Akzo Nobel N.V PO Box 9300
6800 SB Arnhem
The Netherlands Environmental Chemistry Department

1



## CONFIDENTIAL

Title 

### ACUTE TOXICITY OF QUATERNARY C12-C14 ALKYL METHYLAMINE ETHOXYLATE METHYL CHLORIDE [BEROL R648 NG] TO WATER FLEAS,

DAPHNIA MAGNA

Authors M. Kean, I. Garttener, B.Kluskens, J van Dam

Date 26-3-2012

Document number Not Yet Assigned

Study number T12008 ODA



## REPORTT12008 ODA

2



## ABSTRACT

In order to predict the effects of Quaternary C12-C14 Alkyl Methylamine Ethoxylate Methyl Chloride (Berol R648) in an aquatic environment, an acute
immobilization test to Daphnia magna was conducted in accordance with OECD test guidelines and with the OECD Principles of Good Laboratory Practice.

The toxicity of the test chemical to juvenile daphnids was determined in a static system over an exposure period of 48 hours. Nominal concentrations of 0.25, 0.56, 1.2, 2.7 and 6.0 mg/L and were tested. For the highest 2 concentrations a statistically significant effect in comparison to the control was observed.

The NOEC was determined as 1.2 mg/L and the LOEC was determined as 2.7 mg/L. (Measured all concentrations >80% of nominal)

The EC 50

(48h) was calculated as 2.58 mg/L. (Measured all concentrations >80% of nominal)

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

x Immobilization in the control did not exceed 10%

x Oxygen concentration did not fall below 3 mg/L at any point during the study

x The EC 50

value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).

x No daphnids were trapped on the surface for any length of time

x Chemical analysis demonstrated that test substance remained stable throughout the test.

The maximum variation in pH over the full test period was 0.2 pH units in the control which meets the guideline pH variation requirements.

All concentrations reported refer to product as received and have not been corrected for active ingredient.T12008 ODA

3



## STUDY ORGANIZATION

Sponsor AkzoNobel Surface Chemistry AB Stenungsund, S-44485

Sweden

Study monitor Bengt Fjallborg AkzoNobel Surface Chemistry AB Stenungsund, S-44485

Sweden

Location Akzo Nobel N.V Environmental Chemistry Department Velperweg 76

P.O. Box 9300

6824 SB Arnhem

The Netherlands

Study director M. Kean

Quality Assurance Unit H. van Daalen

Management K. van Ginkel

Experimental initiation date 31.1.2012 Experimental completion date 2.2.2012



## ARCHIVING AND STORAGE

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 AkzoNobel 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 AkzoNobel Arnhem archives for a period of ten years. Test material will be stored deep-frozen under the sample code T12008 for ten years or only as long as the quality of the test substance permits evaluation.T12008 ODA

4



## GLP COMPLIANCE STATEMENT

The study was performed in compliance with the OECD Principles of Good Laboratory Practice (as revised in 1997) ENV/MC/CHEM (98)17, 21 January 1998, including all supplements published up to the starting date of the study. 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 following aspects of the study were not conducted under GLP:

x Preliminary testingT12008 ODA

5



## QUALITY ASSURANCE STATEMENT

This report was audited by the Quality Assurance Unit contracted by AkzoNobel Arnhem. 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 phases of the study, 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 of the study Dates of inspection Dates of reporting

Study Plan 31.1.2012 31.1.2012

Test – Calibration Standard Preparation 31.1.2012 31.1.2012

Report 3.4.2012 3.4.2012T12008 ODA

6



## CONTENTS

INTRODUCTION 7

TEST GUIDELINES, MODIFICATIONS AND DEVIATIONS 7

MATERIALS 8

METHODS 10

RESULTS 11

CHEMICAL ANALYSES 12

CONCLUSION 14

QUALITY CRITERIA 12

AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN 13

REFERENCES 14

TABLES AND FIGURES 16

ANNEX 1 18

ANNEX 2 19

ANNEX 3 23

ANNEX 4 24T12008 ODA

7



## INTRODUCTION

Objectives The purpose of this test was to determine the acute toxicity of the test chemical to
the water flea Daphnia magna in fresh water. The acute toxicity to D. magna is usually expressed as the median effective concentration for immobilization. This is the concentration, which immobilizes 50% of the animals in a test batch within a period of continuous exposure (EC 50 ). Furthermore, the concentration causing no significant immobility and the lowest concentration causing significant immobility (NOEC/LOEC) in comparison to the control are also commonly determined during an acute toxicity study.

Principle of the test Juvenile daphnids (<24h old) were exposed to a series of concentrations of the test substance in the test medium, Dutch Standard Water. Under otherwise identical test conditions the effects on the mobility of Daphnia magna exposed to the test substance relation to a control without test substance was recorded over a period of
48 hours.



## TEST GUIDELINES, MODIFICATIONS AND DEVIATIONS

The Daphnia magna immobilization test was performed in accordance with OECD Guideline for testing of chemicals 202 (Ref 1) and Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Annex V, PART C: METHODS FOR THE DETERMINATION OF ECOTOXICITY, C.2: „Daphnia Acute Immobilization test“, Official Journal of the European Union, L 142, Volume 51, 31 May 2008, with no modifications or deviations.

The test was carried out in compliance with the principles of Good Laboratory Practice (GLP).T12008 ODA

8



## MATERIALS

Test material The test substance (project sample code T12008) was received from the sponsor. Data on the handling, stability, composition, purity or other characteristics of the test substance supplied by the sponsor was accepted and used without further
check.

Name Berol R648 NG

Chemical name Quaternary C12-C14 Alkyl Methylamine Ethoxylate Methyl Chloride
Purity / active ingredient 60 % (Approx)

CAS reg. no. Not yet assigned

Appearance Orange/amber viscous liquid

Water solubility Clear stock of 100 mg/L was achieved in preliminary studies

Lot S002420

Stability Will be determined analytically during the test. Preliminary
studies indicate that the test substance is stable under test
conditions.
Storage until required In original container in the dark

Expiry date 2013 06-19

See Annex 4 for CoA

Chemicals All reagents used were of reagent grade quality.

De-ionized water The de-ionized 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 P S/cm. This water was produced from tap water in a water purification system according to the relevant Standard Operation Procedure.T12008 ODA

9 Test medium The test medium was Dutch Standard Water (DSW), having a pH of approximately 8.2, and a conductivity between 550 and 650 P s/cm, containing per liter of deionized water: 200 mg of CaCl 2 ·2H 2 O, 180 mg of MgSO 4 ·7H 2 O, 100 mg of NaHCO 3 and 20 mg of KHCO 3 (Standard Operation Procedure O1) . The guideline criteria requires the CaCO 3 content to be between 10 and 250 mg/L (approximately 1-14 odH). Hardness in the test water was measured once at the start of the study using the appropriate Dr Lange test kit. This was validated by analyzing a CaCl 2 Standard. The dilution water was saturated with oxygen before the start of the test. This was done by thorough aeration for at least 30 minutes before use.

Test flasks As test vessels 50 ml glass beakers were used, containing 50 ml of test solution. All vessels were covered by glass plates during the test.

Test room, temperature control and light regime The test was carried out in a temperature-controlled room. The test temperature was set between 18 and 22°C and the actual temperature should remain constant within ± 1°C. The light regime was 16 h of ambient light per day, provided by
fluorescent tubes.

Other Apparatus Apparatus was used according to the relevant Standard Operation Procedure as detailed in the study plan. The dissolved oxygen concentrations and conductivity were determined electrochemically using an oxygen electrode / conductivity electrode and meter. The pH was determined with a pH meter. The temperature was measured with a temperature sensor and recorder. Total hardness were measured using Dr Lange test kits and checked for accuracy with appropriate
standard solutions.

Source and maintenance of the test organisms
The test animals were taken from a Daphnia magna stock, cultured in conformity with Standard Operation Procedure (E 1). The animals used in the test were less than 24 hours old were obtained from parent animals aged between 2-4 weeks. Daphnia were originally obtained from NOTOX B.V. Hambakenwetering 7 5231 DD ‘s-Hertogenbosch.

Quality control of the test organisms The sensitivity of the Daphnia was checked by performing an inhibition test with a reference compound (potassium dichromate) twice a year. The sensitivity was tested for compliance with the guidelines.T12008 ODA

10



## METHODS

General test principles and procedures The test was performed as a static test for 48 hours. 20 animals divided into 4
batches of 5 animals were tested at each of 5 test concentrations and in the control. Those animals which are not able to swim within 15 seconds after gentle agitation of the test vessel are considered to be immobile. The number of animals being trapped at the surface was determined. These animals were not regarded as immobile and were made to re-submerge. In summary the daphnids were randomly placed in the test fluids and the test vessels were placed in a random manner within each group. The test vessels were not aerated during the test and the animals were not fed. The test was inspected at 0, 24 and 48 hours.

Determination of dissolved oxygen, pH, temperature and water
hardness. The dissolved oxygen and pH was determined before the start and at the end of the test in the highest and lowest test concentrations and in the control. During the test the temperature in the test room was measured continuously in an additional test vessel filled with de-ionized water only under identical conditions as the actual test vessels. The hardness in the batch of water used for testing was measured once only.

Preparation of solutions A stock solution of 109 mg/L of the test substance was prepared by accurately weighing 0.0109 g of test substance, on an analytical balance. The test chemical was then added to approximately 80 mL the test media and was sonicated for 1 minute. A clear homogeneous stock solution was achieved. This solution was then made up to 100 ml with the test media and used to generate test concentrations below. The stock was continually stirred during use. The pH was measured as 8.1 this was not adjusted further.

Test concentrations Following a preliminary test the following test concentrations were chosen. The stock solution be diluted in test media to achieve the following concentrations: 0.25, 0.56, 1.2, 2.7 and 6.0 mg/L. 4 Control replicates containing only test medium
were also included in the test.T12008 ODA

11 Sampling and preparation for analysis Samples of 10 ml were taken at the start of the test at all concentrations, in the stock solution and in the control and diluted at least 1:1 with leaching solution and further if required. At the end of the test a pooled 30ml from all of the replicates of the test concentrations were sampled and diluted 1:1 with leaching solution. All samples were shaken in the leaching solution for 30 minutes at 150 RPM before being diluted further as required (in leaching solution) and filtered as required. See annex 1 for analytical method.

Chemical analysis All samples taken were analysed following the method and apparatus settings as
detailed in Annex 1.



## RESULTS

Preliminary test

A preliminary test was conducted using identical methods and materials as detailed for the definitive test but without chemical analysis and with less test replicates. The basic validity criteria were met during the preliminary study and the results were considered sufficient to determine the test range for the
definitive test.

Definitive test

The NOEC was determined as 1.2 mg/L and the LOEC was determined as 2.7mg/L. (Nominal Concentrations)

The EC 50

(48h) was calculated as 2.58 mg/L. (Nominal Concentrations)

The Williams test was used for the NOEC/LOEC determination and the Trimmed spearman Karber method was used for the EC50 determination. Normality and equality of variance of the data could not be confirmed. All calculations were conducted using the ToxCalc 5.023 software (Ref 3).

A Non Parametric statistical test (Steels many –one rank test) confirmed the NOEC and LOEC results reported above calculated by the Williams test.

Raw data is displayed in Table 6.T12008 ODA

12 pH, temperature, oxygen and hardness.

The pH measurements show that the pH remained stable in the test in all concentrations. The temperature varied from 19.7 to 21.05 °C during the test. The oxygen concentration did not fall below 3 mg/L at any point during the test. All measurements including hardness were in accordance with the required conditions described in the study plan. See tables 4 & 5.

Chemical Analysis

Table 3

Test samples T=0

conc. (mg/L) % Recovery of

Nominal

control <LOD
- 0.25 0.22 88

0.56 0.51 91

1.2 1.09 91

2.7 2.51 93

6.0 5.39 90

Stock 101.08* 93

Test samples T=48h

conc. (mg/L) % Recovery of

Nominal

control 0 - 0.25 0.22 88

0.56 0.51 91

1.2 1.14 95

2.7 2.57 95

6.0 5.76 96

Stock n.d.** Not analyzed.

*= the stock was prepared at an initial concentration of 109 mg/L

**=stock was not determined at the end of the test.

Table 3 A

Parameters Limit Actual Linearity (R 2 )  0.98 0.9997 Repeatability (CV (%)) at lowest standard  20 1.7 Repeatability (CV (%)) at highest standard  20 0.9 LOQ (μg/L) <10 1.24 LOD (μg/L) <10 0.37 System stability (% of nominal)  10 4.0 Recovery from medium (%)*  70 and  120 >88%

*Recovery from medium was determined in the stock at T=0 (table 3) above.T12008 ODA

13 The results of the chemical analysis demonstrate that the main component of the test chemical detected in the test medium remained stable during the test period and was present in concentrations close to that of the nominal concentrations (> 80 % <120 % of the nominal concentrations). The nominal concentrations were therefore used for endpoint determination.



## QUALITY CRITERIA

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

x Immobilization in the control did not exceed 10%

x Oxygen concentration did not fall below 3 mg/L at any point during the study

x The EC 50

value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).

x No daphnids were trapped on the surface for any length of time

x The measured concentration of the test chemical remained stable during the study.



### AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN

Amendments to the study plan

x The study plan was amended. Test medium on page 4 should read “DSW” not “natural spring water”. On page 7 study plan should read “samples should be diluted 50:50 with leaching solution” not “with 50% leaching

solution”.

x The study plan was amended to indicate that should storage stability samples be needed, that triplicate samples would be taken.

Deviations from the study plan

x NoneT12008 ODA

14



## DISCUSSION / CONCLUSION

The toxicity test met the critical validity criteria in the guideline and in the study plan for both the toxicity test and analytical measurements.

A dose response curve with confidence limits was calculated and an EC 50 was determined. NOEC and LOEC values were also calculated but homogeneity and normality of data could not be confirmed. Due to the robustness of the test used (Ref 7) this is not expected to significantly influence these values. This was double checked with a non parametric method that confirmed these results.

Test substance analysis demonstrated that the main component present in the test medium was stable throughout the test period and use of nominal concentrations is justified.

The test can be considered an accurate representation of the test substance

effects to daphnia magna

.T12008 ODA

15



## REFERENCES

Referred to or used in the preparation of the report.

1. OECD, 2004. Daphnia sp., Acute immobilisation test. Guidelines for testing

of chemicals nr. 202, 13 April 2004. Organization for Economic Co-operation

and Development, Paris.

2. EEC, 1989. Directive 79/831, Annex V, part C.: Methods for the

determination of ecotoxicity, part C.2. Acute toxicity to Daphnia magna

(updated version 11/1989).

3. ToxcalcTM User Guide Version 

### V 5.0.23 , 1996

4. Asking the right questions Ecotoxicology and Statistics SETAC 1995T12008 ODA

16



## TABLES AND FIGURES

Table 4 pH measurements

Nominal test

concentration

(mg/L) Time (hours)

0 48

Control 8.0 8.2

0. 25 8.0 8.2

0. 56 8.1 8.3

1.2 8.1 8.3

2.7 8.1 8.3

6.0 8.1 8.3

Table 5 Oxygen measurements

Nominal test

concentration

(mg/L) Time (hours)

0 48

Control 9.1 9.1

0. 25 9.0 9.0

0. 56 9.0 9.0

1.2 9.0 9.1

2.7 9.0 9.1

6.0 9.1 9.0

Note: Test water hardness was measured as 11.9 odH in the batch used for testing.T12008 ODA

17 Table 6 Numbers of Mobile Daphnia

Conc

mg/L 0

hours 24

hours 48

hours

Control I 5 5 5

II 5 5 5

III 5 5 5

IV 5 5 5

Total 20 20 20

0.25 I 5 5 5

II 5 5 5

III 5 5 5

IV 5 5 5

Total 20 20 20

0.56 I 5 5 5

II 5 5 5

III 5 5 5

IV 5 5 5

Total 20 20 20

1.2 I 5 5 5

II 5 5 5

III 5 5 5

IV 5 5 5

Total 20 20 20

2.7 I 5 5 1

II 5 5 3

III 5 5 3

IV 5 5 2

Total 20 20 9

6.0 I 5 5 0

II 5 5 0

III 5 5 0

IV 5 5 0

Total 20 20 0T12008 ODA

18 Statistical Calculations (Including Non parametric confirmation)

Figure 1
* = Statistical difference from the control Daphnia Immobilisation Test according OECD-48 Hr Survival

Test ID: T12008

Lab ID: Env Chem

Protocol: OECD-202-Acute Immobilisation Test DM-Daphnia magna

Comments: Berol R648 Conc-mg/L 1 2 3 4

0 1.0000 1.0000 1.0000 1.0000

0.25 1.0000 1.0000 1.0000 1.0000

0.56 1.0000 1.0000 1.0000 1.0000

1.2 1.0000 1.0000 1.0000 1.0000

2.7 0.2000 0.6000 0.6000 0.4000

6 0.0000 0.0000 0.0000 0.0000

Transform: Arcsin Square Root 1-Tailed Number Total Conc-mg/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Resp Number

0 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 0 20

0.25 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 0.000 2.360 0.1503 0 20

0.56 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 0.000 2.360 0.1503 0 20

1.2 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 0.000 2.360 0.1503 0 20 *2,7 0.4500 0.4500 0.7301 0.4636 0.8861 27.587 4 9.658 2.360 0.1503 11 20

6 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 20 20

Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates non-normal distribution (p <= 1) 0.57538 0.905 -1.3039 7.42774 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 5) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df

Dunnett's Test 1,2 2,7 1.8 0.08473 0.08918 0.30273 0.00811 1.3E-07 4, 15

Trimmed Spearman-Karber

Trim Level EC50 95% CL

0,0% 2.5855 2.1616 3.0924
5,0% 2.5759 2.1121 3.1416
10,0% 2.5664 2.0554 3.2045
20,0% 2.5476 1.9081 3.4014 Auto-0,0% 2.5855 2.1616 3.0924

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0.1 1 10Response

Dose mg/L

Daphnia Immobilisation Test according OECD-48 Hr Survival

Test ID: T12008 ODC Lab ID: Environmental Chemistry

Protocol: OECD-202-Acute Immobilisation Test Test Species: DM-Daphnia magna

Comments: Non Parametric check of NOEC/LOEC Conc-mg/L 1 2 3 4

0 1.0000 1.0000 1.0000 1.0000

0.25 1.0000 1.0000 1.0000 1.0000

0.56 1.0000 1.0000 1.0000 1.0000

1.2 1.0000 1.0000 1.0000 1.0000

2.7 0.2000 0.6000 0.6000 0.4000

6 0.0000 0.0000 0.0000 0.0000

Transform: Arcsin Square Root Rank 1-Tailed Conc-mg/L Mean N-Mean Mean Min Max CV% N Sum Critical

0 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4

0.25 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 18.00 10.00

0.56 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 18.00 10.00

1.2 1.0000 1.0000 1.3453 1.3453 1.3453 0.000 4 18.00 10.00
*2,7 0.4500 0.4500 0.7301 0.4636 0.8861 27.587 4 10.00 10.00

*6 0.0000 0.0000 0.2255 0.2255 0.2255 0.000 4 10.00 10.00

Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates non-normal distribution (p <= 1) 0.52249 0.916 -1.4086 9.12644 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 5) NOEC LOEC ChV TU Steel's Many-One Rank Test 1,2 2,7 1.8T12008 ODA

19



## ANNEX 1

Analytical procedure for the quantification of Berol R648 NG in aqueous samples using an LC-MS/MS system

Introduction

A method is described to determine the concentration of the test substance in aqueous samples (i.e. Dutch standard water) at the beginning and end of the test. Procedures and instrumentation were based on High Performance Liquid Chromatography combined with mass spectrometry (LC-MS/MS). The test substance is a mixture of a component with multiple ethoxy groups. The component visible with the highest response in the test medium was considered the most relevant component for the ecotoxicity testing and was considered to be the main component. Samples were quantified using a calibration curve.

The main component visible in the test medium had a mass of 654.5 and can be represented by the chemical structure as indicated below in figure I.

CH 3 N + O CH 3
CH 3 OH

x

Figure I: Structure of Berol R648 NG x= number of ethoxy groups (main component contains in total 10 ethoxy groups)

Analytical procedure The following conditions were used to determination of the test compound at the start of the test. The conditions for HPLC are given in table I, for MS in table II
and III.

Table I: Parameters for HPLC (Agilent Technologies 1200 series)

Column: Zorbax SB-C18 2.1x30 mm 3.5μm

Column temp.: 50 °C

Flow: 0.5 ml/min

Mobile phase: A= H 2 O + 2% (v/v) Formic acid B= Methanol + 2% (v/v) Formic acid

Gradient mobile phase: 0 min. 100 % A 0 % B

2 min. 0 % A 100 % B

5 min. 0 % A 100 % B

5.1 min. 100 % A 0 % B

7 min. 100 % A 0 % B

Injection volume: 10 μlT12008 ODA

20 Table II: Parameters for MS (Agilent Technologies G 6410B, triple quad)

Ion source: ElectroSpray Polarity: Positive mode

Detector mode: Multiple Reaction Mode

Delta EMV 300 V

Source parameters: Gas temperature: 350 °C
Gas flow: 12 l/min Nebulizer: 60 psi Capillary voltage: 3000 V

Table III: MS acquisition parameters

Compound Precursor
Ion MS 1

RES Product

Ion MS 2

RES Dwell

time (Ms) Frag.

(V) Collision

Energy (V)

Berol R648 NG 654.5 Unit 72.2 Unit 200 218 63

Sample preparation For the standards a stock solution of the test substance was made in leaching solution at a concentration of 764 mg/L. This Leaching solution contains 100 g/L MgCl 2 * 6 H 2 O in a methanol: 2-propanol 50:50 (v/v) solution. From this stock solution dilutions were made in the concentration range 0 –1000 μg/L for the calibration standards (n  5). Control standards from the middle range of the calibration series were analyzed at a minimum rate of one per ten samples and
after each test series.

Aqueous samples Aqueous samples were diluted 50:50 (v/v) with leaching solution. Further dilutions if necessary were done in leaching solution.

Calculation of concentrations Quantification was done by measurement of peak areas. The concentrations of the test substance in the samples were calculated from the relation between concentration of standards (Cs) and peak area (PAs) obtained with linear regression analysis:

PAs = Cs * Slope + constant

slope constant area peak Sample ion concentrat Sample T12008 ODA

21 Method validation

Linearity The analytical method was found to be linear over the concentration range of 0 – 1000 μg/L with an R 2 of at least 0.9997 using the system settings described above (paragraph: Analytical procedure). See figure II, calibration curve 31-01- 2012 (start of the test) as an example of the linearity of the calibration curve.

Figure II: calibration curve 31-01-2012 (start of the test)

Repeatability Six aliquots of the lowest and highest standard concentration prepared from a single homogenous sample were analyzed. Mean values, standard deviations and variation of coefficients are given in the table below. Repeatability of the analytical method was concluded to be acceptable hence the coefficient of variation was below the required 20%.

Table IV repeatability lowest and highest standard concentration Sample Response (counts) of

Main component Sample Response (counts) of main component 25 μg/L # 1 2743.4 1000 μg/L # 1 97753.0 25 μg/L # 2 2669.0 1000 μg/L # 2 96760.1 25 μg/L # 3 2701.4 1000 μg/L # 3 97772.2 25 μg/L # 4 2698.8 1000 μg/L # 4 97076.8

25 μg/L # 5 2616.0 1000 μg/L # 5 97482.5 25 μg/L # 6 2641.5 1000 μg/L # 6 95421.3

mean 2678.4 mean 97044.3

stdev 45.8 stdev 887.7 CV (%) 1.7 CV (%) 0.9

SD = standard deviation
CV = coefficient of variationT12008 ODA

22 The Limit of Detection (LOD) was calculated using the signal to noise ratio (S/N). The LOD is calculated as 3 * S/N whereas the Limit of Quantification (LOQ) is
calculated as 10 * S/N. S/N was determined with the software of the LC-MS/MS (i.e. Mass Hunter) based on peak height. (signal definition; peak height, noise definition; peak to peak, noise region; retention time 5.0 – 5.3 min.) from the lowest concentration standard i.e. 25 μg/L. The LOD and LOQ values are reported in table V.

Table V: LOD and LOQ values (and signal to noise ratio)

Sample Signal/noise
ratio LOD LOQ

25 μg/L 31

- 01

- 2012 202.3 0.37 μg/L 1.24 μg/L 25 μg/L 02

- 02

- 2012 213.7 0.35 μg/L 1.17 μg/L

Accuracy/Precision The control standards analyzed during sample series were all within the specified quality criteria i.e. remaining within 10% of the expected values based on the
calibration curve standard see table VI.

Table VI: Control standards Sample Response (counts) % of

nominal

calibration st. 31

- 01

- 2012 25184.5 --

control st. 31-01-2012 23671.6 94.0

calibration st. 02

- 02

- 2012 25914.4 --

control st. 02-02-2012 24876.0 96.0

Note: 250μg/L Nominal

Example chromatogram

Figure III: calibration standard 100 μg/LT12008 ODA

23



## ANNEX 2T12008 ODA

24



## ANNEX 3