## Final Research Report ECRA F07012 T 05012 ODA Apr i l19, 2007 M.G .J . Geurts , J. van Dam a n d 

### L. Lofgren ACUTE TOXICITY OF WATER FLEAS, 

### DAPHNIA MAGNA

ICS- 103 

### CONFIDENTIAL Project identification Client Subclient Client No Subclient No Technology & Enginee r ing

Page 

### 1 of 

### 26 Project No Task No 859 . 024 Drafted by department Environmental Chemistry and Regulatory AffairsI CS-103 Final Research Report ECRA F07012 T05012 ODA Apri119 , 2007

ABSTRACT 

### The acute toxicity of Page2 Technology & Engineering



### to 

### Daphnia magna (water flea) was tested under static conditions for 48 hours in accordance with OECD and EEC guidelines for testing chemica l s . The test was performed in compliance w ith Good Laboratory Practice (GLP). The 48 h EC50 

### was calculated to be 1.21 mg/L with 95% confidence limits of 1.06 and 1 .38 mg/L. The h ighest test concentration causing no immobility (no observed effect concentration, NOEC) after 48 hours was observed at 0 .8 mg/L . These results are ca lculated 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 a re obtained : 48 h EC 50 

### = 1 mg/L and a NOEC of 0 .66 mg/L. Sub-lethal effects, such as floating at the surface , were not observed in the test conce ntrations where no immobility was observed . The results of the chemical ana lyses show that the initial concentrations in the test solutions were close to the nominal (expected) concentrations . A decrease of< 6% of test substance concentrat ion during the 48 hours of testing was observed. The effects observed are therefore expressed based on their nomina l concentrations.;;; ;,. ICS-103 Final Research Report ECRA F07012 T05012 ODA April 19 , 2007

ACUTE TOXICITY OF Sponsors Study monitor STUDY ORGANIZATION Location

Study director Quality Assurance Unit Management Experimental initiation date Experimental completion date ARCHIVING AND STORAGE 

### DAPHNIA MAGNA Akzo Nobel Surface Chemistry AB Se 44485 Stenungsund Sweden 

# - Page3 Technology & Engineering

Envir onmental Chemistry and Regulatory Affairs Dept.

lng . H. van Daalen lr. A.R. Luttmer 29-01-2007 23-02-2007

The project f ile incl uding the final report, amendmen ts to the final report, the study plan, amendments to the study plan, records of quality assurance inspect ions, a ll letters, memos and notes and raw data perta ining to the study will be retained in the archives of ···············

- for a period of ten years . Other records including master schedule sheet , laboratory notebooks, logbooks, re cords of the maintenance and ca li bration of equipment, summary of tra ining, cu rricula vitae and job descriptions of the personnel involved in the study, records related to l ocation and storage of the test substance w ill also be kept in the ••• archives for a period of ten years . Test material will be stored deep-frozen under the sample code T 05012 for ten years or on ly as long as the quality of the test substance pe rmits evaluation .I CS- 1 0 3 Final Resea rc h Report ECRA F07012 T05012 ODA Apri l 19, 20 0 7

GLP COMPLIANCE STATEMENT Page4 Technology & Engineering

The study was conducted in compliance with the followi ng 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 l aws. regul ations and administrative provis ions relating to the application of the Prin c iples of Good Laboratory Practice and the verification of the i r applications for tests on chemical substances (O .J . No . 

### L 77 of 23 .3 . 1999) . • Staatstoezicht op de Vo l ksgezondheid . Voedsel en Waren Autoriteit. GLP Monitoring Unit. The report contains an accurate description of the results . The test substance data were submitted by the s ponsor (unless stated otherwise) , who accepted full responsib ili ty 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 ana lyses were due to the complexity of the analytical method performed outside the GLP facil ity and the refore no GLP is c l aimed for this part of the resu lts . This has however no impact on the quality of the chem ical analyses and the final outcome of the test. Study director lng ····

Man a gement l r. A . R. Luttmer date

# 2~ 

# ' 

# 0{' - 2eD

# 7

s ignature

# ~--

date signatureI CS-103 F in al Research Report ECRA F07012 T05012 ODA April19 , 2007



### QUALITY ASSURANCE STATEMENT Page 5 Technology & Engineering
This report was audited by the Quality Assurance Unit contracted by ••••••• It is considered to be an accurate presentation of the methods and procedures applied i n the course ofthe study and an accurate reproduct ion of the data recorded. Listed below are the dates of i nspection of this study by the Quality Assurance Un it and the dates on which its findings were reported to Study D i rector and Management. 

### Phase ofthe study Study plan Test (preparation of the test substance stock solution) Report 

### Quality Assu rance Un it lng . 

### H. van Oaalen 

### Dates of inspection 

### 30-01-2007 30-01-2007 04-04-2007 Dates of reporting 

### 30-01-2007 30-01-2007 04-04
-2007ICS-103 Fi nal Research Report ECRA F07012 T050 1 2 ODA April19, 2007 Page6 Technology & Engineering CONT ENTS 

### 1 . INTRODUCTION 

### 7 2. TEST GUIDELINES, MODIFICATIONS AND DEVIATIONS 7 3. MATERIALS 

### 8 3.1 Test substance 

### 8 3.2 Chemicals 

### 8 3.3 De-ionized water 

### 8 3 .4 Test vessels 

### 8 

### 3 .5 Test room and light regime 

### 9 3 .6 Test an i mals 

### 9 4 . METHODS 9 4 . 1 Test principle and procedures 

### 9 4 .2 Prepara1 tion of the test medium 

### 9 4 .3 Preparation of the stock solution 10 4 .4 Preparation of the test solutions 10 4 .5 Chem ical analys is 10 4 .6 Determinatio n of dissolved oxygen, pH, conductivity , water hardness and temperature i n the test sol utions 10 4. 7 Evaluation of data 11 5. RESULTS 11 5.1 Toxicity 11 5.2 Ch e m ical analysis 11 5 .3 Oxyg e n, pH, conductivity, water hardness and temperature 12 6 . QUALITY CR ITERIA 12 7. AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN 7.1 Amendments to the study plan 7.2 Deviations form th e study plan 

### 8. REFERENCES ANNEX 1 ANNEX 

### 2 ANNEX3 12 12 12 13 

### 17 18 24ICS-103 Final Research Report ECRA F07012 T05012 ODA April19. 2007



### 1 . I NTRODU CTION 

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

### (water flea) under continuous exposure during 48 hours in a static test. Page 7 T

ec hnology & En g in ee ring



### to Daphnia



### Acute toxicity is the discernible adverse effect induced in an organism with in a short time of exposure to a substance. The acute toxici ty of a test substance is expressed as the median effective concentration (EC 50), 

### which is the concentration in water, which immobilizes 50% of the test animals within a period of continuous exposure. Those animals, which were not able to swim for 15 seconds after gentle agitation of the test vessel, were cons idered to be immobi le.. Other endpoints that. if possible. were derived from the results are the highest tested concentration causing no immobilization (NOEC), the lowest tested concentration with significant immobility as compared to the control (LOEC) and the lowest concentration causing 100% (EC 100 ) 

### imm obility . The swimming ability and other sub-lethal effects, such as being trapped at the surface, were recorded after 24 and 48 hours, if observed . 2 . T E ST G UIDELINES , MODIFI CATIONS AND DEVIATIONS 

### The study was carried out in accordance with OECD (8.1) and EEC (8.2) guidelines for testing of chemicals with one modification of the test guideline: o The chemica l ana lyses is not performed under GLP conditionsICS-103 Final Resear ch Report ECRA F07012 T05012 ODA April19, 2007

3. MATERIALS 3.1 Test substance A sample PageS Technology & Engineering

(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 unti l required 6506001437 82% Brown viscous liquid Soluble Not available A has a very low vapour pressure Assumed to be stable under test conditions At room temperature in the dark For additional information on the compound see Annex 1 . 3.2 Chemicals and

All reagents used were of reagent grade quality and obtained from J.T. Baker Chemicals BV, Deventer, the Netherlands , ACROS chemicals (formally known as Janssen Chimica), Geel , Belgium and Fluka Chemie, Buchs , Switzerland. 3.3 De-ionized water The de-ionized water used contained not more than 0.01 mg/L of copper, had a N on purgable organic carbon (NPOC) -co ntent of not more than 2.0 mg/L and a conductivity of less than 5 JlS/cm. This water was produced from tap water in a water purification system according to Standard Operation Procedure K 10 (8.8). 3.4 Test vessels As test vessels 50 ml glass beakers were used, containing 50 ml of test solution. They were cove red by glass plates during the test.ICS-103 Final Research Report ECRA F07012 T05012 ODA April 19, 2007



### 3.5 Test room and light regime Page 9 Technology & Engineering



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

# 22oc and the actual temperature was kept constant within 

### ± 1 

# oc. The light regime was 16 h of ambient light per day, provided by fluorescent tubes. 3.6 Test animals 

### The test was performed with Daphnia magna 

### Clone 5 (water fleas), which was obtained from NOTOX at 's Hertogenbosch according to Standard Operation Procedure E 1 (8.3) . The animals used in the test were less than 24 hours old at the beginning of the test and were obtained from parent animals having an age of 2-4 weeks. 

### 4. METHODS 4.1 Test principle and procedures 

### The test was performed as a static test The total duration was 48 hours. 20 daphnids divided into 4 groups of 5 animals were used at the test concentration and control. The daphnids were exposed to the chosen concentrations of the test substance as described below and immobility and sub-lethal effects were recorded at approximately 24 and 48 hours. The daphnids were considered immobile when they were not able to swim for 15 seconds after gentle agitation of the test vessel. In addition to immobi lity, sub-lethal effects such as floating at the surface were recorded. The daphnids were random ly placed in the test solutions and the test vessels were positioned in a random manner. During the test the vessels are covered with glass plates. No aeration of the test substance too k place during the test period. The animals were not fed during the test. 4.2 Preparation of the test medium 

### The test medium, so called Dutch Standard Water (DSW), was used, having a pH of approximately 8 .2, and a conductivity between 550 and 650 1-JS/cm, containing per liter of de-ionized water: 200 mg of CaCI 2

### ·2H2

### 0, 180 mg of MgS04

### ·7H2

### 0, 100 mg of NaHC0 3 

### and 20 mg of KHC0 3 

### (Standard Operation Procedure01 

### (8.12)). The test medium was aerated before being used in the test. The air was water-saturated and purified by an active coal and cotton filter according to Standard Operating Procedure K 12 (8.9). Before the start of the test the pH of the DSW was measured to be 8.6 and adjusted to 8.2 using hydrochloric acid.JCS-103 Final Research Report ECRA F070 12 T05012 ODA April 19, 2007



### 4 .3 Preparation of the stock solution Page 10 Technology & Engineering



### The test substance is soluble in water . A test substance solution of 

### 1 .004 g/L was prepared by loading 

### 0. 100 4 g of test substance, we ighed out on an analytical balance (Standard Operation Procedure K 

### 4 (8 .6)) in a 

### 100 ml volumetric flask. The flas k was filled up to the mark with test medium . The stock solution was manually shake n until a homogenous solution was obtained. The pH of the stock solution was checked and found to be 8 .2 and not adjusted . 4 . 4 Preparation of the test solutions The test solutions were prepared by additio n of the adequate amounts of stock solution to the test vessels . Based on the results of the preliminary test the following final dilutions were prepared: 

### 0 .2- 0.4 - 0 .8 - 1 .6 and 3 .22 mg/L of the test substance . Controls contain ing on l y test medium were i ncluded in the test. 4.5 Chemical analysis At the start of the test, a sampl e 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 . Th e sample was transferred into a preweighed 

### 100 ml glass bottle and weighed. Then 

### 2.5 ml of methanol was added and the bottle was again weighed . For the samples at the end of the test, the solutions with the same test concentration were pooled in a beaker and 

### 50 ml of sample was transferred without filtration into a prewe ighed 

### 100 ml glass bottle. After adding lhe sample the weight of the bottle was recorded and 

### 2.5 ml of methanol was added and again the weight was recorded . This was also done for the control. The sample bottles were stored in a refrigerator prior to transport for chemical analysis . The amount of Ethoquad MC 

### 1214/25 present in the test concentrations was quantified outside the GLP facility. Chemical ana l yses were performed according to the analytical procedure described in Annex 2 . 4.6 Determination of dissolved oxygen , pH , conductivity, water hardness and temperature in the test solutions The dissolved oxygen and pH of all test concentrations and controls were measured at the beginning and at the end of the test. Conductivity were measured only at the beginning of the test in the control solution whether its values are within limits. The temperature was measured continuously in an additional test vessel filled with DSW and kept under identical conditions as the actual test vessels. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter according to Standard Operation Procedure K 2 (8 .5) . The pH was measured using a microcomputer pH-meter according to Standard Operation Procedure K 1 (8.4) . The conductivi ty wasICS-103 Final Research Report ECRA F07012 T05012 ODA April 19,2007 Page 11 T

e chnology & E ngin ee rin g measured using a portable conductivity meter according to Standard Operation Procedure K 24 (8.10). The temperature was measured w ith a thermocouple according to Standard Operation Procedu re K 6 (8 .7). 

### 4 .7 Eva lu at i on of data If possible , the EC 50 was calculated with the computer program TOXCALCT Mversion 5.0 according to Standard Operation Procedure 

### l 2 (8 . 11 ), using the trimmed Spearman- Karber method (8 . 13). The concentrations causing zero and 100% immobility was. if possible, deri ved directly from the test observations. 

### 5 . R E SULTS The results presented in this chapter are based on the performed definitive test. 

### 5 .1 To x ici ty Pr

e l i m ina ry t est Durin g the preliminary range finding test, which was performed as a non-GLP test, effects were observed which lead to the conclusion that an EC 50 for the test substance should be found close to 1 mg/L. 

### Defin i ti ve t est The results of the test on mobility are summarized Table 1 . The results a re reported based on nominal concentrations . The 48 h EC 50 - was calculated to be 1.21 mg/L with 95% confidence lim its of 1.06 and 1.38 mg/L. The highest test concentrat ion where no effect were observed was 0.8 mg/L. These results are calculated based on the test substance as received . According to the CoA the test substance contains 82% trimethylalkyl amine ammonium salt. After correction to 100% trimethylal kyl am i ne ammonium salt (active ingredient) the following effect data are obtained: 48 h EC50 

### = 1 mg/L and a NOEC of 0 . 66 mg/L. T he dose-effect curve presenting the percent immobil ization against the concentration after 48 hours of exposure is presented in figure 1 . 

### 5.2 Chemic al anal ys i s The resu l ts of the chemical analyses are presented i n tab le 4. Due to the complexity of the method of ana lyses , the analyses were performed outside the G L P facility. Details on the method applied are described in Annex 2 of this report. The sample weights wh ich were used to compensate for dilution of the samples by methanol are presented in Table 5 .ICS-103 Fi nal Research Report ECRA F07012 T05012 ODA April19,2007 Page 12 Technology & Engineering 

### The results of the chemical analyses show that no test substance was detected in the control samples; the result also show that the concentration of the test compound during the test remained within 80% of the concentration at the beginning of the test. Because the test substance was also used as the sta ndard for chemical analyses, the results as presented are calculated based on the test substance as received . Based on these arguments the effects observed are expressed based on nominal concentrations. 5.3 Oxygen, pH , conductivi ty, water hardness and temperature 

### The maximum variation in oxygen concentration during the test was 0 . 3 mg/L. The oxygen concentration during the test ranged from 8.9 to 9.2 mg**L** 

### and the values observed are summarized i n table 2. The initial p H value observed of DSW was 8.2. The maximum variation in pH during the test was 0.4 units . The pH observed during the test ranged from 8.2 to 7 .8 units and the values observed are summarized in table 3. The conductivity of DSW was 590**~s/cm.** 

### The temperature in t he test vessels during the test period ranged from 20 .25 to 20.40° C . 6. QUALITY CRITERIA 

### The following quality criteria have been met in the present study: • Immobi lization i n the co ntrol did not exceed 10% at the end of the test. • In accordance with OECD guideline 202 (8.1) the oxygen concentration was not lower than 3 mg/L at the end of the test. • There were no test anima l s trapped at the surface of the water in the control group. • In accordance with OECD guidelin e 202 (8.1) and EEC guideline C2 (8.2), the che m ical analyses carried out showed that the concentration of the test compound during the test remained within 80% of the concentration at the beginning of the test. 7. AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN 

### 7. 1 Ame ndments to the study plan 

### There were no amendments to the study plan 7 .2 Deviations form the study plan 

### There were no deviations to the study planICS-103 Final Research R· eport ECRA F07012 T05012 ODA April 19,2007



### 8. REFERENCES Page 13 Technology & Engineering



### 8.1 OECD, 2004. Daphnia sp . , Acute im mobilisation test. Guidelines for testi ng of chemicals nr. 202, 13 April2004. Organization for Economic Co-operation and Development. Paris. 8.2 Directive 67/548/EEC, Annex V, part C: Methods for the determination of ecotoxicity Part C .2 . Acute toxicity to Daphnia magna (updated version 11 /1989). 8.3 Standard Operation Procedure E 1: Culturing of Daphnia magna 

### 8.4 Standard Operation Procedure K 1: pH-meter 8.5 Standard Operation Procedure K 2: Oxygen meters 8.6 Standard Operation Procedure K 4: Balansen 8. 7 Standard Operation Procedure K 6: Thermo meters 8..

8 Standard Operation Procedure K 10: Deionizer 

### 8.9 Standard Operation Procedure K 12: Lu chtfilters 8.10 Standard Operation Procedure K 24: Geleidbaarheidsmeter 8.11 Standard Operation Procedure L 2: Acute toxicity test of fish and Daphnia magna 

### (calculation of effects with the TOXCALCTM computer program) 8. 12 Standard Operation Procedure 

### 0 1 : Preparation method for culture and test media 8.13 M.A. Hamilton, P.C. Russo and R.V. Thurston, 1977: 'Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays'. Env.Sci. 

### & Techno!. 11, 714- 719. Correction, 12 (1978) 417 8.14 C.E. Stephan, 1977: 'Methods for calculating an LC 50'. 

### In: Aquatic toxicology and hazard evaluation, F .L. Mayer and J.L. Hamel ink (Eds.), 65-84 8.15 OECD Guideline no. 1 (1998), Directives 88/320/EEC and 1999 /12/EC.ICS-103 Fina l Research R, eport ECRA F07012 T05012 ODA April19, 2007

T a ble 1 : Test res u lts Nominal test substance Batches concentrat ion (mg/L) Batch I Batch II 0 (cont rol) Batch Ill Batch IV Total Batch I Batch II 0.2 Batch Ill Batch IV Total Batch I Batch II 0.4 Batch Ill Batch IV Total ! Batch I Batch II 0 .8 Batch Ill Batch IV Total Batch I Batch II 1 .6 Batch Ill Batch IV Total Batch I Batch II 

### 3.2 Batch Ill Batch IV Total I 0 hours 5 5 5 

### 5 20 5 5 5 5 

### 20 5 5 5 5 20 5 5 5 5 20 5 5 5 5 20 5 5 5 5 20 T echnology & Engi n ee r i ng

Number of mobile animals 24 hours 48 hours 5 5 5 5 5 5 

### 5 5 20 20 5 5 5 5 5 5 5 5 

### 20 20 5 5 5 5 5 5 5 5 20 20 5 5 5 5 5 4 5 5 20 19 5 2 5 0 5 1 5 0 20 

### 3 5 0 5 0 5 0 5 0 20 0

# .. " ICS-103 Final Research Report ECRA F07012 T05012 ODA April19, 2007

1 .0 0 .9 0 .8 0.7 Q) 0 .6 fl) r:: 

## &. 0 . 5 fl)

**~** 0.4 0.3 02 0 . 1 0.0 0 . 1 1 Dose m g/L 10 

### Fi gur e 

### 1 . Dose-response curve at 48h exposure to Table 2 : Oxygen measurements ( mg/1 ) nominal test substance 0 hours 48 hours concen tration (mq/l) 0 9 . 1 9.0 0.2 9.2 8.9 0.4 9 . 2 9 . 0 0 . 8 9 . 1 9.0 1 . 6 9 . 1 9.0 3 .2 9 . 1 8 .9 

### Tabl e 3: pH m eas u re m e nts 

### 1 Nominal test

substance 0 hours 48 hours concentration (mg/l) 0 8 .0 7.7 0.2 8.1 7.8 0.4 8 . 1 7.8 0 . 8 8.2 7.8 1 . 6 8 .2 7.8 3.2 8.2 7 .8 Page 15 Technology & Engi neeringICS-103 Final Research Report ECRA F07012 T05012 ODA April19. 2007

Table 4: The results of the chemical ana lyses

Sampl e c od e I Test numbe r T 05012 ODA Control t=Oh T 050 1 2 ODA Co ntrol t=48h < 0. 1 T 05012 ODA 0.2 mg/L t=Oh 0. 1 7 T 050 1 2 ODA 0.2 mg/L t=48h 0. 1 7

T 05012 O DA 0.8 mg/L f;;Oh 0.76 T 05012 ODA 0.8 mg/L t=48h 0.79

T 05012 ODA 3.2 mg/L t=Oh 3.1 T 05012 ODA 3.2 mg/L t=48h 3.3 < 0.1 0. 17 0. 1 5 0.78 0 .80

3.3 3.3 All Control sam p les were below detection l i m it wh ich is 0. 02 mg/L. T a bl e 5 : Sample weight Page 16 Technology & Engi n e ering

<0.1 0.17 5.9 0. 1 6 0.77 -3.2 0.795 3.2

- 3.1 3.3

## .:_:~.-~:;.~**~~ :~'.** **;~t~~~**

# ..**~~~;~ ?~0\::.,.·::~:~:~,::** **~:·;: ~~~;r-r~:f:·'~i** **Tn~~r~: ~::J(;_(;_:·::** **~;~:~:·~ :r:;_~~~;::::i,::**  

# ..:}.

·**~~: :·,:;** **!~:** · ..

## ~:·,:':::.. ::

## =:~·.::~:~::;**-~:** . T 05012 ODA Contol t=Oh 30 - 1-2007 85.06 13 4 .71 136.72 T050 1 2 O DA Control t=48h 1-2-2007 84.99 134.86 136.83 T 05012 ODA 0.2 mQ/L t=Oh 30-1-2007 85.55 134.72 136.79 T050 1 2 ODA 0.2 mg!L t=48h 1-2-2007 85.4 135.33 1 37.34 T050 1 2 ODA 0.8 mg/L t ;;Oh 30- 1 -2007 85.35 134 .48 136.57 T05012 ODA 0.8 mg/L t=48h 1-2-2007 84.87 134.74 136.74 T05012 ODA 3.2 mq/L t=Oh 30-1 -2007 85.02 134.22 1 36.28 T 05012 ODA 3.2 mg/L t=48h 1-2-2007 85.02 134.81 136.79ICS-103 Final Research Report ECRA F07012 T05012 ODA April 19, 2007

PEG 1000-1500 F,...11mine Alcottol ethoX)'Iate F ally amine ethoxylate 

# - Certificate of Analyze Page 17 Technology & Engineering 

### ANNEX 1ICS-103 Final Resear ch Report ECRA F07012 T05012 ODA April19, 2007



### Method of a n a lyses

Su rfactant• - Analytical M ethods for R&D AM46 Re¥ised by (pc>Yian.**~hn)** Analytical M anager Ajlp'owed by {position. siglgture) QHSE Manager 

### o- IOno 2007..02-01 AM46-2309 S'-Sanhfetene• Proc:4Ks 1509001 s.ction End_ 3**Pa~** 1 of 6 Validunli 2010..02..01 

### No . 309 

### trace levels in wate r Procedure Sign Usa Lofgren Purpose/Backgrou nd

Remarlt literature Principle

Safety

Apparatus The method is used forthe determination o at low levels in OECO water (freshwater algae medium) and OSW water (daphnia test medium) .
ANL07002
The samples are analyzed rot 1 OECD water (freshwater algae medium) and OSW water (daphnia test med ium) . The samples are analyzed by revened phase liquid c:hromatography and mas. spectrometric detection . The mass spectrometer is run in selective ion monitoring (SJM) with eleclroapray ionization (ESt) in positive mode . Extemal standard calibration is used . Acetonitri and methanol are highly flammable . They are harmful by inhalation, In contactwith sltin and ifswalowed . They are ifrit111ing to eyes. See safety data sheebl 5-3072 and Safewin 40247. Acetic acid and can cause severe bums. See safety data sheet Safewin 40293 . Wear suitable protective gloves and handle in fume hood . Liquid Chromatograph-Mass spectrometer (LCQ Duo, Thermofinnigan), lon trap, ESI..sourca Pipette (Eppendotf Multiplpette Pro) Standard laboratory glassware. Page 18 T

echno l ogy & Engi n eerin g 

### ANNE X2ICS-103 Final Research Report ECRA F07012 T05012 ODA April19, 2007

Rw!Hd by**(po~** signalln) Analytical Manager Aflprowd by (position. signa(ure) QHSE Man119!'r

LCMS data Column : Column temp : Surfactan ta - Analytica l Methods for R&D AM-46 

### o-. IOno 2007-02-01 AM46-2309 Standovohference Process 1509001 $ed;on End. 3

HyPURITY C18 {5 urn, 2.1'50 mm (Thermo)) 40 " C 

# p. 2of6 V:alid..mil 2010-02-01

Mobile phase : A : acetonitrile/water 5:95 (vfv) + 0 . 5 % acetic acid B: acetonitril + 0 . 5 ')(, acetic acid Gradient

Flow : Pump: 0.2 mllmin Gradient Time (min) 0 . 0 20 25 27 40 A(%) 100 0 0 100 100 B (%) 0 100 100 0 0
Detector: Injection: Mass spectrometer LCQ Duo, ESJ-soorce, + mode 5JJI Detector parameters Man spectrometer: MS SIM parameters: ions 522, 566,610, 6S4, 698, 742, 786, 830, 874, 918**~** Mode: + Sheath gas 

### now (arb): 40 Aux gas floW (arb) : 5 Spray vottaga (kV) : 4 . 5 Capilary temp ("C) : 200**Cap~lary** voltage (V) : 25 Tube lens otflet {V) : 0 Cbemicals/reage n ts Acetonitrile, pa Deionized and distilled water Methanol pa 2-i>f'Opanol pa Acetic acid (1 • 00 1ii

% iii

)

# l

pa

ill•••• Reference of • 1 Page 19 T

echn o l ogy & E ngi n eeri ngICS-103 Final Research Report ECRA F 070 12 T05012 ODA April 19,2007

Rftised by (poUion. oignalln ) Analytical Manager Appn)ved by (position ,**s~re)** QHSE Manager Calibration Surfactants -Analytical Methods for R&D**A~6** Ou IOno 2007-02-01 AM46-2309**s~~rence** f'loocess 1 509001 s.ctian End. 3 Is sue 1**Pa~** 3 of6 Valid umil 2010..02-01 Prepare calibration solutions at five levels in a concentration range close to the expected concentrations in the samples. Prepare the calibnltion solutiona in the same matrix as for the samples. Sample preparation The sample is refrigerated and 5% (v/V) ot methanol is added . The temperature ofthe sample is adjusted to room temperature prior to analysis. Procedure The samples and calibration solutions are injected Into the LCMS. Calculation A linear ealibration graph is constructed from the calibration samples, from which the amount of Ethoquad MC1214125 can be calculated . XCalibur software for LCO Duo is used for the calculations. Precision and accuracy Six repetitive injections of one control sample in OECD water containing 0 .95 mg/l of Ethoqued gave an RSO of 1 . 8 % . Six repetitive Injections of one control •ample in OSW containing 0 . 95 moll of Ethoquad gave an RSO of 3 .7 % . Six repetitive injections of one standard solution in OECD water containing 0.21 mgJl of Ethoquad gave an RSD of 2 . 9 % . Six repetitive Injections of one standard solution in OSW cont.Ullng 0.21 mg/1.. of Ethoqued gave an RSO of 1 . 8 % . Six repetitive Injections of one standard solution in OECO water containing 10. 6 mgJL of Ethoqued gave an RSO of 3.5 % . Limit of quantitation T he lower limit of quantitation is 0 . 1 mg/1 . The detection limit is 0 .02 mg/1 .

Linearity of calibration curve The calbration curve i s linear w ithin the range of 0 . 1 - 2 mg/1 and in the range of 1-100 mgJI. Page 20 Technology & EngineeringI CS- 1 0 3 Fina l Resea rch Re port ECRA F070 1 2 T0 50 1 2 O DA April 19, 20 07

Surfactant. -Analytical Methods for R&D AM46 R@Vised by (posilion. signature) Analytic:al Manager**~d** by (position. sigN!ure) QHSE Manager Reporting D• IOno 2007-02 ..01 AM46- 2 309 Sbnd ... ...,..,oce Process 1509001

The result is reported os amount

# or t•••• numbers. Others Section End. 3**Pa~** 4 of6 V<llid.nil 2010..02..01

in mg/1 with two significant 

### The samples 

### are considered to be stable when 

### refrioera ii

ted

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a i dd

i tio

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### ~orl**········** Full sun. positive mode Pag e 2 1 Technology & E n gineeringICS-103 Final Research Report ECRA F07012 T05012 ODA April19, 2007

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### "' ICS-103 Fi nal Research Report ECRA F07012 T05012 ODA April 19,2007

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### Determination of---inwater from Toxicity studies; Study plans T 05012 AL; T 05012 ODA; T 05012 OFA Introduction: 24 water samples from toxicity studies: • T 05012 AL "Effects pseudokirchnieriella**su"'~""'nit<>t<>"** • T 05012 ODA "Acute toxicity • T 05012 OFA "Acute toxicity of were received from Marc Geurts for th e analysis 2309. Experimental: 24 samples were received on 21-2-2007. See Table 1. Table 1 Sample code 

### I Test number Sample date T 05012 AL Control t=Oh 29-1-2007 T 05012 AL Control t=72h 1-2-2007 T 05012 AL 0.0625 mg/L t=Oh 29-1-2007 T 05012 AL 0.0625 mg/L t=72h 1-2-2007 T 05012 AL 0.25 mg/L t=Oh 29-1-2007 T 05012 AL 0.25 mg/L t=72h 1-2-2007 T 05012 AL 1 .0 mg/L t=Oh 29-1-2007 T 05012 AL 1 .0 mg/L t=72h 1-2-2007 T 05012 ODA Control t=Oh 30-1-2007 T 05012 ODA Control t=48h 1-2-2007 T 05012 ODA 0.2 mg/L t=Oh 30-1-2007 T 05012 ODA 0.2 mg/L t=48h 1-2-2007 T 05012 ODA 0.8 mg/L t=Oh 30-1-2007 T 05012 ODA 0.8 mg/L t=48h 1-2-2007 T 05012 ODA 3.2 mg/L t=Oh 30-1-2007 T 05012 ODA 3.2 mg/L t=48h 1-2-2007 T 05012 OFA Control t=Oh 12-2-2007 T 05012 OFA Control t=96h 16-2-2007 T 05012 OFA 6.25 mg/L t=Oh 12-2-2007 T 05012 OFA 6.25 mg/L t=96h 16-2-2007 T 05012 OFA 25 mg/L t=Oh 12-2-2007 T 05012 OFA 25 mg/L t=96h 16-2-2007 T 05012 OFA 100 mg/L t=O h 12-2-2007 T 05012 OFA 100 mg/L t=96h 16-2-2007 n the growth of the freshwater green alga water fleas, daphnia magna" danio rerio" method AM46-

Sample storage: The samples were refrigerated prior to analysis. Date of analysis: 27-2-2007 to 5-3-2007. Laboratory:ICS-103 Final Research Report ECRA F07012 T05012 ODA April19 , 2007



### Shipment address : Page25 T

e chno l o gy & E ngin eer in g



### Head of Laboratory: - Equipment: See method AM46-2309 Ca l ibration samples : prepared from the refe rence labeled T05012 sub-sample 20-02-2007 JD . The reference was sent together with the samples . Procedure: See method AM46-2309 Prior to ana lysis by LCMS, each of the refrigerated samples was briefly mixed and 1 .5 ml aliquots were transferred to a HPLC vial and stored at room temperatu re in the auto samp ler. All samples were in j ected within 24 hours. Deviations from method AM46-2309: The limit of quantification was defined as 0.1 mg/L for the method. Therefore samples T 05012 AL 0 .0625 mg/L t=Oh and T 05012 AL 0 .0625 mg/L t=72h , were concentrated five times by evaporation at 30°C under a gentle stream of nitrogen . The samples were reconstituted in methanoi/OECD water (5/95) (v/v) before analys i s. Observations: I n samples with sample codes: T 05012 AL control t=72'h T 050 1 2 AL 0.0625 mg/L t=72 h T 05012 AL 0.25 mg/L t=72h T 05012 AL 1 .01 mg/L t=72h A green layer was observed at the bottom of the sample bottles .ICS-103 Final Research Report ECRA F07012 T05012 ODA April 19,2007

Results : Page 26 Technology & Engineering

The concentrations methanol content of 5 measured are given in table 2 below. A correction for the

*Samples T 

### 05012 AL 

### 0.0625 mg/L t=Oh and T 

### 05012 AL 

### 0.0625 mg/L t=72h, were concentrated five times by evaporation before analysis. All Control samples were below detection limit which is 0.02 mg/L.