ICS-103 

17---

AKZONOBEL 

Final Research Report 

ECRA F07013 T 05012 OFA April 19, 2007 

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

ACUTE TOXICITY OF ETHOQUAD MC 1214/25 

TO DANIO RERIO 

CONFIDENTIAL 

Project identification 

Client Akzo Nobel Surface Chemistry AB 

Subclient 

Client No 

Subclient No 

Technology 

& Engineering 

Page 1 of 27 

Project No 

Task No 859.024 

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

ECRA F07013 T 05012 OFA 

April 19.2007 

ABSTRACT 

~~ AKZONOBEL 

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& Engineering 

The acute toxicity of Ethoquad MC 1214/25 to the fresh water fish Dania rerio (zebra fish) was tested 

under static conditions for 96 hours in accordance with OECD and EEC guidelines for testing of 

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

The 96 h LC50 was calculated to be 35.36 mg/L. Based on the test results a NOEC (no observed effect 

concentration) after 96 hours was observed at 25 mg/L. No Sub-lethal effects were observed at 

concentrations where no mortality was observed after 96 hours of testing. 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: 96 h LC50 = 29 mg/L and a highest test 

concentration causing no effects after 96 hours is 20.5 mg/L. 

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 < 4% of test substance concentration 

during the 96 hours of testing was observed. The effects observed are therefore expressed based on 

their nominal concentrations. 
ICS-103 Final Research Report 

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f! ~~ 

AKZONOBEL 

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FISH ACUTE TOXICITY TEST WITH ETHOQUAD MC 1214/25 

Sponsors 

Study monitor 

STUDY ORGANIZATION 

Location 

Study director 

Quality Assurance Unit 

Management 

Experimental initiation date 

Experimental completion date 

ARCHIVING AND STORAGE 

Akzo Nobel Surface Chemistry AB 

Se 44485 Stenungsund 

Sweden 

M. Company 

Akzo Nobel Technology & Engineering 

Environmental Chemistry and Regulatory Affairs Dept. 

P.O. Box 5136 

6802 EC Arnhem 

The Netherlands 

Ing. M.G.J. Geurts 

Ing. H. van Daalen 

Ir. A.R. Luttmer 

12-02-2007 

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 Akzo Nobel Technology & Engineering Arnhem 

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 Akzo Nobel Technology & Engineering Arnhem 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 F07013 T 05012 OFA 

April 19,2007 

GLP COMPLIANCE STATEMENT 

~~ AKZONOBEL 

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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 

Ing. M.G.J. Geurts 

Management 

Ir. A.R. Luttmer 

date .. J.~.~!P.k:2~1 

~/ 

.~ ......... . 

date ..... ~?I"fft.7 
ICS~ 103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19, 2007 

QUALITY ASSURANCE STATEMENT 

{J 

~r-~ 

AKZONOBEL 

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The Quality Assurance Unit contracted by Akzo Nobel Technology and Engineering Arnhem audited 

this report. 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 and phases of inspection of this study by the Quality Assurance Unit and 

the dates on which its findings were reported to Study Director and Management. 

Dates of inspection 

12.02.2007 

12.02.2007 

04.04.2007 

Quality Assurance Unit 

Ing. H. van Daalen 

Phase of study 

Study Plan 

test (fish distribution and history) 

report 

Dates of reporting 

12.02.2007 

12.02.2007 

04.04.2007 

date.;)&:.{;(.: .. ~ 
ICS-103 Final Research Report 

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April 19, 2007 

fJ ~~ 

AKZONOBEL 

CONTENTS 

1. INTRODUCTION 

2. TEST GUIDELINES, MODIFICATIONS AND DEVIATIONS 

3. MATERIALS 

3.1 Test substance 

3.2 Chemicals 

3.3 Deionized water 

3.4 Test vessels 

3.5 Test incubator and light regime 

3.6 Test animals 

4. METHODS 

4.1 Test principle and procedure 

4.2 Preparation of the stock solution 

4.3 Preparation of the test solutions 

4.4 Preparation of the test medium 

4.5 Chemical analyses 

4.6 Dissolved oxygen, pH and temperature measurements in the test solutions 

4.7 Evaluation of data 

5. RESULTS 

5.1 Toxicity 

5.2 Chemical analyses 

5.3 Oxygen, pH and temperature 

6. QUALITY CRITERIA 

7. AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN 

7.1 Amendments to the study plan 

7.2 Deviations from the study plan 

8. REFERENCES 

9. APPENDICES 

ANNEX 1 

ANNEX 2 

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7 

7 

8 

8 

8 

8 

8 

9 

9 

10 

10 

10 

10 

11 

11 

11 

11 

12 

12 

12 

13 

13 

13 

13 

13 

13 

14 

18 

25 
ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19,2007 

1. INTRODUCTION 

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AKZONOBEL 

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The purpose of this study was to determine the acute lethal toxicity of Ethoquad MC 1214/25 to the 

freshwater fish Dania rerio (zebra fish) under continuous exposure during 96 hours in a static test. 

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 normally expressed as the median lethal concentration (LC50). 

That is the concentration in water which kills 50% of a test batch of fish within a period of continuous 

exposure. Lethality of the fish is defined as the lack of opercular movement. 

Sublethal effects such as deviations in the behavior or the appearance of the fish were recorded, if 

observed. 

2. TEST GUIDELINES, MODIFICATIONS AND DEVIATIONS 

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

chemicals without modification of the test guideline and with the following deviations: 

• There are no modifications and deviations from the test guidelines. 
ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

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3. MATERIALS 

3.1 Test substance 

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A sample of Ethoquad MC 1214/25 (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 

Ethoquad MC 1214/25 

6506001437 

82% 

Brown viscous liquid 

Soluble 

Not available 

A quaternary ammonium compound is a salt and 

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 

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 Deionized water 

The deionized water used contained not more than 0.01 mg/L of copper, had a TOC-content of not 

more than 2.0 mg/L and a conductivity of less than 5 IJS/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, 5-L aquaria containing 3 L of test solution were used. They were covered with glass 

plates during the test. 
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AKZONOBEL 

3.5 Test incubator and light regime 

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The test was carried out in a temperature controlled room, the light regime was 16 h of ambient light 

per day, provided by fluorescent tubes. 

3.6 Test animals 

The test was performed with juvenile Danio rerio (zebra fish), which were bred in-house and were 

accepted according to the quality control criteria of the guidelines and Standard Operation Procedure 

E 2 (8.3). The fish in stock were fed one to three times per day with commercially available dry, 

deep-frozen food, or nauplii. The feeding was stopped 24 hours before the test was started, and the 

animals were not fed during the test. From five test fish the length was measured at the beginning of 

the test and showed the length of animals to be ~3 cm. The biomass loading was verified at the 

beginning of the test based on visual inspection. Shortly after the test was finished the fish were killed 

and their wet weight and length measured. Fish that died during the test were subjected to this 

procedure shortly after they were removed from the test aquarium. The average biomass load of 0.32 

gIL was calculated from the average weight of all fish and the volume of test water in the aquaria. 
ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19,2007 

4. METHODS 

4.1 Test principle and procedure 

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The test was performed as a static test which means the test media was not replaced during 96 hours. 

7 Fish were used per test concentration and control. 

Under otherwise identical test conditions, the fish were exposed to the chosen concentrations of the 

test substance as described below and mortality and sub-lethal effects were recorded at least within 

the first six hours and then at approximately 24,48, 72 and 96 hours. The fish were considered dead 

when a lack of opercular movement was observed and touching of the caudal peduncle produced no 

reaction. Dead fish were removed from the test vessels directly after being observed, which was 

checked at least twice each day. In addition to death, sub-lethal effects such as erratic swimming, loss 

of reflex, increased excitability, lethargy, changes in physiology, discoloration, pigmentation, excessive 

mucous production, hyperventilation, opaque eyes, curved spine or hemorrhaging were recorded. Fish 

that were convulsing or showing other severe forms of distress not considered transient in nature and 

likely to become more severe before the exposure is terminated, were sacrificed for humane reasons. 

These fish were treated as having died in the test. 

The fish were randomly placed in the test vessels which were positioned in the test room in a random 

manner. During the test the vessels were covered with glass plates. The test solutions present in the 

test vessels were aerated with water-saturated air purified by an active coal filter and a cotton filter 

during testing. 

4.2 Preparation of the stock solution 

A stock solution was prepared as following: an accurate measured amount of 0.9999 g test substance 

was transferred into 1 L volumetric flask which was filled up the mark with test medium. The pH was 

checked and found to be 8.1 and therefore not adjusted 

4.3 Preparation of the test solutions 

The stock solution was diluted into freshly prepared test medium to the following test concentrations: 

6.25 - 12.5 - 25 - 50 and 100 mg/L. A control containing only test medium was included in the test. 
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4.4 Preparation of the test medium 

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As test medium so called Dutch Standard water (DSW) was used, with a pH of approximately B.2 and 

a conductivity between 550 and 650 I-lS'cm-1

, containing per liter of de-ionized water: 100 mg of 

NaHC03, 20 mg of KHC03• 200 mg of CaCI2·2H20 and 1BO mg of MgS04·7H20 (Standard Operation 

Procedure 0 1 (B.12)). The test medium was aerated before being used in the test. The air was watersaturated and purified by an active coal and cotton filter (Standard Operation Procedure K 12 (B.9)). 

4.5 Chemical analyses 

At the start and end 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. 

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 Dissolved oxygen, pH and temperature measurements in the test solutions 

The dissolved oxygen and pH were measured daily of the test concentration and control vessel when 

living fish were present. Conductivity was measured only at the beginning of the test in the control 

solution whether its value is within limits. The temperature was measured continuously in the control 

vessel. The dissolved oxygen concentrations were determined electrochemically using an oxygen 

electrode and meter according to Standard Operation Procedure K 2 (B.5). The pH was measured 

using a microcomputer pH-meter according to Standard Operation Procedure K 1 (B.4). The 

conductivity was measured using a portable conductivity meter according to Standard Operation 

Procedure K 24 (B.10). The temperature was measured with a digital thermocouple according to 

Standard Operation Procedure K 6 (B.7). 

4.7 Evaluation of data 

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

Standard Operation Procedure L 2 (B.11), using the trimmed Spearman-Karber method (B.13). The 

concentrations causing zero and 100% immobility were derived directly from the test observations. 
ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19, 2007 

5. RESULTS 

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In this chapter the result of a definitive test are presented. Prior to the definitive test a preliminary 

range finding test was performed to assess the level of toxicity. 

5.1 Toxicity 

Preliminary test 

During the preliminary range finding test, performed as a non-GLP test, no effect was observed after 

96 hours of testing at 1 mg/L and 10 mg/L and 100% mortality at 100 mg/L. 

Definitive test 

The results on survival and sub-lethal effects are summarized in Table 1. All results are calculated based 

on the test substance as received thus not corrected for the active ingredient content. The results show 

that the test substance had lethal effect at 100 mg/L of Ethoquad MC 1214/25 after 24 hours and for 5 

out of 7 fish at 50 mg/L after 48 hours. No lethal or sub-lethal effects were observed at the other test 

concentrations of respectively 6.25 - 12.5 and 25 mg/L of test substance. 

The 96 h LCso was calculated to be 35.36 mg/L. The highest test concentration where no effect were 

observed was 25 mg/L 

The dose-effect curve presenting the percent immobilization against the concentration after 96 hours of 

exposure is presented in figure 1. 

5.2 Chemical analyses 

The test medium had a conductivity of 637 I-IS/cm and a pH of 8.1. 

The results of the chemical analyses are presented in table 4. 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. 

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. 

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. 

Based on these arguments the effects observed are expressed based on nominal concentrations. 
" .::r 

ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19,2007 

5.3 Oxygen, pH and temperature 

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The maximum variation in oxygen concentration during the test was in the range of 8.1 to 8.6 mg/L and 

are presented in Table 2. The pH variation during the test was in the range of 7.8 to 8.1. The results of 

the pH measurements are summarized in Table 3. 

The temperature in the control test vessels during the test period ranged from 22.9 to 23.1 ac. 

6. QUALITY CRITERIA 

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

• Mortality in the control did not exceed one fish at the end of the test. 

• The dissolved oxygen concentration was >60% of the air saturation value throughout the test. 

• The measured concentration of test substance was more than 80% of the nominal concentration 

throughout the test and at the end of the test the measured concentration was more than 80% of 

that at the beginning. 

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 from the study plan 

8. 

8.1 

8.2 

8.3 

8.4 

8.5 

8.6 

8.7 

8.8 

8.9 

8.10 

The fish were not obtained from a retailer as stated in the SP but from in lab breeding. This 

deviation is not considered to have an impact on the outcome of the test. 

REFERENCES 

OECD Guidelines for testing of chemicals, 203: Fish, acute toxicity test (7/1992). 

EEC Directive 79/831, Annex V, part C: Methods for the determination of ecotoxicity, part 

C.1. Acute toxicity to fish (update 11/1989). 

Standard Operation Procedure E 2: Obtaining and maintaining of fish 

Standard Operation Procedure K 1: pH-meters 

Standard Operation Procedure K 2: Oxygen meters 

Standard Operation Procedure K 4: Balansen 

Standard Operation Procedure K 6: Thermometers 

Standard Operation Procedure K 10: Deionizer 

Standard Operation Procedure K 12: Luchtfilters 

Standard Operation Procedure K 24: Geleidbaarheidsmeter 
ICS-103 Final Research Report 

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AKZONOBEL 

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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 medial lethal concentrations in toxicity bioassays'. Env.Sci. & Technol.ll, 714-719. 

Correction, 12 (1978)417. 

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

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

8.15 OECD Guideline no. 1 (1998), Directive 88/320/EEC and 1999/12/EC 

9. APPENDICES 

Tables 1 to 5 
ICS-103 Final Research Report 

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April 19. 2007 

f! 4~ 

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Table 1: Survival and sub-lethal effects of fish 

Conc. 

(mg/L) 

No. of 0 hours 4 hours 24 hours 

fish N Effects No. Effects No. Effects 

exposed dead dead 

7 7A 7A 7A 

7 7A 7A 7A ~----------r------7-----~~----------~-----7 7A 7A 

7 7A 7A 7A 

i i~ =i~===:===7===:=1=A=6=R=: 

Conc. No. of 48 hours 72 hours 96 hours 

(mg/L) fish No. Effects No. Effects No. Effects 

exposed dead dead dead 

O(Control) 7 7A 7A 7A 

6.25 7 7A 7A 7A 

12.5 7 7A 7A 7A 

25 7 7A 7A 7A 

50 7 5 1A1R 2 

100 7 

Observed effects: 

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A = appearing normal; E = erratic swimming; A = surfacing; N = loss of equilibrium; R = lying on the bottom 

1.0 'T'"""'"------......jT.-... ~-------. 

0.9 

0.8 

0.7 

: 0.6 c &. 0.5 

Ih 

~ 0.4 

0.3 

0.2 

0.1 . 

10 100 1000 

Dose mglL 

Figure 1. Dose-response curve at 96h exposure to ethoquad Me 1214/25 
ICS-103 Final Research Report 

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AKZONOBEL 

Table 2: Oxygen measurements test 

Dilution (mg/L) o hours 24 hours hours 

Control 8.5 8.4 8.5 

6.25 8.6 8.5 8.5 

12.5 8.5 8.4 8.4 

25 8.5 8.3 8.3 

50 8.5 8.4 8.1 

100 8.6 8.3 * 

* no fish left In test vessel 

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72 hours 96 hours 

8.4 8.5 

8.5 8.5 

8.4 8.4 

8.2 8.3 

8.4 8.5* 

* 8.5* 
ICS-103 Final Research Report 

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April 19, 2007 

Table 3: pH-measurements 

Nominal 

concentration o hours 

(mg/L) 

Control 8.1 

6.25 8.1 

12.5 8.1 

25 8.1 

50 _ .. 

100 _. 

* no fish left in test vessel 

fJ ~;-=---

AKZONOBEL 

24 hours 48 hours 

7.8 7.9 

7.9 7.9 

7.9 7.9 

7.9 7.9 

7.9 7.8 

7.9 * 

Table 4: The results of the chemical analyses 

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72 hours 96 hours 

7.8 7.9 

7.9 7.9 

7.9 ( .9 

7.8 7.8 

7.9 7.9* 

* 7.9* 

Sample code 1 Test number Ethoquad Me 1214/25 (mg/L) decrease 

Injection 1 Injection 2 Average (%) 

T 05012 OF A Control t=Oh < 0.1 < 0.1 <0.1 - T 05012 OF A Control t=96h < 0.1 < 0.1 <0.1 

T 05012 OFA 6.25 mg/L t=Oh 6.4 6.4 6.4 

3.9 

T 05012 OF A 6.25 mg/L t=96h 5.9 6.4 6.15 

T 05012 OFA 25 mg/L t=Oh 26 26 26 

-1.9 

T 05012 OF A 25 mg/L t=96h 27 26 26.5 

T 05012 OFA 100 mg/L t=Oh 102 104 103 

-1 

T 05012 OFA 100 mg/L t=96h 105 103 104 

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

Table 5: Sample weight 
ICS-103 Final Research Report 

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Method of analyses 

Ethoquad MC1214125 

Lot nr: 6506001437 

AKZONOBEL 

Certificate of Analyze 

N,N-dimethyl-N-slkyl + 15 mol EO 821)6 " 

Tri me1hyl alkyl amine ammonium salt <0.5% 

PEG 1000-1500 14% 

Free amine UIIMI 

Alcohol ethoxyla1e <1'M1 

Fatty amine ethoxylate <2% 

Alkyl = C tl1~ 

\\S&dn38.d2IUntra\U&et'i$\maCO\My Ooctmen15\HydiOtiOPi\Hml\CoA _6506001437_ ElhDquad MCI214_25.dac 1 {1} 

Pnnt Inta: Maccr.2006-t2-18 AuIhor: Matmn COIIlpanyt2001j..12·18 

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ANNEX 1 
ICS-103 Final Research Report 

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f) ~~ 

AKlONOBEL 

~~ AKlONOBEL Surfactants - Analytical Methods for R&D AM46 

Revised by {position, signature) 

Analytical Manager 

Approved by (position, signature) 

QHSE Manager 

03te 10 no 

2007-02-01 A1VI46-2309 Stand~ Process 

1509001 

Section Encl. 

3 

Issue 

1 

Page 

10fS 

Valid until 

2010-02-01 

No. 309 Ethoquad MC1214125 at trace levels in water 

Procedure 

Sign 

lisa Lofgren 

Pu~enBackground 

Remark 

literature 

Principle 

Safety 

Apparatus 

The method is used for the determination of Ethoquad MC1214125 at low levels 

in OECD water (freshwater algae medium) and DSW water (daphnia test 

medium). 

ANL07002 

The samples are analyzed for Ethoquad MC1214125 in OECO water (freshwater 

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

The samples are analyzed by reversed phase liquid chromatography and mass 

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

(SIM) with eledrospray ionization (ESI) in positive mode. External standard 

calibration is used. 

Acetonitril and methanol are highly flammable. They are harmful by inhalation. in 

contact with skin and if swa.owed. They are initating to eyes. See &afeiy data 

sheets S...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 (Lea Duo, ThermoFinnigan), Ion trap. 

ESI...source 

Pipette (Eppendorf Multipipette Pro) 

Standard laboratory glassware. 

Page 19 

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ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19, 2007 

~~ AKZONOBEL 

Revised by (position, signature) 

Analytical Manager 

Approved by (posilicln. signature) 

QHSe Manager 

lCMS data 

Column: 

Column temp: 

fJ ~r=----

AKZONOBEL 

Surfactants - Analytical Methods for R&D AM46 

Date 10 no 

2007-02-01 AM46-2309 

Standardreference Process 

1509001 

Section End. 

3 

Issue 

1 

HyPURITY C18 (5 urn, 2.1·50 mm (Thermo)) 

Page 

20f6 

Valid \Inti! 

2010-02-01 

Mobile phase: A: acetonitrilelwater 5:95 (v/v) + 0.5 % acetic acid 

B: acetonitril + 0.5 % acetic acid 

Gradient 

Flow: 0.2 mllmin 

Pump: Gradient 

Time (min) 

0.0 

20 

25 

27 

40 

A(%) 

100 

o 

o 

100 

100 

B(%) 

o 

100 

100 

o 

o 

Detector: Mass spectrometer LCQ Duo, ESI-source, + mode 

Injection: 5 ~I 

Detector parameters 

Mass spectrometer: 

MS SIM parameters: ions 522, 566,610, 654, 698, 742, 786, 830,874, 918 

ESI 

Mode: + 

Sheath gas flow (arb): 40 

Aux gas flow (arb): 5 

Spray voltage (tV): 4.5 

Capillary temp (·C): 200 

CapDlary voltage (V): 25 

Tube lens offset (V): 0 

Chemicals/reagents 

Acetonitrile, pa 

Deionized and distilled water 

Methanolpa 

2..propanol pa 

Acetic acid (100%) pa 

Reference of Ethoquad MC121412S 

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ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19,2007 

--Y~ AKlONOBEL 

Revised by (position. signature) 

Analytical Manager 

Approved by (position. signature) 

QHSE Manager 

Calibration 

Surfactants - Analytical Methods for R&D AM46 

Date. IOna 

2007-02-01 AM46-2309 

Standardreference Process 

1509001 

Section End. 

3 

Issue 

1 

Pagl! 

30f6 

Valid until 

2010-02-01 

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

concentrations in the samples. 

Prepare the calibration solutions in the same matrix as for the samples. 

Sample preparation 

The sample is refrigerated and 5% (v/v) of 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 calibration graph is constructed from the calibration samples, from which the 

amount of Ethoquad MC1214125 can be calculated. XCaJibur software for LCQ Duo is 

used for the calculations. 

Precision and accuracy 

Six repetitive injections of one control sample in OECO water containing 0.95 mgll of 

Ethoquad gave an RSO of 1.8 %. 

Six repetitive injections of one control sample in OSW containing 0.95 mgIl of 

Ethoquad gave an RSO of 3.7 %. 

Six repetitive injections of one standard solution in OECO water containing 0.21 mgJI.. 

of Ethoquad gave an RSD of 2.9 %. 

Six repetitive injections of one standard solution in OSW containing 0.21 mgl1.. of 

Ethoquad gave an RSD of 1.8 %. 

Six repetitive injections of one standard solution in OECD water containing 10.6 mglL. 

of Ethoquad gave an RSD of 3.5 %. 

limit of quantitation 

The lower limit of quantitation is 0.1 mgn. 

The detection limit is 0.02 mgn. 

linearity 01 calibration curve 

The calibration curve is linear within the range of 0.1-2 mgn and in the range of 1-100 

mgn. 

Page 21 

Technology 

& Engineering 
'CS~103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19, 2007 

---I~ 

1~ AKZONOBEL 

AKZONOBEL Surfactanls ~ Analytical Methods for R&D AM46 

Revised by (position, ~nature) Dabt 10 no Section Encl 

Analytical Manager 2007 -O2~01 AM4~2309 3 

Approved by (position. signature) Standardrel'erence Process 

QHSE Manager 1509001 

Reporting 

Page 

4016 

Valid until 

2010-02-01 

The result is reported as amount of Ethoquad MC1214125 in mgJI with two significant 

numbers. 

Others 

The samples are considered to be stable when refrigerated, with the addition of 5% 

methanol, for at least 3 months at the level of 1 mgn of Ethoquad MC1214125. 

\!If' a,CO-24ft 

,00 

a 

i atl 

1. 

~40 ;21 

~ /I .. It ut ", 

. " 

:a 

j 

<C 

i IE 

Ftg i.Total ion current (TIC) chromatogram and mass spectrum for Ethoquad MC1214125; 

Full scan. positive mode 

Page 22 

Technology 

& Engineering 
ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19, 2007 

~~ AKZONOBEL 

Revised by (position, 5ignawre) 

Analytical Manager 

Apptowed by (position. signatuf&) 

QHSE Manager 

1~ AKZONOBEL 

Surfactants - Analytical Methods for R&D AM46 

Oatft 10 no Section EncL Page 

2007-02-01 AM46-2309 3 50f6 

Validvntil 

2010''()2-O1 

Standardreference Process 

IS09OO1 

N.; ""GE7 

Issue 

1 

TIC f: + et ESI SlIII ftoI' !i:I2,oo.SUJlO. 

~J,IliI; ll1it)lll411,Oit. esc,oo.ess.oo, ... 0NItt ... 742,00.:14'.00; 11I8,oo.:1l7.llO: 

~1," e14.Q0.47!lPG', D1$,lIO-iHO,lIOt liS (16.0317 .. ,. 

IIII-DSlht4185-H RT: 1:\,51.1.,10 All, Il Ill.; 1.D19 

'f, • t E-"SI SIIoI "",-r sn.oo.&n)lO.see,IlI]..8S7.00. 1110.00.$11<(00, es ... ooaSs,oo. MJlIJ..8lQ,oo; 142,00-143.00; T8II,OOo-I'I7.{I!). 8~QO.ti1 ... 

I 

i 

i 

i 

~ 

,,4'$ 

7 ." 

rig 2. SIM chromatogram and mass spectrum for Ethoquad MC1214125 at 0.8 mgIL 

Elhoquad_C/12 

Y = 2.19082e+007+8.58756e+O08t X RA2 = 0.9952 W: Equal 

1150000000 

41(100000000 

50000000 

0.0 0.5 1.0 1.5 2.0 2.5 

ughnl 

rtg 3. Calibration graph for Eihoquad MC121412!S: cone.range 0.1-2.1 mgIL 

Page 23 

Technology 

& Engineering 
ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19,2007 

~7--

{J 

~r---. 

AKZONOBEL 

AKZONOBEL Surfactants - Analytical Methods for R&D AM46 

Revised by (position. signall.U'e) 

Analytical Manager 

Approved by (position, signature) 

QHSE Manager 

DatR ID no Section End. 

2007-02-01 AM46-2309 

Standan:lreference Process 

IS09001 

C12-Ouatemary 

3 

Y = 2.84B0ge+009+323232e+00S*X RA2 = 0.9929 W. Equal 

4000000000 

3000000000 

co 

~ 2000000000 

1000000000 

o 50 

ug/ml 

100 

Ftg 4. Calibration graph for Ethoquad MCt214125; conc.range 1-100 mgll. 

Issue 

1 

Page 

60f6 

Validun.lil 

2010-02-01 

Page 24 

Technology 

& Engineering 
ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19, 2007 

fJ ~~ 

Page 25 

AKZONOBEL Technology 

Determination of Ethoquad Me 1214/25 in water from Toxicity studies; 

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

Introduction: 

24 water samples from toxicity studies: 

& Engineering 

ANNEX 2 

• T 05012 AL "Effects of Ethoquad MC 1214/25 on the growth of the freshwater green alga 

pseudokirchnieriella subcapitata" 

• T 05012 ODA "Acute toxicity of Ethoquad MC 1214/25 to water fleas, daphnia magna" 

• T 05012 OFA "Acute toxicity of Ethoquad MC 1214/25 to danio rerio" 

were received from Marc Geurts for the analysis of Ethoquad MC1214/25 with method AM46-

2309. 

Experimental: 

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

Table 1 

Sample code I Test num 

T 05012 AL Control t=Oh 29-1-2007 ~~~----~~~----- -------~ T 05012 AL Control t=72h 07 

T 05012 AL 0.0625 m IL t 29-1-2007 

T 05012 AL 0.0625 m IL t=72h 1-2-2007 

T 05012 AL 0.25 m IL t=Oh 29-1-2007 

T 05012 AL 0.25 m IL t=72h 1-2-2007 

T 05012 AL 1.0 m IL t=Oh 29-1-2007 

T 05012 AL 1.0 m IL 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 m IL t=Oh 30-1-2007 

T 05012 ODA 0.2 m IL t=48h 1-2-2007 

T 05012 ODA 0.8 m IL t=Oh 30-1-2007 

T 05012 ODA 0.8 m IL t=48h 1-2-2007 

T 05012 ODA 3.2 30-1-2007 

T 05012 ODA . 1-2-2007 

T 05012 OFA 2-2-2007 

T 05012 OFA Co 16-2-2007 

T 05012 OFA 6.25 m IL t=Oh 12-2-2007 

T 05012 OFA 6.25 m IL t=96h 16-2-2007 

T 05012 OFA 25 m IL t=Oh 12-2-2007 

T 05012 OFA 25 m IL t=96h 16-2-2007 

T 05012 OFA 100 m IL t=Oh 12-2-2007 

T 05012 OFA 100 m IL t=96h 16-2-2007 

Sample storage: 

Date of analysis: 

Laboratory: 

The samples were refrigerated prior to analysis. 

27-2-2007 to 5-3-2007. 

Analytical Laboratory 

R&D SBU SE 

Akzo Nobel Surface Chern istry AB 

Stenungsund 
ICS-103 Final Research Report 

ECRA F07013 T 05012 OFA 

April 19,2007 .-,r~ 

AKZONOBEL 

Shipment address: Akzo Nobel Surfactants Europe 

Stenungsundsfabriker 

444 85 Stenungsund 

Sweden 

Head of Laboratory: Lisa Lofgren 

Equipment: See method AM46-2309 

Page 26 

Technology 

& Engineering 

Calibration samples: Calibration samples were prepared from the reference labeled T05012 

Ethoquad MC1214/25 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 brieny 

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 

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

Observations: In samples with sample codes: 

T 05012 AL control t=72h 

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 F07013 T 05012 OFA 

April 19.2007 

Results: 

Page 27 

Technology 

& Engineering 

The concentrations of Ethoquad MC 1214/25 measured are given in table 2 below. A correction for the 

methanol content of 5 % (v/v) has been done. 

Table 2 

Sample code 1 Test number Ethoquad Me 1214/25 (mg/L) 

Injection 1 Injection 2 

T 05012 AL Control t=Oh < 0.1 < 0.1 

T 05012 AL Control t=72h < 0.1 < 0.1 

T 05012 AL 0.0625mg/L t=Oh 0.03 * 0.03 * 

T 05012 AL 0.0625mg/L t=72h 0.03 * 0.03 * 

T 05012 AL 0.25mg/L t=Oh 0.22 0.25 

T 05012 AL 0.25mg/L t=72h 0.16 0.17 

T 05012 AL 1.0 mg/L t=Oh 1.1 1.1 

T 05012 AL 1.0 mg/L t=72h 0.95 1.0 

T 05012 ODA Control t=Oh < 0.1 < 0.1 

T 05012 ODA Control t=48h < 0.1 < 0.1 

T 05012 ODA 0.2 mg/L t=Oh 0.17 0.17 

T 05012 ODA 0.2 mg/L t=48h 0.17 0.15 

T 05012 ODA 0.8 mg/L t=Oh 0.76 0.78 

T 05012 ODA 0.8 mg/L t=48h 0.79 0.80 

T 05012 ODA 3.2 mg/L t=Oh 3.1 3.3 

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

T 05012 OFA Control t=Oh < 0.1 < 0.1 

T 05012 OFA Control t=96h < 0.1 < 0.1 

T 05012 OFA 6.25 mg/L t=Oh 6.4 6.4 

T 05012 OFA 6.25 mg/L t=96h 5.9 6.4 

T 05012 OF A 25 mg/L t=Oh 26 26 

T 05012 OFA 25 mg/L t=96h 27 26 

T 05012 OFA 100 mg/L t=Oh 102 104 

T 05012 OFA 100 mg/L t=96h 105 103 

* 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.