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 

REPORT
T12008 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 EC50 (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 EC50 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 testing 
T12008 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 3.4.2012 
T12008 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 24
T12008 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 (EC50). 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 PS/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 Ps/cm, containing per liter of deionized water: 200 mg of CaCl2·2H2O, 180 mg of MgSO4·7H2O, 100 mg of NaHCO3

and 20 mg of KHCO3 (Standard Operation Procedure O1) . The guideline criteria 

requires the CaCO3 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 CaCl2 

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 EC50 (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 (R2)  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 EC50 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 None 
T12008 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 EC50 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 1995 
T12008 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 0 
T12008 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 10

Response

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 1.8
T12008 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. 

3CH

N

O +

CH3

OH CH3

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= H2O + 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 μl 
T12008 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 

MgCl2 * 6 H2O 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

Sample peak area constant

Sample concentration



 
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 R2 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 variation 
T12008 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/L 
T12008 ODA 

23 

ANNEX 2 
T12008 ODA 

24 

ANNEX 3