, 2007 

4.3 Preparation of the stock solution 

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The test substance is soluble in water. A test substance solution of 1.004 g/L was prepared by loading 

0.1004 g of test substance, weighed out on an analytical balance (Standard Operation Procedure K 4 

(8.6)) in a 100 ml volumetric flask. The flask was filled up to the mark with test medium. The stock 

solution was manually shaken until a homogenous solution was obtained. The pH of the stock solution 

was checked and found to be 8.2 and not adjusted. 

4.4 Preparation of the test solutions 

The test solutions were prepared by addition of the adequate amounts of stock solution to the test 

vessels. Based on the results of the preliminary test the following final dilutions were prepared: 0.2-

0.4 - 0.8 - 1.6 and 3.22 mg/L of the test substance. Controls containing only test medium were 

included in the test. 

4.5 Chemical analysis 

At the start of the test, a sample of 50 ml was taken from the lowest, middle and highest test 

concentration and the control solution just before the distribution of the test concentration and the 

control solution over the test vessels and adding the test animals. The sample was transferred into a 

preweighed 100 ml glass bottle and weighed. Then 2.5 ml of methanol was added and the bottle was 

again weighed. 

For the samples at the end of the test, the solutions with the same test concentration were pooled in a 

beaker and 50 ml of sample was transferred without filtration into a preweighed 100 ml glass bottle. 

After adding lhe sample the weight of the bottle was recorded and 2.5 ml of methanol was added and 

again the weight was recorded. This was also done for the control. The sample bottles were stored in 

a refrigerator prior to transport for chemical analysis. The amount of Ethoquad MC 1214/25 present in 

the test concentrations was quantified outside the GLP facility. 

Chemical analyses were performed according to the analytical procedure described in Annex 2. 

4.6 Determination of dissolved oxygen, pH, conductivity, water hardness and temperature 

in the test solutions 

The dissolved oxygen and pH of all test concentrations and controls were measured at the beginning 

and at the end of the test. Conductivity were measured only at the beginning of the test in the control 

solution whether its values are within limits. The temperature was measured continuously in an 

additional test vessel filled with DSW and kept under identical conditions as the actual test vessels. 

The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode 

and meter according to Standard Operation Procedure K 2 (8.5). The pH was measured using a 

microcomputer pH-meter according to Standard Operation Procedure K 1 (8.4). The conductivity was 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19,2007 

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measured using a portable conductivity meter according to Standard Operation Procedure K 24 (8.10). 

The temperature was measured with a thermocouple according to Standard Operation Procedure K 6 

(8.7). 

4.7 Evaluation of data 

If possible, the EC50 was calculated with the computer program TOXCALCTM version 5.0 according to 

Standard Operation Procedure l 2 (8.11 ), using the trimmed Spearman-Karber method (8.13). The 

concentrations causing zero and 100% immobility was. if possible, derived directly from the test 

observations. 

5. RESULTS 

The results presented in this chapter are based on the performed definitive test. 

5.1 Toxicity 

Preliminary test 

During the preliminary range finding test, which was performed as a non-GLP test, effects were 

observed which lead to the conclusion that an EC50 for the test substance should be found close to 1 

mg/L. 

Definitive test 

The results of the test on mobility are summarized Table 1. The results are reported based on nominal 

concentrations. 

The 48 h EC50 - was calculated to be 1.21 mg/L with 95% confidence limits of 1.06 and 1.38 mg/L. 

The highest test concentration where no effect were observed was 0.8 mg/L. These results are 

calculated based on the test substance as received. According to the CoA the test substance contains 

82% trimethylalkyl amine ammonium salt. After correction to 100% trimethylalkyl amine ammonium salt 

(active ingredient) the following effect data are obtained: 48 h EC50 = 1 mg/L and a NOEC of 0.66 mg/L. 

The dose-effect curve presenting the percent immobilization against the concentration after 48 hours 

of exposure is presented in figure 1. 

5.2 Chemical analysis 

The results of the chemical analyses are presented in table 4. Due to the complexity of the method of 

analyses, the analyses were performed outside the GLP facility. Details on the method applied are 

described in Annex 2 of this report. The sample weights which were used to compensate for dilution of 

the samples by methanol are presented in Table 5. 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19,2007 

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The results of the chemical analyses show that no test substance was detected in the control samples; 

the result also show that the concentration of the test compound during the test remained within 80% 

of the concentration at the beginning of the test. Because the test substance was also used as the 

standard for chemical analyses, the results as presented are calculated based on the test substance as 

received. Based on these arguments the effects observed are expressed based on nominal 

concentrations. 

5.3 Oxygen, pH, conductivity, water hardness and temperature 

The maximum variation in oxygen concentration during the test was 0.3 mg/L. The oxygen 

concentration during the test ranged from 8.9 to 9.2 mg L and the values observed are summarized 

in table 2. 

The initial pH value observed of DSW was 8.2. The maximum variation in pH during the test was 0.4 

units. The pH observed during the test ranged from 8.2 to 7.8 units and the values observed are 

summarized in table 3. The conductivity of DSW was 590 ~s/cm. 

The temperature in the test vessels during the test period ranged from 20.25 to 20.40°C. 

6. QUALITY CRITERIA 

The following quality criteria have been met in the present study: 

• Immobilization in the control did not exceed 10% at the end of the test. 

• In accordance with OECD guideline 202 (8.1) the oxygen concentration was not lower than 3 mg/L 

at the end of the test. 

• There were no test animals trapped at the surface of the water in the control group. 

• In accordance with OECD guideline 202 (8.1) and EEC guideline C2 (8.2), the chemical analyses 

carried out showed that the concentration of the test compound during the test remained within 

80% of the concentration at the beginning of the test. 

7. AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN 

7.1 Amendments to the study plan 

There were no amendments to the study plan 

7.2 Deviations form the study plan 

There were no deviations to the study plan 
ICS-103 Final Research R·eport 

ECRA F07012 T05012 ODA 

April 19,2007 

8. REFERENCES 

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8.1 OECD, 2004. Daphnia sp., Acute immobilisation test. Guidelines for testing of chemicals nr. 202, 

13 April2004. Organization for Economic Co-operation and Development. Paris. 

8.2 Directive 67/548/EEC, Annex V, part C: Methods for the determination of ecotoxicity Part C.2. 

Acute toxicity to Daphnia magna (updated version 11 /1989). 

8.3 Standard Operation Procedure E 1: Culturing of Daphnia magna 

8.4 Standard Operation Procedure K 1: pH-meter 

8.5 Standard Operation Procedure K 2: Oxygen meters 

8.6 Standard Operation Procedure K 4: Balansen 

8. 7 Standard Operation Procedure K 6: Thermometers 

8 .. 8 Standard Operation Procedure K 10: Deionizer 

8.9 Standard Operation Procedure K 12: Luchtfilters 

8.10 Standard Operation Procedure K 24: Geleidbaarheidsmeter 

8.11 Standard Operation Procedure L 2: Acute toxicity test of fish and Daphnia magna (calculation 

of effects with the TOXCALCTM computer program) 

8. 12 Standard Operation Procedure 0 1: Preparation method for culture and test media 

8.13 M.A. Hamilton, P.C. Russo and R.V. Thurston, 1977: 'Trimmed Spearman-Karber method for 

estimating median lethal concentrations in toxicity bioassays'. Env.Sci. & Techno!. 11, 714-

719. Correction, 12 (1978) 417 

8.14 C.E. Stephan, 1977: 'Methods for calculating an LC50'. In: Aquatic toxicology and hazard 

evaluation, F .L. Mayer and J.L. Hamelink (Eds.), 65-84 

8.15 OECD Guideline no. 1 (1998), Directives 88/320/EEC and 1999/12/EC. 
ICS-103 Final Research R,eport 

ECRA F07012 T05012 ODA 

April19, 2007 

Table 1: Test results 

Nominal test 

substance Batches 

concentration 

(mg/L) 

Batch I 

Batch II 

0 (control) Batch Ill 

Batch IV 

Total 

Batch I 

Batch II 

0.2 Batch Ill 

Batch IV 

Total 

Batch I 

Batch II 

0.4 Batch Ill 

Batch IV 

Total 

!Batch I 

Batch II 

0.8 Batch Ill 

Batch IV 

Total 

Batch I 

Batch II 

1.6 Batch Ill 

Batch IV 

Total 

Batch I 

Batch II 

3.2 Batch Ill 

Batch IV 

Total 

I 0 hours 

5 

5 

5 

5 

20 

5 

5 

5 

5 

20 

5 

5 

5 

5 

20 

5 

5 

5 

5 

20 

5 

5 

5 

5 

20 

5 

5 

5 

5 

20 

Technology 

& Engineering 

Number of mobile animals 

24 hours 48 hours 

5 5 

5 5 

5 5 

5 5 

20 20 

5 5 

5 5 

5 5 

5 5 

20 20 

5 5 

5 5 

5 5 

5 5 

20 20 

5 5 

5 5 

5 4 

5 5 

20 19 

5 2 

5 0 

5 1 

5 0 

20 3 

5 0 

5 0 

5 0 

5 0 

20 0 
.. 

" 

ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19, 2007 

1.0 

0.9 

0.8 

0.7 

Q) 0.6 fl) 

r:: &. 0.5 fl) 

~ 0.4 

0.3 

02 

0.1 

0.0 

0.1 1 

Dose mg/L 

10 

Figure 1. Dose-response curve at 48h exposure toTable 2: Oxygen measurements (mg/1) 

nominal test 

substance 0 hours 48 hours 

concentration 

(mq/l) 

0 9.1 9.0 

0.2 9.2 8.9 

0.4 9.2 9.0 

0.8 9.1 9.0 

1.6 9.1 9.0 

3.2 9.1 8.9 

Table 3: pH measurements 

1 Nominal test 

substance 0 hours 48 hours concentration 

(mg/l) 

0 8.0 7.7 

0.2 8.1 7.8 

0.4 8.1 7.8 

0.8 8.2 7.8 

1.6 8.2 7.8 

3.2 8.2 7.8 

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Technology 

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ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19. 2007 

Table 4: The results of the chemical analyses 

Sample code I Test number 

T 05012 ODA Control t=Oh 

T 05012 ODA Control t=48h < 0.1 

T 05012 ODA 0.2 mg/L t=Oh 0.17 

T 05012 ODA 0.2 mg/L t=48h 0.17 

T 05012 ODA 0.8 mg/L f;;Oh 0.76 

T 05012 ODA 0.8 mg/L t=48h 0.79 

T 05012 ODA 3.2 mg/L t=Oh 3.1 

T 05012 ODA 3.2 mg/L t=48h 3.3 

< 0.1 

0.17 

0.15 

0.78 

0.80 

3.3 

3.3 

All Control samples were below detection limit which is 0.02 mg/L. 

Table 5: Sample weight 

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<0.1 

0.17 

5.9 

0.16 

0.77 

-3.2 

0.795 

3.2 -3.1 

3.3 

.:_:~.-~:;.~ ~~ :~'. ;~t~~~ .. ~~~;~ ?~0\::.,.·::~:~:~,:: ~:·;: ~~~;r-r~:f:·'~i Tn~~r~:~::J(;_(;_:·:: ~;~:~:·~ :r:;_~~~;::::i,:: .. :}.· ~~: :·,:; !~: · .. ~:·,:'::: .. :: =:~·.::~:~::; -~: . 

T05012 ODA Contol t=Oh 30-1-2007 85.06 134.71 136.72 

T05012 ODA Control t=48h 1-2-2007 84.99 134.86 136.83 

T05012 ODA 0.2 mQ/L t=Oh 30-1-2007 85.55 134.72 136.79 

T05012 ODA 0.2 mg!L t=48h 1-2-2007 85.4 135.33 137.34 

T05012 ODA 0.8 mg/L t;;Oh 30-1-2007 85.35 134.48 136.57 

T05012 ODA 0.8 mg/L t=48h 1-2-2007 84.87 134.74 136.74 

T05012 ODA 3.2 mq/L t=Oh 30-1 -2007 85.02 134.22 136.28 

T05012 ODA 3.2 mg/L t=48h 1-2-2007 85.02 134.81 136.79 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19, 2007 

PEG 1000-1500 

F,...11mine 

Alcottol ethoX)'Iate 

F ally amine ethoxylate 

-

Certificate of Analyze 

Page 17 

Technology 

& Engineering 

ANNEX 1 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19, 2007 

Method of analyses 

Surfactant• - Analytical Methods for R&D AM46 

Re¥ised by (pc>Yian. ~hn) 

Analytical Manager 

Ajlp'owed by {position. siglgture) 

QHSE Manager 

o- IOno 

2007..02-01 AM46-2309 

S'-Sanhfetene• Proc:4Ks 

1509001 

s.ction End_ 

3 

Pa~ 

1 of 6 

Validunli 

2010..02..01 

No. 309 trace levels in water 

Procedure 

Sign 

Usa Lofgren 

Purpose/Background 

Remarlt 

literature 

Principle 

Safety 

Apparatus 

The method is used for the determination o at low levels 

in OECO water (freshwater algae medium) and OSW water (daphnia test 

medium). 

ANL07002 

The samples are analyzed rot 1 OECD water (freshwater 

algae medium) and OSW water (daphnia test medium). 

The samples are analyzed by revened phase liquid c:hromatography and mas. 

spectrometric detection. The mass spectrometer is run in selective ion monitoring 

(SJM) with eleclroapray ionization (ESt) in positive mode. Extemal standard 

calibration is used. 

Acetonitri and methanol are highly flammable. They are harmful by inhalation, In 

contact with sltin and if swalowed. They are ifrit111ing to eyes. See safety data 

sheebl 5-3072 and Safewin 40247. Acetic acid and can cause severe bums. See 

safety data sheet Safewin 40293. 

Wear suitable protective gloves and handle in fume hood. 

Liquid Chromatograph-Mass spectrometer (LCQ Duo, Thermofinnigan), lon trap, 

ESI..sourca 

Pipette (Eppendotf Multiplpette Pro) 

Standard laboratory glassware. 

Page 18 

Techno logy 

& Engineering 

ANNEX2 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19, 2007 

Rw!Hd by (po~ signalln) 

Analytical Manager 

Aflprowd by (position. signa(ure) 

QHSE Man119!'r 

LCMS data 

Column: 

Column temp: 

Surfactanta - Analytical Methods for R&D AM-46 

o-. IOno 

2007-02-01 AM46-2309 

Standov ohference Process 

1509001 

$ed;on End. 

3 

HyPURITY C18 {5 urn, 2.1'50 mm (Thermo)) 

40"C 

p. 

2of6 

V:alid..mil 

2010-02-01 

Mobile phase: A: acetonitrile/water 5:95 (vfv) + 0.5 % acetic acid 

B: acetonitril + 0.5 ')(, acetic acid 

Gradient 

Flow: 

Pump: 

0.2 mllmin 

Gradient 

Time (min) 

0.0 

20 

25 

27 

40 

A(%) 

100 

0 

0 

100 

100 

B (%) 

0 

100 

100 

0 

0 

Detector: 

Injection: 

Mass spectrometer LCQ Duo, ESJ-soorce, + mode 

5JJI 

Detector parameters 

Man spectrometer: 

MS SIM parameters: ions 522, 566,610, 6S4, 698, 742, 786, 830, 874, 918 

~ 

Mode: + 

Sheath gas now (arb): 40 

Aux gas floW (arb): 5 

Spray vottaga (kV): 4.5 

Capilary temp ("C): 200 

Cap~lary voltage (V): 25 

Tube lens otflet {V): 0 

Cbemicals/reagents 

Acetonitrile, pa 

Deionized and distilled water 

Methanol pa 

2-i>f'Opanol pa 

Acetic acid (1•001ii%iii) lpaill•••• Reference of• 1 

Page 19 

Technology 

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ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19,2007 

Rftised by (poUion. oignalln) 

Analytical Manager 

Appn)ved by (position, s~re) 

QHSE Manager 

Calibration 

Surfactants -Analytical Methods for R&D A~6 

Ou IOno 

2007-02-01 AM46-2309 

s~~rence f'loocess 

1509001 

s.ctian End. 

3 

Issue 

1 

Pa~ 

3 of6 

Valid umil 

2010..02-01 

Prepare calibration solutions at five levels in a concentration range close to the expected 

concentrations in the samples. 

Prepare the calibnltion solutiona in the same matrix as for the samples. 

Sample preparation 

The sample is refrigerated and 5% (v/V) ot methanol is added. The temperature of the 

sample is adjusted to room temperature prior to analysis. 

Procedure 

The samples and calibration solutions are injected Into the LCMS. 

Calculation 

A linear ealibration graph is constructed from the calibration samples, from which the 

amount of Ethoquad MC1214125 can be calculated. XCalibur software for LCO Duo is 

used for the calculations. 

Precision and accuracy 

Six repetitive injections of one control sample in OECD water containing 0.95 mg/l of 

Ethoqued gave an RSO of 1.8 %. 

Six repetitive Injections of one control •ample in OSW containing 0.95 moll of 

Ethoquad gave an RSO of 3.7 % . 

Six repetitive injections of one standard solution in OECD water containing 0.21 mgJl 

of Ethoquad gave an RSD of 2.9 %. 

Six repetitive Injections of one standard solution in OSW cont.Ullng 0.21 mg/1.. of 

Ethoqued gave an RSO of 1.8 %. 

Six repetitive Injections of one standard solution in OECO water containing 10.6 mgJL 

of Ethoqued gave an RSO of 3.5 % . 

Limit of quantitation 

The lower limit of quantitation is 0 .1 mg/1. 

The detection limit is 0.02 mg/1. 

Linearity of calibration curve 

The calbration curve is linear within the range of 0 .1-2 mg/1 and in the range of 1-100 

mgJI. 

Page 20 

Technology 

& Engineering 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19, 2007 

Surfactant. -Analytical Methods for R&D AM46 

R@Vised by (posilion. signature) 

Analytic:al Manager 

~d by (position. sigN!ure) 

QHSE Manager 

Reporting 

D• IOno 

2007-02..01 AM46-2309 

Sbnd ... ...,..,oce Process 

1509001 

The result is reported os amount ort•••• 

numbers. 

Others 

Section End. 

3 

Pa~ 

4 of6 

V<llid.nil 

2010..02..01 

in mg/1 with two significant 

The samples are considered to be stable when refrioeraiitediii, iiWll.thiiilhl eiiiaiddi iitio·i niio.f 5% 

methanol, for at least 3 months at the level of 1 mgJI of 

~--- "" ._ .. ~ .... ... a. 

!. 

~ ' .. i .... ., • 

J 

j 

0: 

I 

I 

I 

I 

• 

... 

... 

"" '"- TC ... 

-.. 

F"og I.To~l Full sun. ion current positive (TIC) mode chromatogram and mass spectrum ~orl········ 

Page 21 

Technology 

& Engineering 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19, 2007 

Surroctants -Analytical Methods for R&D AM"6 

R"iHd by (IJ'O'ilicn. signatu~) 

Analytical Manager 

Approve<l by (position, signature) 

QHSE Manager 

AT. O.Oli•2S,OOI 

I 

I 

I 

Dabt 

2007-02-01 

StandanRfenMIC& 

1509001 

t S,\i) t.H,. t1~ t2.,~ " 

•• 

c:e.ttS17~t4 •5o-11 RT 1U1-14.7't AV 0 tt.. 1,t1£e 

IDno 

AM46-2309 

~s 

11..~)11£> 

Section 

3 

End. 

ls.sue 

1 

Page 

5of6 

Valid unti 

2010-02..01 

UCF! • t£,&a !M .... i22,-C0-52)J)O, 

51& 00-617,011. 110,.00411.00. 1'-4,00-IS"OO~ 

-..oo.en.ao.742,oo.l43.00.: , •• D!).ll1.00j 

no_oNSt,OQ; 174,.064npcl. 011-,00.0,._00t 

wa -..ot17e14 

'f • • c UISJWw•r m ,I)0.6:n.OO. 586..<&6111.oo. t1~GO.en !O t6..e ~s..oa et.e.ooeo~.oo. l•Z.00.1CS..OO; IM,f4.1t'1.oo. a)O,OG-&:tt ~ 

I 

I 

j 

! • ; 

,., 

...... ...... 

.... Fog 2. SIM chromatogram and mau sp.adrum rort•••••••••l 0.11 mg/L 

Y=2.19082e+007+8.58756e+OQ8•X RA2 "'0.9952 W: Equal 

200000000 

1150000000 

<100000000 

50000000 

0.0 0.5 1.0 1.5 2.0 2.5 

ughnl 

F'IQ 3. Calibration graph ,., ......... COf\C.range 0.1-2.1 mg/L 

Page 22 

Technology 

& Engineering 
·• 

"' 

ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19,2007 

RIMHcl by (~ signatl.n) 

Analytical Manager 

Approved by {positicn. signatln) 

QHSE Manager 

Surfactant. -Analytical Methods for R&D AM46 

OJte 10 no 

2007-02-01 AM46-2309 

Stlndanhfetence Process 

1509001 

s.ction End. 

3 

Issue 

1 

Y = 2.84809e+009+323232e+008"X R"2 = 0.9929 W. Equal 

4000000000 

3000000000 

«< 

~ 2000000000 

100000000 

0 50 

UO/Illl 

1 0 

F"tg 4. Calibration graph for Ethoquad MC1214/25: canc.range 1-100 mg/L 

Pa~ 

6of6 

Valid unlil 

2010-02-01 

Page 23 

Technology 

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ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19,2007 

Technology 

& Engineering 

Annex 3 

Determination of---in water from Toxicity studies; 

Study plans T 05012 AL; T 05012 ODA; T 05012 OFA 

Introduction: 

24 water samples from toxicity studies: 

• T 05012 AL "Effects 

pseudokirchnieriella su"'~""'nit<>t<>" 

• T 05012 ODA "Acute toxicity 

• T 05012 OFA "Acute toxicity of 

were received from Marc Geurts for the analysis 

2309. 

Experimental: 

24 samples were received on 21-2-2007. See Table 1. 

Table 1 

Sample code I Test number Sample date 

T 05012 AL Control t=Oh 29-1-2007 

T 05012 AL Control t=72h 1-2-2007 

T 05012 AL 0.0625 mg/L t=Oh 29-1-2007 

T 05012 AL 0.0625 mg/L t=72h 1-2-2007 

T 05012 AL 0.25 mg/L t=Oh 29-1-2007 

T 05012 AL 0.25 mg/L t=72h 1-2-2007 

T 05012 AL 1.0 mg/L t=Oh 29-1-2007 

T 05012 AL 1.0 mg/L t=72h 1-2-2007 

T 05012 ODA Control t=Oh 30-1-2007 

T 05012 ODA Control t=48h 1-2-2007 

T 05012 ODA 0.2 mg/L t=Oh 30-1-2007 

T 05012 ODA 0.2 mg/L t=48h 1-2-2007 

T 05012 ODA 0.8 mg/L t=Oh 30-1-2007 

T 05012 ODA 0.8 mg/L t=48h 1-2-2007 

T 05012 ODA 3.2 mg/L t=Oh 30-1-2007 

T 05012 ODA 3.2 mg/L t=48h 1-2-2007 

T 05012 OFA Control t=Oh 12-2-2007 

T 05012 OFA Control t=96h 16-2-2007 

T 05012 OFA 6.25 mg/L t=Oh 12-2-2007 

T 05012 OFA 6.25 mg/L t=96h 16-2-2007 

T 05012 OFA 25 mg/L t=Oh 12-2-2007 

T 05012 OFA 25 mg/L t=96h 16-2-2007 

T 05012 OFA 100 mg/L t=Oh 12-2-2007 

T 05012 OFA 100 mg/L t=96h 16-2-2007 

n the growth of the freshwater green alga 

water fleas, daphnia magna" 

danio rerio" 

method AM46-

Sample storage: The samples were refrigerated prior to analysis. 

Date of analysis: 27-2-2007 to 5-3-2007. 

Laboratory: 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April19, 2007 

Shipment address: 

Page25 

Technology 

& Engineering 

Head of Laboratory: -

Equipment: See method AM46-2309 

Calibration samples: prepared from the reference labeled T05012 

sub-sample 20-02-2007 JD. The reference was 

sent together with the samples. 

Procedure: See method AM46-2309 

Prior to analysis by LCMS, each of the refrigerated samples was briefly 

mixed and 1.5 ml aliquots were transferred to a HPLC vial and stored at 

room temperature in the auto sampler. All samples were injected within 

24 hours. 

Deviations from method AM46-2309: 

The limit of quantification was defined as 0.1 mg/L for the method. 

Therefore samples T 05012 AL 0.0625 mg/L t=Oh and T 05012 AL 

0.0625 mg/L t=72h, were concentrated five times by evaporation at 30°C 

under a gentle stream of nitrogen. The samples were reconstituted in 

methanoi/OECD water (5/95) (v/v) before analysis. 

Observations: In samples with sample codes: 

T 05012 AL control t=72'h 

T 05012 AL 0.0625 mg/L t=72h 

T 05012 AL 0.25 mg/L t=72h 

T 05012 AL 1.01 mg/L t=72h 

A green layer was observed at the bottom of the sample bottles. 
ICS-103 Final Research Report 

ECRA F07012 T05012 ODA 

April 19,2007 

Results: 

Page 26 

Technology 

& Engineering 

The concentrations 

methanol content of 5 

measured are given in table 2 below. A correction for the 

*Samples T 05012 AL 0.0625 mg/L t=Oh and T 05012 AL 0.0625 mg/L t=72h, were concentrated five 

times by evaporation before analysis. 

All Control samples were below detection limit which is 0.02 mg/L.