### 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 ChemistryAB Subclient Client No Subclient No Technology & Engineering



### Page 1 of27 Project No Task No 859.024 Drafted by department 

### ECRA
- Environmental Chemistry and RegulatoryAffairs Dept.ICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19.2007



### ABSTRACT

# ~~ AKZONOBEL Page 2 Technology & 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 oftesting. 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 ECRA F07013 T 05012 OFA April 19.2007 

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FISH ACUTE TOXICITYTESTWITH 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 ChemistryAB Se 44485 Stenungsund Sweden M. Company

Akzo Nobel Technology 

### &Engineering Environmental Chemistry and RegulatoryAffairs 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 ofAkzo Nobel Technology 

### &Engineering Arnhem for a period of ten years. Other records including master schedule sheet, laboratory notebooks, logbooks, records ofthe maintenance and calibration of equipment, summary of training. curricula vitae and job descriptions ofthe personnel involved in the study, records related to location and storage ofthe 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 oronly as long as the qualityofthe test substance permits evaluation.ICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19,2007

GLP COMPLIANCE STATEMENT

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The studywas 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 of23.3.1999). • 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 ofthese 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 forthis part ofthe results. This has however no impact on the qualityofthe chemical analyses and thefinal outcome ofthe test.


### Studydirector 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-~** 

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Technology & Engineering

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 ofthe study and an accurate reproduction ofthe 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 ofstudy 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 ECRA F07013 T 05012 OFA April 19, 2007 

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CONTENTS 1. INTRODUCTION 2. TEST GUIDELINES, MODIFICATIONSAND DEVIATIONS 3. MATERIALS 3.1 Test substance 3.2 Chemicals 3.3 Deionized water 3.4 Testvessels 3.5 Test incubator and light regime 3.6 Test animals 4. METHODS 4.1 Test principle and procedure 4.2 Preparation ofthe stock solution 4.3 Preparation ofthe test solutions 4.4 Preparation ofthe test medium 4.5 Chemical analyses 4.6 Dissolved oxygen, pH and temperature measurements in the test solutions 4.7 Evaluation ofdata 5. RESULTS 5.1 Toxicity 5.2 Chemical analyses 5.3 Oxygen, pH and temperature 6. QUALITY CRITERIA 7. AMENDMENTS TOAND 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 Page 6 Technology & Engineering

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 25ICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19,2007



### 1. INTRODUCTION 

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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 ofa test substance is normally expressed as the median lethal concentration (LC50). 

### That is the concentration in water which kills 50% of a test batch offish within a period of continuous exposure. Lethality ofthe fish is defined as the lack ofopercular 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 ofthe 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 April 19,2007



### 3. MATERIALS 3.1 Testsubstance

# 1~ AKZONOBEL PageS Technology & Engineering



### 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 validitythereof. 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 avery lowvapour pressure Assumed to be stable undertest 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 awater purification system according to Standard Operation Procedure K 10 (8.8)


### 3.4 Testvessels As test vessels, 5-L aquaria containing 3 L of test solution were used. They were covered with glass plates during the test.ICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19, 2007

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3.5 Test incubatorand light regime Page 9 Technology & Engineering



### The test was carried out in a temperature controlled room, the light regime was 16 h of ambient light per day, provided byfluorescent 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 ofthe 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 ofthe 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 ofall fish and the volume oftest 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 ofthe caudal peduncle produced no reaction. Dead fish were removed from the test vessels directly after being observed, which was checked at leasttwice 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 ofthe stocksolution A stock solution was prepared as following: an accurate measured amount of0.9999 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 ofthe 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 onlytest medium was included in the test.ICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19,2007 

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4.4 Preparation ofthe test medium Page 11 Technology & Engineering



### 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

### ·2H2

### 0 and 

### 1BO mg of MgS04

### ·7H2

### 0 (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 ofthe 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 ofthe 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 ofdata If possible, the EC50 

### was calculated with the computer program TOXCALCTM 

### 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% immobilitywere derived directlyfrom 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 testwas performed to assess the level oftoxicity. 5.1 Toxicity Preliminarytest During the preliminary range finding test, performed as a non-GLP test, no effect was observed after 96 hours oftesting 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 oftest 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 of637 I-IS/cm 

### and a pH of8.1. 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 ofthe samples by methanol are presented in Table 5. The results ofthe chemical analyses showthat no test substance was detected in the control samples; the result also show that the concentration ofthe test compound during the test remained within 80% ofthe concentration at the beginning ofthe 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 22.9to 23.1

# ac.
6. QUALITY CRITERIA Thefollowing qualitycriteria have been met in the present study: • Mortality in the control did not exceed one fish atthe end ofthe test. • The dissolved oxygen concentration was >60% ofthe airsaturation 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 atthe beginning.


### 7. AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN

7.1 Amendments tothe 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 ofthe test.


### REFERENCES

OECD Guidelines for testing ofchemicals, 203: Fish, acute toxicitytest (7/1992). EEC Directive 79/831, Annex V, part C: Methods for the determination of ecotoxicity, part C.1. Acute toxicityto fish (update 11/1989). Standard Operation Procedure E2: Obtaining and maintaining offish 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: GeleidbaarheidsmeterICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19, 2007 

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8.11 Standard Operation Procedure L 2: Acute toxicity test offish and daphnia magna (calculation ofeffects 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. Mayerand J.L. Hamelink (Eds.). 65-84. 8.15 OECD Guideline no. 1 (1998), Directive 

### 88/320/EEC and 

### 1999/12/EC
9. APPENDICES Tables 1to 5ICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19. 2007 

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Table 1: Survival and sub-lethal effects offish

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: Page 15
Technology & Engineering

A = appearing normal; E = erratic swimming; A = surfacing; N = loss ofequilibrium; 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 1214/25ICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19, 2007 

# .---,f7---- AKZONOBEL

Table 2: Oxygen measurements test

Dilution (mg/L) 

## ohours 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 Page 16
Technology & Engineering

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 ECRA F07013 T 05012 OFA April 19, 2007



### Table 3: pH-measurements

Nominal concentration 

## ohours (mg/L) Control 8.1 6.25 8.1 12.5 8.1 25 8.1 50 

# _ .. 100 

## _. * nofish left in test vessel 

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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 ofthe chemical analyses Page 17
Technology & Engineering

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 1Test number Ethoquad Me 1214/25 (mg/L) decrease Injection 1 Injection 2 Average 

### (%) T 05012 OFA Control t=Oh < 0.1 < 0.1 <0.1



### - T 05012 OFA Control t=96h < 0.1 < 0.1 <0.1 T 05012 OFA 6.25 mg/Lt=Oh 6.4 6.4 6.4 3.9 T 05012 OFA 6.25 mg/Lt=96h 5.9 6.4 6.15 T 05012 OFA 25 mg/L t=Oh 26 26 26 -1.9 T 05012 OFA 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 limitwhich is 0.02 mg/L. Table 5: Sample weightICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19, 2007 

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Method ofanalyses

Ethoquad MC1214125 

### Lotnr: 6506001437 

# AKZONOBEL
Certificate ofAnalyze


# " N,N-dimethyl-N-slkyl + 15 mol EO 821)6 Tri me1hyl alkyl amine ammonium salt <0.5% PEG 1000-1500 14% Freeamine UIIMI Alcohol ethoxyla1e <1'M1 Fattyamine 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: MatmnCOIIlpanyt2001j..12·18 Page 18
Technology & Engineering ANNEX 1ICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19,2007 

# f)**~~** 

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

Revised by{position,signature) Analytical Manager Approvedby(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 attrace levels in water

Procedure Sign lisa Lofgren**Pu~enBackground**

Remark
literature Principle

Safety

Apparatus The method is usedforthedetermination ofEthoquad MC1214125 at low levels in OECDwater(freshwateralgaemedium) and DSWwater (daphnia test medium).

ANL07002

The samplesare analyzed forEthoquad MC1214125in OECOwater(freshwater algae medium)and DSWwater(daphniatestmedium). The samples areanalyzed byreversed phase liquid chromatographyand mass spectrometricdetection. Themassspectrometeris run in selective ion monitoring (SIM)witheledrospray ionization (ESI) in positive mode. External standard calibration is used.
Acetonitril and methanol are highlyflammable. Theyare harmful byinhalation. in contactwith skin and ifswa.owed.Theyare initatingtoeyes. See &afeiydata sheets S...3072 andSafewin40247. Acetic acid and can cause severe bums. See safetydatasheetSafewin40293. Wearsuitable protective glovesand handlein fume hood. liquidChromatograph-Massspectrometer(Lea Duo,ThermoFinnigan), Iontrap. ESI...source Pipette (EppendorfMultipipette Pro) Standard laboratory glassware. Page 19
Technology & EngineeringICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19, 2007

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Revised by(position, signature) Analytical Manager Approvedby(posilicln. signature) QHSe Manager

lCMS data Column: Column temp: 

# fJ**~r=----** 

# AKZONOBEL

Surfactants
- Analytical Methods for R&DAM46

Date 10 no 2007-02-01 AM46-2309 Standardreference Process 1509001 Section End. 3 Issue 1

HyPURITYC18 (5 urn, 2.1·50 mm (Thermo)) Page 20f6 Valid\Inti! 2010-02-01

Mobile phase: A:acetonitrilelwater5:95 (v/v) + 0.5% aceticacid B: acetonitril + 0.5% aceticacid 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: Massspectrometer LCQ Duo, ESI-source, + mode Injection: 5**~I** Detector parameters Massspectrometer: MS SIM parameters: ions522, 566,610, 654, 698, 742, 786, 830,874, 918 ESI Mode: + Sheathgasflow (arb): 40 Aux gasflow (arb): 5 Spray voltage (tV): 4.5 Capillarytemp(·C): 200 CapDlaryvoltage (V): 25 Tube lensoffset (V): 0 Chemicals/reagents Acetonitrile, pa Deionized and distilledwater Methanolpa 2..propanol pa Acetic acid (100%) pa Reference of Ethoquad MC121412S Page 20
Technology & EngineeringICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19,2007

# --Y~ AKlONOBEL

Revised by(position. signature) Analytical Manager Approvedby(position.signature) QHSE Manager
Calibration Surfactants
- Analytical Methodsfor R&D AM46
Date. IOna 2007-02-01 AM46-2309 Standardreference Process 1509001 Section End. 3 Issue 1 Pagl! 30f6 Validuntil 2010-02-01

Prepare calibration solutions atfive levels in a concentration range closeto theexpeckKI concentrations in thesamples. Preparethe calibration solutions in thesame matrix asforthe samples. Samplepreparation The sample is refrigerated and 5% (v/v)ofmethanol is added. Thetemperature ofthe sampleis adjustedto room temperature priortoanalysis.

Procedure Thesamples and calibration solutions are injected into the LCMS.
Calculation A linearcalibration graph is constructed from the calibration samples, fromwhichthe amountofEthoquad MC1214125 can be calculated. XCaJibur softwarefor LCQ Duois usedfor the calculations. Precision and accuracy Six repetitive injections ofone control sample in OECOwatercontaining 0.95mgllof Ethoquad gavean RSO of1.8%. Six repetitive injections ofonecontrol sample inOSWcontaining 0.95mgIlof Ethoquad gavean RSO of3.7%. Six repetitive injectionsofonestandard solution in OECOwater containing 0.21 mgJI.. ofEthoquad gavean RSD of2.9 %. Six repetitive injectionsofonestandardsolution in OSWcontaining 0.21 mgl1.. of Ethoquad gavean RSDof 1.8 %. Six repetitive injectionsofonestandardsolution in OECDwater containing 10.6mglL. ofEthoquadgave an RSD of3.5 %. limitofquantitation The lowerlimitofquantitation is0.1 mgn. The detection limit is0.02 mgn. linearity01calibration curve The calibration curve is linearwithin the rangeof0.1-2 mgnand in the range of1-100 mgn. Page 21
Technology & Engineering**'CS~103** Final Research Report ECRA F07013 T 05012 OFA April 19, 2007

# ---I~

# 1~ AKZONOBEL

AKZONOBEL Surfactanls**~**Analytical Methodsfor R&D AM46

Revised by(position,**~nature)** Dabt 10 no Section Encl Analytical Manager 2007**-O2~01 AM4~2309**  3 Approvedby(position. signature) Standardrel'erence Process QHSE Manager 1509001
Reporting Page 4016 Validuntil 2010-02-01

The result is reported as amountofEthoquad MC1214125 in mgJI with two significant numbers. OthersThesamplesare considered to be stable when refrigerated, with the addition of5% methanol,foratleast3 months atthe level of 1mgnof Ethoquad MC1214125.

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### 1.**~40** ;21**~** /I

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Ftg i.Total ioncurrent (TIC)chromatogramand mass spectrumfor Ethoquad MC1214125; Full scan. positive mode Page 22
Technology & EngineeringICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19, 2007

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Revised by(position,5ignawre) Analytical Manager Apptowedby (position. signatuf&) QHSE Manager

# 1~ AKZONOBEL

Surfactants
- Analytical Methodsfor R&D AM46

Oatft 10 no Section EncL Page 2007-02-01 AM46-2309 3 50f6 Validvntil 2010''()2-O1 Standardreference Process IS09OO1

N.;

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rig 2. SIMchromatogramand mass spectrum for Ethoquad MC1214125 at0.8 mgIL Elhoquad_C/12 Y=2.19082e+007+8.58756e+O08tX 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 graphfor Eihoquad MC121412!S: cone.range0.1-2.1 mgIL Page 23
Technology & EngineeringICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19,2007

# ~7-- {J**~r---.** 

# AKZONOBEL

AKZONOBEL Surfactants
- Analytical Methodsfor R&DAM46

Revised by(position. signall.U'e) Analytical Manager Approvedby (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. Calibrationgraph for EthoquadMCt214125; conc.range 1-100mgll. Issue 1 Page 60f6 Validun.lil 2010-02-01 Page 24
Technology & EngineeringICS-103 Final Research Report ECRA F07013 T 05012 OFA April 19, 2007 

# fJ**~~** Page 25 

# AKZONOBEL Technology



### Determination of Ethoquad 

## Me 1214/25 in waterfrom Toxicitystudies; 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 1214/25on the growth ofthe freshwater green alga pseudokirchnieriella subcapitata" • T 05012 ODA "Acute toxicity of Ethoquad MC 1214/25to waterfleas, daphnia magna" • T 05012 OFA "Acute toxicity of Ethoquad MC 1214/25to 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 

### ITest 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 ODA0.8 m 

### IL t=Oh 30-1-2007 T 05012 ODA0.8 m 

### IL t=48h 1-2-2007 T 05012 ODA3.2 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 ofanalysis: Laboratory: The samples were refrigerated prior to analysis. 27-2-2007 to 5-3-2007. Analytical Laboratory R&D 

### SBU SE Akzo Nobel Surface ChernistryAB StenungsundICS-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 ofthe 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 of5 % (v/v) has been done. Table 2

Sample code 

### 1Test 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/Lt=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 05012AL 1.0 mg/Lt=Oh 1.1 1.1 T 05012AL 1.0 mg/L t=72h 0.95 1.0 T 05012 ODA Control t=Oh < 0.1 < 0.1 T 05012 ODAControl t=48h < 0.1 < 0.1 T 05012 ODA0.2 mg/Lt=Oh 0.17 0.17 T 05012 ODA0.2 mg/L t=48h 0.17 0.15 T 05012 ODA 0.8 mg/Lt=Oh 0.76 0.78 T 05012 ODA0.8 mg/Lt=48h 0.79 0.80 T 05012 ODA 3.2 mg/Lt=Oh 3.1 3.3 T 05012 ODA3.2 

### 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/Lt=Oh 6.4 6.4 T 05012 OFA 6.25 mg/Lt=96h 5.9 6.4 T 05012 OF A 25 mg/Lt=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/Lt=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 byevaporation before analysis. All Control samples were below detection limitwhich is 0.02 mg/L.