DR.U.NOACK-LABORATORIEN

Report Page 4 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## Personnel Involved

Study Director: Martina Noack (Biologist)

Scientist: Karin Petersen (Analytical part) (Food chemist)

Technical Staff: Alexandra Donath Ulrike Lehmann Thomas Nowakowski Carmen Rack Katharina Warnecke

Quality Assurance Unit: Gwendolin Kraetzig (Biologist)

Dr. Ines Stanetzek (Chemist)

Test Facility Management: Dr. Udo Noack (Biologist)

Monitor Ing. Marc Geurts on behalf of the sponsor

Address Akzo Nobel Technology & Engineering Department ECRA Velperweg 76 6802 EC Arnhem The NetherlandsDR.U.NOACK-LABORATORIEN

Report Page 5 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## Table of Contents

Page

Title Page 1
Statement of GLP Compliance 2
Statement of Quality Assurance Unit 3

Personnel Involved 4

Table of Contents 5

List of Tables 6
List of Figures 6

1 Summary 7

2 Characterisation Data of the Test Item 9

3 Method 10

4 Results 16

4.1 Results of the non GLP Preliminary Range Finding Test 16

4.2 Results of the Definitive Test 17

4.2.1 Biological Data 17

4.2.2 Measured Exposure Concentrations during the Definitive Test 18

4.3 Test of the Reference Item 19

4.4 Water Quality Parameters 19

5 Validity Criteria 19

6 Conclusions 20

7 Literature / References 20

8 Graphs: Results of Statistical Analysis 21

9 Physicochemical Data 22

10 GC-MS Analysis of Oleyln itrile (CAS No. 112-91-4) 24

10.1 Method 24

10.2 Method Validation 28

10.2.1 Linearity 28

10.2.2 Repeatability of Injections 28

10.2.3 Limit of Quantification (LOQ) 29

10.3 Representative Calibration Curve 30

10.4 Representative Chromatograms 31

11 Certificate of Test Item Analysis 37

12 GLP-Certificate of D R .U.N OACK -L ABORATORIEN 38DR.U.NOACK-LABORATORIEN

Report Page 6 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## List of Tables

Page

Table 1: EC 10 -, EC 50 - (with Confidence Interval), EC 100 - Values, NOEC and LOEC 8

Table 2: Immobilization Rates of the Preliminary Range Finding Test 16

Table 3: Immobilization Rates of the Definitive Test after 24 and 48 hours of Exposure 17

Table 4: Concentrations and Recovery Rates of Oleylnitrile (CAS No. 112-91-4) 18

Table 5: Water Parameters at 0 and 24 h (Fresh Media) 22

Table 6: Water Parameters at 24 h (Old Media) 22

Table 7: Water Parameters at 48 h (Old Media) 23

Table 8: Water Parameters of the Dilution Water 23

Table 9: GC Temperature Programme 24

Table 10: Enrichment and Dilution Steps 26

Table 11: Repeatability of Injections of Ol eylnitrile (CAS No. 112-91-4) 28

Table 12: Recovery Rates of Fortified Samples of Oleylnitrile (CAS no. 112-91-4) 29



## List of Figures

Figure 1: Concentration-Effect Relationship of Oley lnitrile (CAS No. 112-91-4) after 24 h 21 Figure 2: Concentration-Effect Relationship of Oley lnitrile (CAS No. 112-91-4) after 48 h 21 Figure 3: Calibration Curve of the Standard 30 Figure 4: Chromatogram & Spectrum of a Standard 31 Figure 5: Chromatogram & Spectrum of the Test Item (Fresh Medium, 0 h) 32 Figure 6: Chromatogram & Spectrum of the Test Item (Fresh Medium, 0 h) 33 Figure 7: Chromatogram & Spectrum of the Control (Fresh Medium, 0 h) 34 Figure 8: Chromatogram & Spectrum of the Test Item (Old Medium, 24 h) 35 Figure 9: Chromatogram & Spectrum of the Control (Old Medium, 24 h) 36DR.U.NOACK-LABORATORIEN

Report Page 7 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## 1 Summary

In the acute immobilization test with Daphnia magna (STRAUS) the effects of the test item Oleylnitrile (CAS No. 112-91-4, batch number : S001149) were determined according to OECD 202 (2004) from 2010-04-26 to 2010-05-31, with the definitive exposure phase from 2010-05-19 to 2010-05-21, at D R .U.N OACK -L ABORATORIEN , Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany.

The study was conducted with natural river wa ter under semi-static conditions with a water soluble fraction (WSF) with a nominal loading of 10 mg/L and further 5 dilution levels, with nominal loadings in the range of 0.032 to 3.2 mg/L , set up in a geometric series with a dilution factor of √ 10 , over a period of 48 h. 20 test organism s were exposed to each test concentration and control.

Oleylnitrile (CAS No. 112-91-4) is insoluble in water and also has a strong tendency to adsorb to suspended matter, algae and test vessels or organic material (including dissolved organic matter such as humic acids). Many poorly solubl e substances in general but long chain nitriles in particular rank among the most difficult subs tances to test in environmental toxicology. Standard guideline studies are inappropriate to te st substances with such properties and the current REACH Guidance Documents do not provide sufficient guidance concerning bioavailability and exposure assessment for difficult substances like the nitriles as these were written with normal hydrophobic chemicals in mind, failing to take into account the lack of bioavailability that occurs in the environment with these substances. The aquatic ecotoxicity tests with nitriles were therefore performed in river water to allow a PEC aquatic,bulk /PNEC aquatic,bulk approach and is considered to be conservative but more environmentally realistic than the standard met hod. This approach is based on PEC estimations representing ‘total aquatic concent rations’. To characterize the risk to the aquatic compartment the PEC aquatic,bulk is compared with the PNEC aquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2001).

In order to class a standard laboratory toxicity study valid, it is of particular importance that - besides information on test substance, test method / conditions and test organism used - suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations ar e based upon measured levels. For ecotoxicity tests performed using the bulk approach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effe ct relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult s ubstances is that in the presence of suspended matter, humic acids and/or algae, the residual sorption to glassware will be negligible. The results of these bulk approach test s are therefore much easier to interpret, more environmental realistic, and if compared to PEC bulk clearly provide a more appropr iate assessment of risks for the environment.DR.U.NOACK-LABORATORIEN

Report Page 8 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

Nominal loadings of the test item Oleylnitr ile were selected after a non GLP preliminary range finding test as follows: 0.032 - 0.100 - 0.320 - 1.00 - 3.20 - 10.0 mg/L. The test item Oleylnitrile was analytically verified in the fresh media (0 and 24 h) of all tested loading levels and control and in the old media (after 24 and 48 h) at the loading levels 0.100 and 1.00 mg/L and the control via GC-MS. The samples of the fresh media (0 and 24 h) show ed recoveries in the range of 44 to 62 % of the nominal values. The recoveries in the old media (after 24 and 48 h) were in the range of 34 to 66 % of the nominal values. Adsorption of the te st item to the glassware was checked after an exposure period of 24 h at a test item concent ration of 1.00 mg/L. The observed concentration after applying the concentration procedure (s. Pr eparation of Adsorption sample) was determined to be below LOQ S showing that less than 5.7 % of the observed initial geometric mean concentration of 0.542 mg/L was sorbed to glasswar e. All effect values given are therefore based on the geometric mean of the initially measured concentrations of the test item in the fresh media (0 and 24 h), calculated to be: 0.0160 – 0.0569 – 0.155 – 0.542 – 1.90 – 5.91 mg/L. The mean recovery rates were in the range of 48 to 59 %.

The water quality parameters (pH-value and disso lved oxygen concentration), measured in the fresh media (0 and 24 h) and in the old media (24 and 48 h), were determined to be within acceptable limits. The validity criteria of the test guideline were fulfilled.

Table 1: EC 10 -, EC 50 - (with Confidence Interval), EC 100 - Values, NOEC and LOEC (based on the geometric mean initia lly measured concentrations of the test item Oleylnitrile)

Test Duration

[h] Oleylnitrile
Geometric Mean Initially Measured
Test Item Concentration [mg/L] Confidence Interval p = 95% [mg/L]

EC 10 24 0.0160 ---

48 0.0160 ---

EC 50 24 0.542 --

48 0.0302 0.0262 – 0.0348

EC 100 24 > 5.91 ---

48 0.155 ---

NOEC 48 0.0160 ---

LOEC 48 0.0569 ---DR.U.NOACK-LABORATORIEN

Report Page 9 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## 2 Characterisation Data of the Test Item

TEST ITEM Oleylnitrile (CAS No. 112-91-4)

Commercial name Arneel O

Batch number S001149

Content 101.1 % (w/w), active content nitriles C-chain distribution of active content: 1. C14 part: 4 % (w/w) 2. C16 part: 10 % (w/w) 3. C17 part: 2 % (w/w) 4. C18 part: 84 % (w/w)

Appearance Light yellow, liquid

Water solubility Insoluble

Solubility under test Not specified conditions

Expiry date 2018-01-30

Recommended storage Avoid elevated temperatures.

Storage at test facility Room temperatur e, protected from moisture and light

Retention of test item At least 1 g has been retained and stored at 6 ± 2 °C.

Identification parameter Name, batch number, state and colour at test facility

The test item and the information concerning t he test item were provided by the sponsor.DR.U.NOACK-LABORATORIEN

Report Page 10 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## 3 Method

TEST GUIDELINE OECD-Guideline for Testing of Chemicals 202,

“ Daphnia sp., Acute Immobilization Test”(adopted 13 April, 2004)

TYPE AND PURPOSE Acute Immobilization Test to Daphnia magna to determine the OF THE STUDY EC 10 -, EC 50 - and EC 100 -values after 24 and 48 h of exposure under semi-static conditions.

TEST SYSTEM

Test organism Daphnia magna STRAUS (Clone 5)

Reason for the selection Daphnia magna is the preferred species in accordance with the of the test organism testing guideline and is bred at the test facility.

Origin Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)

Breeder D R .U.N OACK -L ABORATORIEN
, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany

Culture In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 μE

⋅ m -2

⋅ s -1

.
Culture medium Elendt M4, according to E LENDT (1990), modified to a total hardness of 160 to 180 mg CaCO 3 /L.

Culture feeding At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata

, Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 10 6 cells/mL.DR.U.NOACK-LABORATORIEN

Report Page 11 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

TEST ITEM Oleylnitrile (CAS No. 112-91-4)

Preparation of the A water soluble fractions (WSF) with a nominal loading Water Soluble Fractions of 10 mg/L was prepared with dilution water two days prior to (WSF) application (0 h) and water renewal (after 24 h). The WSF will be shaken with 20 rpm for 24 h at room temperature. After a separation phase of at least 24 h the WSF was taken from the homogeneous liquid phase. There will be no filtration applied to prepare the WSF.

Tested loading levels 0.032 - 0.100 - 0.320 - 1.00 - 3.20 - 10.0 mg/L of the WSF (dilution factor √ 10)

CONTROL Four replicates of dilution water without test item tested under the same conditions as the test groups

REFERENCE TEST A reference test was conducted as a static acute immobilization test (acc. to DIN 38412 L11) with a test duration of 24 h once per month in order to prove the valid ity of the test system and test conditions at the test facility. The results of the most recent test are presented in part 4.3.

Reference item Potassium dichromate p.a. (S IGMA )

Batch No. MKBC1858

Purity ≥ 99.5 %

Storage stability 2012-01-29

Test concentrations 0.625 – 1.00 – 1.60 – 2.56 mg/L

Ranges of validity 1.0 mg/L < EC 50 (24 h) < 2.5 mg/L, according to AQS P 9/2 (clone 5) 0.6 mg/L < EC 50 (24 h) < 2.1 mg/L, according to OECD 202 (clone A)

Test duration 2010-05-04 to 2010-05-05

TEST METHOD Semi-static (water renewal after 24 h)

Test duration 48 h

Test vessels Glass beakers (4 cm ID x 7 cm H), 50 mL capacity

Test volume 20 mLDR.U.NOACK-LABORATORIEN

Report Page 12 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

Dilution water Natural river water of the river Leine was used. Specifications of the natural river are presented in the table below. The natural river water was filtered with a membrane filter (0.45 μm, RC, M ACHEREY -N AGEL ) prior to use in the test.

River: Leine

Location Marienbergstraße, D-31171 Nordstemmen, Germany

(52° 10’ 14.98‘’, 9° 46’ 7.64’’) Sampling Date 2009-12-15 Weather on Day of Sampling Cloudy, ca. -1 °C Colour yellowish, clear

pH 7.97 Conductivity [μS/cm] 386 Dissolved Oxygen [mg O 2 /L] 8.62 DOC [mg C/L] 3.9 TOC [mg C/L] 3.9 Ammonium-N [mg N/L] 0.042

Nitrate-N [mg N/L] 2.62 Total Nitrogen [mg N/L] 3.53 o-Phosphate-p [mg P/L] 0.062 Total Phosphate [mg P/L] 0.053 Suspended Matter [mg/L] 16.2 Total Hardness [mg CO 3 /L] 154 Storage conditions 6 ± 2 °C

Number of study organisms 20 animals, divided in to 4 parallel samples, each with 5 animals per loading level and control

Age of test organisms 2 to 24 h old daphnids were used for the definitive study. They were obtained by removing the mother animals twice within 22 h.

Acclimatization At least 2 h in dilution water

Application 20 g of the test solutions each were weighed out into the test vessels. The test organisms were transferred with a small amount of dilution water (1 st application, 0 h) or test solution (water renewal, 24 h) by using a pipette.

Test temperature 18 - 22 °C, ± 1 °C constant

Illumination Diffuse light, illumination strength max. 20 μE

⋅ m -2

⋅ s -1

Photoperiod 16/8 h light/dark cycle

Feeding The daphnids were not fed during the study.DR.U.NOACK-LABORATORIEN

Report Page 13 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

DETERMINATION OF All loading levels and the control were analytically verified THE TEST ITEM in the fresh media (0 and 24 h) by GC-MS. For the fresh media, samples of the freshly prepared test solutions were taken and analysed. The old media were analytically verified at two loading levels 0.100 and 1.00 mg/L and the control, respectively. For the old media samples, additional replicates were prepared without test organisms and incubated under test conditions until analysis. The sorption of the test item to the glassware was quantified once during the test period after the first sampling interval. Therefore, one test vessel of the test concentration 1.00 mg/L, containing daphnids, was emptied after 24 h of exposure and rinsed twice with demineralised water. Thereafter an extraction of the test vessel was carried out. Details of the analytical method are presented in part 10. Analytical results are presented in part 4.2.2.

TYPE AND FREQUENCY Prior to application (0 h) and water renewal after 24 h the water OF MEASUREMENT AND quality parameters (pH value, dissolved oxygen concentration, OBSERVATIONS temperature, conductivity and to tal hardness) of the dilution water were measured. At the beginning of the test and after 24 h the water quality parameters of the fresh media (p H value, oxygen concentration) were measured in one additional replicate per loading level and control. After 24 h and 48 h the water parameters in the old media were measured in all replicates per loading level and control. The dissolved O 2 concentration at the end should be ≥ 3 mg/L. The deviation of the pH-value from the initial value should not be more than 1.5 units. The room temperature was recorded throughout the test with a hygrothermograph. Immobility was determined in all groups after 24 and 48 h. An animal was considered to be imm obile, if it is not able to swim within 15 seconds after gentle agitation of the test vessel.

Equipment Oximeter, HQ 40d multi (H ACH L ANGE ) pH-Meter, inoLab pH Level 1 (WTW) Thermohygrograph (L UFFT ) Conductometer, Cond 340i (WTW) Spectrophotometer, DR 5000 (H ACH L ANGE ) Analytical balance (S ARTORIUS ) and balances (M ETTLER and K ERN ) Rotating shaker, 3040 (GFL) Incubator, Rumed (R UBARTH A PPARATE G MB H) with TimerDR.U.NOACK-LABORATORIEN

Report Page 14 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

STATISTICAL METHODS EC 10 - and EC 100 - values after 24 and 48 h were deduced directly OF EVALUATION from the observation data wit hout mathematical calculation.

EC-values and The EC 50 -values for the reference test and for the definitive test statistical analyses after 48 h were calc ulated by using sigmoidal dose-response regression. The confidence intervals for both EC 50 -values werecalculated from the best-fit values, the standard error and the tdistribution with the software GraphPad prism. The EC 50 -value for the definitive test after 24 h was deduced directly from the observati on data without mathematical calculation. The concentration e ffect relationships are presented graphically.

Software All data were computer processed and rounded for presentation from the full derived data. Cons equently if calculated manually based on the given data minor variations may occur from these figures. Calculations were carried out using the following software: - GraphPad Prism4, G RAPH P AD S OFTWARE
, I NC

. - Microsoft ® Office Excel, M ICROSOFT C ORPORATION

DEFINITIONS

NOEC (No observed The highest tested concentra tion level at which the test item is effect concentration) observed to have no bi ologically significant effect when compared to the control. Immobilization rates of ≤ 10 % are seen to be not biologically significant according to the guideline.

LOEL (Lowest observed The lowest tested concentration level at which the test item is effect concentration) observed to have a bi ologically significant effect when compared to the control. Immobilization rates of > 10 % are seen to be biologically significant according to the guideline.

CHRONOLOGICAL - Preparation of the WSF (experimental starting) TEST DESCRIPTION - Hatching and acclimatization of the juvenile daphnids - Preparation of the test solutions - Application - Incubation - Water renewal after 24 h (transferring of the daphnids to the fresh media) - Determination of the water quality parameters in the fresh media (after 0 and 24 h) and in the old media (after 24 and 48 h) - Determination of immobilization after 24 and 48 h - Determination of the test item concentrations in the fresh media (after 0 and 24 h) and in the old media (after 24 and 48 h) - Evaluation of dataDR.U.NOACK-LABORATORIEN

Report Page 15 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

VALIDITY CRITERIA The test is unacceptable if more than 10 percent of the control organisms are immobilised or show other signs of disease or stress, e.g. discoloration or unusual behaviour such as trapping on the surface of the water, during the 48 hour test period. The dissolved O 2 concentration at the end should be ≥ 3 mg/L.

DATES

Study initiation 2010-04-14 Experimental starting 2010-04-26 Experimental completion 2009-05-31 Study completion Please refer to page 1

DEVIATIONS FROM None THE GUIDELINE

DEVIATIONS FROM None THE STUDY PLAN

ARCHIVING The following will be retained in the archive of the test facility for at least 15 years:

• all raw data

• study plan

• final report

• all records performed by the quality assurance programme including master schedules

• sample of test and reference item Additionally microfilms will be retained in a safe-deposit by Volksbank Sarstedt, D-31157 Sarstedt.DR.U.NOACK-LABORATORIEN

Report Page 16 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## 4 Results

4.1 Results of the non GLP Preliminary Range Finding Test

A non GLP preliminary range finding test was conduc ted under semi-static conditions with the water soluble fraction (WSF) with a nominal loadi ng of 10 mg/L and further two dilution levels of 1 and 0.1 mg/L. The percentage immobility was deter mined in all test item and control groups after 24 and 48 h of exposure. The immobiliz ation rates are given in Table 2.

Table 2: Immobilization Rates of the Pr eliminary Range Finding Test (n = 20, divided into 2 replicates with 10 daphnids each)

IMMOBILIZATION [%]

24 h 48 h
Replicates Replicates Oleylnitrile

Nominal Loading Level of
the WSF*

[mg/L] 1 2 MV 1 2 MV

10 100 100 100 100 100 100

1 80 90 85 100 100 100

0.1 50 60 55 80 80 80

Control 0 0 0 0 0 0

MV = Mean valueDR.U.NOACK-LABORATORIEN

Report Page 17 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

4.2 Results of the Definitive Test

4.2.1 Biological Data

The percentage immobility, determined in all te st item and control groups after 24 and 48 h under semi-static conditions, is given in Table 3.

Table 3: Immobilization Rates of the Definitive Test after 24 and 48 hours of Exposure (n = 20, divided into 4 replicates with 5 daphnids each)

Oleylnitrile IMMOBILIZATION [%]

24 h 48 h

Replicates Replicates Nominal Loading

Level of the
Water Soluble Fraction (WSF) [mg/L] Geometric Mean Initially
Measured
Concentrations [mg/L] 1. 2. 3. 4. MV 1. 2. 3. 4. MV

10.0 5.91 60 40 100 100 75 100 100 100 100 100

3.20 1.90 60 80 100 60 75 100 100 100 100 100

1.00 0.542 60 20 60 60 50 100 100 100 100 100

0.320 0.155 60 60 40 20 45 100 100 100 100 100

0.010 0.0569 40 40 60 20 40 100 80 80 100 90

0.032 0.0160 0 20 0 20 10 0 20 0 20 10

Control 0 0 0 0 0 0 0 0 0 0

MV = Mean value

The concentration-effect-relationships after 24 and 48 h of exposure are presented graphically (Figure 1 and Figure 2). The EC 50 -value after 48 h was calculated by sigmoidal dose-response regression (Table 1). The EC 50 -value after 24 h was taken from the observed data.DR.U.NOACK-LABORATORIEN

Report Page 18 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

4.2.2 Measured Exposure Concentrations during the Definitive Test

The test item was visually clearly dissolved at all concentration levels throughout exposure. The concentrations and recovery rates, determi ned via GC-MS, are presented in Table 4.

The concentrations of Oleylnitrile (CAS No. 112- 91-4) were analysed at all concentration levels from freshly prepared media (0 h and 24 h) and at 2 concentration levels from 24 h old media (24 h and 48 h) via GC-MS analysis. The sample s of the fresh media (0 h and 24 h) showed recoveries in the range of 44 to 62 % of the nomi nal values. The recoveries in the old media (24 and 48 h) were in the range of 34 to 66 % of the nominal values. The measurement of the adsorption sample of the test item concentrati on 1.00 mg/L was after a process of concentrating determined below the LOQ S
.

Table 4: Concentrations and Recovery Rates of Oleylnitrile (CAS No. 112-91-4)

Sampling date 2010-05-19 0 h (fresh medium) 2010-05-20 24 h (old medium) 2010-05-20 24 h (fresh medium) 2010-05-21 48 h (old medium) Start of analysis 2010-05- 19 2010-05-20 2010-05-20 2010-05-21
Oleylnitrile (CAS No. 112-91-4) Nominal Loading Level of the Water Soluble Fraction (WSF) [mg/L] Meas.

conc. [mg/L] RR

[%] Meas.

conc. [mg/L] RR

[%] Meas.

conc. [mg/L] RR

[%] Meas.

conc. [mg/L] RR

[%] Geometric
Mean Meas.
Conc. [mg/L] Mean
RR

[%]
10.0 5.96 60 -- -- 5.86 59 -- -- 5.91 59

3.2 1.98 62 -- -- 1.83 57 -- -- 1.90 59

1.0 0.620 62 0. 608 61 0.474 47 0.658 66 0.542 54

0.32 0.151 47 -- -- 0.160 50 -- -- 0.155 48

0.1 0.0551 55 0. 0343 34 0.0588 59 0.0382 38 0.0569 57

0.032 0.0141* 44 -- -- 0.0181 57 -- -- 0.0160 50
Adsorption 1.0 -- -- < LOQ S -- -- -- -- Control < LOQ M < LOQ M < LOQ M < LOQ M

Meas. conc. = Measured concent ration of test item, enrichment and dilution taken into account; mean value of 2 injections
Geometric Mean Meas. Concentration = Geometric Mean Meas ured Concentration, calculated from the initially measured values of the fresh media (0 and 24 h) RR = Recovery rate related to the nominal test item concentrations LOQ S = Limit of quantification of the analytical system (1.0 mg/L) LOQ M = Limit of quantification of the anal ytical method (2.0 μg/L, enrichment factor taken into account) * = Re-injected on 31.05.2010 -- = Not analysedDR.U.NOACK-LABORATORIEN

Report Page 19 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

4.3 Test of the Reference Item

The percentage immobility for the reference item potassium dichromate was determined after 24 h. The 24-hour EC 50 -value with 95 % confidence limits (CI ) was calculated by sigmoidal dose-response regression to be:
EC 50 : 1.70 mg/L (CI 1.51 - 1.97 mg/L)
The EC 50 -value of the reference item potassium dich romate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC 50 -value of the reference item is also within the prescribed range of 0.6 - 2. 1 mg/L according to OECD-Guideline 202.

4.4 Water Quality Parameters

The water quality parameters of the fresh m edia (pH-value, dissolved oxygen concentration) were measured in one additional replicate per concentration level and control at the beginning of the test (0 h) and at the water renewal after 24 h (Table 5). After 24 and 48 h the water quality parameters of the old media were measured in all replicates per concentration level and control (Table 6 and Table 7). The water quality parameters (pH-value, oxygen concentration, temperature, conductivity and total hardness) of the dilution water were meas ured at experimental starting (0 h) and water renewal after 24 h (Table 8). During the test the room temperature was in the range of 20 - 21 °C.



## 5 Validity Criteria

The study was performed according to OECD Gui deline 202 (2004). The validity criteria were fulfilled:

• In the control group no Daphnia were immobiliz ed or showed other signs of disease or stress, e.g. discoloration or unusual behaviour su ch as trapping on the surface of the water, during the 48 h test period.

• The dissolved O 2 concentration was > 3 mg/L throughout the test.DR.U.NOACK-LABORATORIEN

Report Page 20 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## 6 Conclusions

In this study the test item Oley lnitrile was found to be toxic to Daphnia magna after 48 h at concentrations of the test item of 0.0569 mg/L ( LOEC 48 h ) and higher. Based on the geometric mean of the initially measured test item concentrations the 48-hour EC 50 for Daphnia magna was calculated to be 0.0302 mg/L (95 % confidence limits: 0.0262 – 0.0348 mg/L). The NOEC after 48 h was determined to be 0.0160 mg/L as the highest tested concentration level with no biologically significant effect (Immobilization rates of ≤ 10 % are considered to be not significant according to the guideline).

The concentrations of Oleylnitrile (CAS No. 112- 91-4) were analysed at all concentration levels from freshly prepared media and at 2 concentrati on levels from old media (24 h and 48 h) via GC-MS analysis. The samples of the fresh media (0 and 24 h) showed recoveries in the range of 44 to 62 % of the nominal values. The recoveries in the old media (after 24 and 48 h) were in the range of 34 to 66 % of the nominal values. Adsorption of the test item to the glassware was checked after an exposure period of 24 h at a test item concentration of 1.00 mg/L. The observed concentration after applying the c oncentration procedure (s. Preparation of Adsorption sample) was determined to be below LOQ S showing that less than 5.7 % of the observed initial geometric mean concentration of 0.542 mg/L was sorbed to glassware. All effect values are therefore given based on the geometric mean of the initially measured concentrations of the test item in the fres h media (0 and 24 h), calculated to be: 0.0160 – 0.0569 – 0.155 – 0.542 – 1.90 – 5.91 mg/L. The mean recovery rates were in the range of 48 to 59 %.



## 7 Literature / References

(1) AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium: Bestimmung der nicht akut giftigen Wirkung von Abwasser gegenüber Daphnien über Verdünnungsstufen (DIN 38412 - L 30)
(2) DIN-Richtlinie 38 412 Teil 11 (1982): Best immung der Wirkung von Wasserinhaltsstoffen auf Kleinkrebse
(3) E LENDT B. P. (1990): Selenium Deficiency in Crustacea, Protoplasma 154, 25 - 30
(4) ICH Harmonised Tripartite Guideline, Va lidation of analytical procedures: Methodology, Q2B (1996)
(5) OECD-Guideline 202 for Testing of Chemicals (adopted 13. April 2004): Daphnia sp., Acute Immobilization Test
(6) SANCO/3029/99 rev.4, Residues: Guidanc e for generating and reporting methods of analysis in support of pre-registration data r equirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive 91/414 (11/07/00)DR.U.NOACK-LABORATORIEN

Report Page 21 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## 8 Graphs: Results of Statistical Analysis

-2.00 -1.75 -1.50 -1.25 -1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 60 70 80 90 100

log conc.Immobilization [%]

Figure 1: Concentration-Effect Relationship of Oleylnitrile (CAS No. 112-91-4) after 24 h (based on the geometric mean initially m easured test item concentrations [mg/L])

-2.00 -1.75 -1.50 -1.25 -1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 60 70 80 90 100

log conc.Immobilization [%]

Figure 2: Concentration-Effect Relationship of Oleylnitrile (CAS No. 112-91-4) after 48 h (based on the geometric mean initially m easured test item concentrations [mg/L])DR.U.NOACK-LABORATORIEN

Report Page 22 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## 9 Physicochemical Data

Table 5: Water Parameters at 0 and 24 h (Fresh Media) (measured in one additional replic ate per loading level and control)

0 h 24 h Oleylnitrile Nominal Loading
Level of the
Water Soluble Fraction (WSF) [mg/L] pH-Value Dissolved
O 2 -Concentration [mg/L] pH-Value Dissolved
O 2 -Concentration [mg/L]

10.0 7.76 8.43 7.69 8.66

3.2 7.77 8.98 7.70 9.22

1.0 7.81 9.07 7.72 9.45

0.32 7.77 9.01 7.81 9.41

0.1 7.80 8.96 7.79 9.28

0.032 7.75 8.95 7.81 9.13

Control 7.85 9.63 7.78 9.43

Table 6: Water Parameters at 24 h (Old Media) (measured in all replicates)

pH-Values Dissolved O 2 -Concentration [mg/L]

Replicates Replicates Oleylnitrile Nominal Loading
Level of the
Water Soluble Fraction (WSF) [mg/L] 1 2 3 4 1 2 3 4

10.0 7.69 7.74 7.75 7. 68 8.33 8.52 8.46 8.48

3.2 7.65 7.62 7.62 7. 60 8.45 8.24 8.28 8.26

1.0 7.61 7.60 7.59 7. 59 8.44 8.39 8.35 8.29

0.32 7.65 7.55 7.61 7.61 8.56 8.47 8.48 8.42

0.1 7.67 7.70 7.68 7. 64 8.67 8.65 8.63 8.69

0.032 7.63 7.63 7. 60 7.75 8.73 8. 72 8.71 8.61

Control 7.68 7.68 7.63 7.56 8.67 8.68 8.74 8.75DR.U.NOACK-LABORATORIEN

Report Page 23 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

Table 7: Water Parameters at 48 h (Old Media) (measured in all replicates )

pH-Values Dissolved O 2 -Concentration [mg/L]

Replicates Replicates Oleylnitrile Nominal Loading
Level of the
Water Soluble Fraction (WSF) [mg/L] 1 2 3 4 1 2 3 4

10.0 7.75 7.78 7.73 7. 87 8.42 8.43 8.42 8.39

3.2 7.73 7.74 7.76 7. 77 8.52 8.58 8.58 8.49

1.0 7.65 7.72 7.71 7. 69 8.54 8.50 8.57 8.55

0.32 7.67 7.53 7.55 7.65 8.48 8.42 8.56 8.53

0.1 7.81 7.59 7.67 7. 63 8.51 8.44 8.52 8.31

0.032 7.73 7.72 7. 70 7.62 8.62 8. 60 8.52 8.61

Control 7.66 7.72 7.71 7.73 8.85 8.76 8.75 8.73

Table 8: Water Parameters of the Dilution Water

Dilution Water
Dated: pH-Value Dissolved
O 2 -Concentration [mg/L] Temperature

[°C] Conductivity

[μS/cm] Total Hardness

[mg CaCO 3 /L]

2010-05-19 7.85 9.63 19.2 266 92

2010-05-20 7.78 9.43 19.0 218 83DR.U.NOACK-LABORATORIEN

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## 10 GC-MS Analysis of Oleylnitrile (CAS No. 112-91-4)

10.1 Method

METHOD OF Various concentrations of Ol eylnitrile (CAS No. 112-91-4) DETERMINATION were analysed via GC-MS with liquid injection on a VF 5 ms capillary column using an external standard calibration. Detection was performed with a mass selective detector (MS).

Equipment Gas chromatograph : CP-3800, V ARIAN Autosampler : CTC Combi PAL, CTC A NALYTICS Detector : MS, Saturn 2000, V ARIAN Software : Saturn GC/MS Workstation 6.8 (SP1), V ARIAN

Additional equipment Chromabond cartridges HR-X 3mL/200mg, batch 60.042 and 59.328, M ACHEREY &N AGEL

Evaporator, vapotherm mobil s, B ARKEY Rotary evaporator, B ÜCHI

Reagents tert-Butylmethylether (M TBE), Rotisolv, HPLC grade, R OTH

Standard The test item was used as external standard.

CONDITIONS OF ANALYSIS

Column VF 5 ms, 30 m, ID: 0.25 mm, film thickness: 0.25 μm, batch: 9238249, V ARIAN Injector Splitless for 1 min Injector temperature 280 °C

Table 9: GC Temperature Programme

Carrier gas Helium 1.0 mL/min Run time 18.0 min Retention time Approx.12.7 min Temperature [°C] Heating rate [°C/min] Hold time [min]

40 0 2

300 20 3DR.U.NOACK-LABORATORIEN

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MS-DETECTOR

Ionisation mode Electron Impact (EI) Scan mode centroid Ion polarity positive Scan method Fullscan Scan Range m/z 75 - 250 Quantification ions m/z 122.0 ; 136.0; 150.0 (most intense ion underlined)

PREPARATION OF Stock solutions of 500 mg/L in MTBE were diluted with MTBE THE STANDARD to 8 concentrations in the range from 0.5 to 50 mg test item/L.

PREPARATION OF The content of the test vessels ( 20mL) of level 1.0 mg/L (24 h) THE ADSORPTION SAMPLE was poured out and the inner surface was extracted three times with appr. 30 mL MTBE. Afterwards, the pooled extract was collected, evaporated to dryness, diluted with MTBE to a volume of 2 mL and analysed. This process was necessary to reach the calibration range.

PREPARATION OF For fresh medium all concentration levels and the control, SAMPLES for old media (24h) the concentration levels 0.1 mg/L, 1.0 mg/L and the control were enriched with Chromabond cartridges HR-X. All were eluted with 10 mL MTBE, evaporated to a volume of app. 0.5 mL and diluted with MTBE to a volume of 2 mL. Further dilution steps were made with MTBE as listed in Table 10.

.DR.U.NOACK-LABORATORIEN

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Table 10 : Enrichment and Dilution Steps

Nominal
Test Item
Concentration [mg/L] Enrichment
Faktor Dilution
Factor Sample
Volume

[mL] Final
Volume

[mL]

50 -- 100 2.0 10.0

-- 20 0.10 1) /0.50 2) 1.0 1) /1.0 2)

50 -- 100 2.0 3.2

-- 10 0.1 1.0

50 -- 100 2.0 1.0

-- 5 0.2 1.0

500 -- 1000 2.0 0.32

-- 5 0.2 1.0

500 -- 1000 2.0 0.10

-- 2 0.5 1.0
0.032 500 1000 2.0
Control 500 -- 1000 2.0
1) first dilution step 2) second dilution step

SAMPLE STORAGE All samples were stored at 6 ± 2 °C until start of analysis, if necessary. The prepared samples were stored on the autosampler until measuring.DR.U.NOACK-LABORATORIEN

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EVALUATION Quantification of the test item was calculated by peak area based on the external standard.

METHOD VALIDATION Following SANCO 3029/99 rev.4 (2000)

Linearity Linearity of detector res ponse was checked by analysis of standards and plotting a calibration graph of peak area versus concentration. The coefficient of determination was calculated.

Repeatability of injections 6 sub-samples of the lowest and highest concentration prepared from a single homogeneous sample were analysed. Mean values, standard deviations and coefficients of variation were calculated.

LOQ Based on the requirements of the study design the limit of quantification of the anal ytical system (LOQ S ) was fixed at 1 mg test item/L. With enrichment factor taken into account, the limit auf quantification of the analytical method (LOQ M ) was fixed at 2 μg test item/L and checked by means of accuracy.

Accuracy Five replicates of algae* dilution water fortified with (Fortified samples) 2 μg test item/L (1 x LOQ M ) and 40 μg test item/L (20 x LOQ M ) as well as two blank replicates cont aining no test item were prepared as described before for the test samples (enrichment factor 500 for 1 x LOQ M and blanks and factor 50 for 20x LOQ M ) followed by analysis. The mean recovery of the fortified samples should be between 70 and 110 % (ideally with the mean in the range 80 – 100 %), to verify the LOQ M values.

Precision Relative standard deviations lower than 20 % were necessary to demonstrate sufficient precisi on of the analytical method.

Specificity Two dilution water blank sa mples were used to prove specificity and blank values being < 30 % of the LOQ M
.

*) The fortified samples were prepared once for the validation of the analytical method. Algae dilution water (river water + 1⁄2 DIN, according to OECD 201) was used to prepare these samples. Since no interferences were observed, it can be excluded for the daphnia dilution water, too.DR.U.NOACK-LABORATORIEN

Report Page 28 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

10.2 Method Validation

10.2.1 Linearity

The analytical system gave linear response in range of nominal of 0.5 to 50 mg/L of the test item. Linearity (R 2 ) of the calibration curve was > 0.992. A representative calibration curve and representative chromatograms are given in Figure 3 to Figure 9.

10.2.2 Repeatability of Injections

The results of the repeatability test are provided in the table below.

Table 11: Repeatability of Injections of Oleylnitrile (CAS No. 112-91-4) Peak area [counts*s]

Oleylnitrile (CAS No. 112-91-4)

Serial No. 0.5 mg/L 50.0 mg/L

1 3706 579290

2 3840 572047

3 3918 573021

4 3919 589294

5 3673 579989

6 3838 570715

Mean ± SD 3816 ± 104 577393 ± 6989

CV [%] 2.74 1.21

SD = Standard deviation

CV = Coefficient of variationDR.U.NOACK-LABORATORIEN

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10.2.3 Limit of Quantification (LOQ)

The limit of quantification (LOQ M ) of the analytical method (including enrichment steps) was fixed at 2 μg Oleylnitrile (CAS no. 112-91-4)/L. The 1 x LOQ M and 20 x LOQ M were checked by means of accuracy. A minimum signal to noise ratio of nine was provided.

Table 12: Recovery Rates of Fortified Samples of Oleylnitrile (CAS no. 112-91-4) Fortified concentrations: 2 μg/L (1 x LOQ M ) and 40 μg/L (20 x LOQ M )

Oleylnitrile (CAS no. 112-91-4)

1 x LOQ M 20 x LOQ M
Meas. conc. [μg/L] RR [%] Meas. conc. [μg/L] RR [%]
Replicate 1 1.67 84 35.5 89
Replicate 2 1.67 83 34.8 87
Replicate 3 1.58 79 38.2 96
Replicate 4 1.85 92 32.4 81
Replicate 5 1.73 87 38.3 96

Mean 1.70 85 35.8 90

SD ± 0.100 2.50

CV [%] 5.9 7.0

Meas. conc. = Measured conc entration, enrichment factor taken into account RR = Recovery rate related to the fortified concentration

SD = Standard deviation

CV = Coefficient of variation

The results of the method validation are shown in Table 12. A mean recovery of 85% with a coefficient of variation (CV) of 5. 9% was obtained for the samples series fortified on 1 x LOQ M level indicating sufficient accuracy and precision according to SANCO 3029/99 rev.4 (2000). Furthermore validation series spiked on 20 x LOQ M level fulfilled requirements of the corresponding guideline with a mean reco very of 90% and a CV of 7.0%. Specificity of the analytical method was demons trated because the test item was not detected in the blank samples.DR.U.NOACK-LABORATORIEN

Report Page 30 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

10.3 Representative Calibration Curve

Figure 3: Calibration Curve of the Standard (dated 2010-05-20) Calibration Curve Report

File: c:\varianws\data\090916ab\13441_liq_dai_a0_promth_100519.mth Detector: 2000 Mass Spec, Address: 40

13441

Curve Fit: Linear, Origin: Include, Weight: None Resp. Fact. RSD: 14.83% Coeff. Det.(r2): 0.999938 y = +1.498791e+4x -3802.787109 Level: 1, Replicate: 1, Deviation: 37.28%Peak Size

Amount (mg/L) 10 20 30 40 50 0 100 200 300 400 500 600 700

k Replicates 21 1 1 1 1 1 1DR.U.NOACK-LABORATORIEN

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10.4 Representative Chromatograms

Target Compound Report for #1 from 13441_0,5mgl_1001.sms

Sample ID: 13441_0,5mgL Operator: Da Instrument ID: GCMSGC1 Last Calibration: 26.05.2010 16:55 Measurement Type: Area Calibration Type: External Standard Acquisition Date: 20.05.2010 10:14 Data File: ...3441_0,5mgl_1001.sms Calculation Date: 26.05.2010 16:55 Method: ...a0_promth_100519.mth Sample Type: Calibration Inj. Sample Notes: [090916AB] Standard mg/l in MTBE Divisor: 1.000000 Multiplier: 1.000000

Parameter Specification Actual Status Compound Number 1 Retention Time 12.705 +/- 0.300 12.700 min. Pass RT Offset 0.005 min. Peak Name 13441 CAS Number None Quan Ions 122.0+136.0+150.0 Area >=2 5067 Pass Height 1491 Amount (RF) 10134.404 Reverse Match N-R >= 900 993 Pass Calibration Equation Linear, Include, None y = +1.0134e+4x +0.0 Baseline Code BV Status Pass Error None

Match Types: N-R : Normal-Reverse

12.25 12.50 12.75 13.00 minutes 0.00 0.25 0.50 0.75 1.00 1.25 1.50 kCounts Quan

Start Search End Search Start Integration End Integration 75 100 125 150 175 200 225 250 m/ z 0% 25% 50% 75% 100%

83 226

91 114 108 137 122 515

123 102 138 83 150

193 178 105 193 35 220 213

221 40 Sample Spectrum for Scan: 1162 RT: 12.715 min. BC BP 122 (515=100%) 13441_0,5mgl_1001.sms

75 100 125 150 175 200 225 250 m/ z 0% 25% 50% 75% 100%

82 456 97 371 122 999

123 270 150 350 192 229 220 389 Reference Spectrum for 13441 Scan: 1169 RT: 12.793 min. 13441_mtbe_std_10,0_1.sms

75 100 125 150 175 200 225 250 m/z -100% -50%

0% 50% 100% Sample Spectrum for Scan: 1162 RT: 12.715 min. BC minus

Seg 2, Scan, Time: 5.00-18.00, EI-Auto-Full, 75-250 m/z Figure 4: Chromatogram & Spectrum of a Standard 0.5 μg/L (dated 2010-05-20)DR.U.NOACK-LABORATORIEN

Report Page 32 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

Target Compound Report for #1 from dai13441_0,032_vf1__1.sms

Sample ID: DAI13441_0,032_VF1_ Operator: Da Instrument ID: GCMSGC1 Last Calibration: 01.06.2010 10:01 Measurement Type: Area Calibration Type: External Standard Acquisition Date: 31.05.2010 18:44 Data File: ...441_0,032_vf1__1.sms Calculation Date: 01.06.2010 10:01 Method: ...q_dai_wdh_100531.mth Sample Type: Analysis Inj. Sample Notes: [090916AB] DAI 134412 A0 in MTBE 500:1 A Divisor: 1.000000 Multiplier: 1.000000

Parameter Specification Actual Status Compound Number 1 Retention Time 12.672 +/- 0.300 12.673 min. Pass RT Offset 0.001 min. Peak Name 13441 CAS Number None Quan Ions 122.0+136.0+150.0 Area >=2 86940 Pass Height 30033 Amount >= 0.000 mg/L 7.083 mg/L Pass Reverse Match N-R >= 900 997 Pass Calibration Equation Linear, Include, None y = +1.3613e+4x -9480.3330 Baseline Code BB Status U Pass Error None

Match Types: N-R : Normal-Reverse Status and Errors: U : User-defined EndPoints.

12.25 12.50 12.75 13.00 minutes 0 5 10 15 20 25 30 kCounts Quan

Start Search End Search Start Integration End Integration 75 100 125 150 175 200 225 250 m/z 0% 25% 50% 75% 100%

93 985

97 2515 122 5057

123 1141 150 1903

152 563 192 1297 220 2210

221 318 248 276 Sample Spectrum for Scan: 1161 RT: 12.701 min. BC BP 122 (5057=100%) dai13441_0,032_vf1__1.sms

75 100 125 150 175 200 225 250 m/z 0% 25% 50% 75% 100%

82 456

94

226 122 999

123 270 150 350 192 229 220 389 Reference Spectrum for 13441 Scan: 1169 RT: 12.793 min. 13441_mtbe_std_10,0_1.sms

75 100 125 150 175 200 225 250 m/z -100% -50%

0% 50% 100% Sample Spectrum for Scan: 1161 RT: 12.701 min. BC minus

Seg 2, Scan, Time: 5.00-18.00, EI-Auto-Full, 75-250 m/z

Figure 5: Chromatogram & Spectrum of the Test Item (Fresh Medium, 0 h) 0.032 mg/L, enrichment fa ctor 500 (dated 2010-05-31)DR.U.NOACK-LABORATORIEN

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Target Compound Report for #1 from dai13441_0,1_vf2_1.sms

Sample ID: DAI13441_0,1_VF2 Operator: Da Instrument ID: GCMSGC1 Last Calibration: 26.05.2010 16:55 Measurement Type: Area Calibration Type: External Standard Acquisition Date: 20.05.2010 13:38 Data File: ...i13441_0,1_vf2_1.sms Calculation Date: 26.05.2010 16:55 Method: ...a0_promth_100519.mth Sample Type: Analysis Inj. Sample Notes: [090916AB] DAI 134412 A0 in MTBE 500:1 Divisor: 1.000000 Multiplier: 1.000000

Parameter Specification Actual Status Compound Number 1 Retention Time 12.700 +/- 0.300 12.698 min. Pass RT Offset 0.002 min. Peak Name 13441 CAS Number None Quan Ions 122.0+136.0+150.0 Area >=2 204278 Pass Height 89432 Amount >= 0.000 mg/L 13.883 mg/L Pass Reverse Match N-R >= 900 998 Pass Calibration Equation Linear, Include, None y = +1.4988e+4x -3802.7871 Baseline Code BB Status Pass Error None

Match Types: N-R : Normal-Reverse

12.25 12.50 12.75 13.00 minutes 0 25 50 75 kCounts Quan

Start Search End Search Start Integration End Integration 75 100 125 150 175 200 225 250 m/ z 0% 25% 50% 75% 100%

83 21885

93 9062 122 46029

123 12798 150 15274 192 10937 220 18756

221 5234 Sample Spectrum for Scan: 1162 RT: 12.697 min. BC BP 122 (46029=100%) dai13441_0,1_vf2_1.sms

75 100 125 150 175 200 225 250 m/ z 0% 25% 50% 75% 100%

82 456

94

226 122 999

123 270 150 350 192 229 220 389 Reference Spectrum for 13441 Scan: 1169 RT: 12.793 min. 13441_mtbe_std_10,0_1.sms

75 100 125 150 175 200 225 250 m/z -100% -50%

0% 50% 100% Sample Spectrum for Scan: 1162 RT: 12.697 min. BC minus

Seg 2, Scan, Time: 5.00-18.00, EI-Auto-Full, 75-250 m/z

Figure 6: Chromatogram & Spectrum of the Test Item (Fresh Medium, 0 h) 0.1 mg/L, enrichment factor 500, dilution factor 2 (dated 2010-05-20)DR.U.NOACK-LABORATORIEN

Report Page 34 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

Target Compound Report for #1 from dai13441_ktr_vf1_1.sms

Sample ID: DAI13441_KTR_VF1 Operator: Da Instrument ID: GCMSGC1 Last Calibration: 26.05.2010 16:55 Measurement Type: Area Calibration Type: External Standard Acquisition Date: 21.05.2010 01:06 Data File: ...i13441_ktr_vf1_1.sms Calculation Date: 26.05.2010 16:55 Method: ...a0_promth_100519.mth Sample Type: Analysis Inj. Sample Notes: [090916AB] DAI 134412 A0 in MTBE 500:1 Divisor: 1.000000 Multiplier: 1.000000

Parameter Specification Actual Status Compound Number 1 Retention Time 12.700 +/- 0.300 12.700 min. Fail RT Offset 0.000 min. Peak Name 13441 CAS Number None Quan Ions 122.0+136.0+150.0 Area >=2 0 Fail Height 0 Amount >= 0.000 mg/L 0.000 mg/L Reverse Match N-R >= 900 684 Fail Calibration Equation Linear, Include, None y = +1.4988e+4x -3802.7871 Baseline Code Status X MY Error Error Y

Match Types: N-R : Normal-Reverse Status and Errors: X : Error M : Missing Peak. Y : Peak not detected, < Size Threshold, or not in Search Window.

12.25 12.50 12.75 13.00 minutes 50 75 100 125 150 Counts Quan

Start Search End Search Start Integration End Integration 75 100 125 150 175 200 225 250 m/z 0% 25% 50% 75% 100%

79 34 83 69

92 8 98 37

107 103

115 24 133 89

134 16 147

47 162 29 185 9 189 34 207 59

221 25 238 49

248 23 Sample Spectrum for Scan: 1160 RT: 12.703 min. BC BP 107 (103=100%) dai13441_ktr_vf1_1.sms

75 100 125 150 175 200 225 250 m/z 0% 25% 50% 75% 100%

82 456 97 371 122 999

123 270 150 350 192 229 220 389 Reference Spectrum for 13441 Scan: 1169 RT: 12.793 min. 13441_mtbe_std_10,0_1.sms

75 100 125 150 175 200 225 250 m/z -100% -50%

0% 50% 100% Sample Spectrum for Scan: 1160 RT: 12.703 min. BC minus

Seg 2, Scan, Time: 5.00-18.00, EI-Auto-Full, 75-250 m/z

Figure 7: Chromatogram & Spectrum of the Control (Fresh Medium, 0 h) < LOQ, enrichment factor 500 (dated 2010-05-21)DR.U.NOACK-LABORATORIEN

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. Target Compound Report for #1 from dai13441_0,1_vf1_1001.sms

Sample ID: DAI13441_0,1_VF1 Operator: Da Instrument ID: GCMSGC1 Last Calibration: 27.05.2010 12:11 Measurement Type: Area Calibration Type: External Standard Acquisition Date: 22.05.2010 11:07 Data File: ...441_0,1_vf1_1001.sms Calculation Date: 27.05.2010 12:11 Method: ...e0_promth_100520.mth Sample Type: Analysis Inj. Sample Notes: [090916AB] DAI 134412 E0 in MTBE 500:1 Divisor: 1.000000 Multiplier: 1.000000

Parameter Specification Actual Status Compound Number 1 Retention Time 12.702 +/- 0.300 12.697 min. Pass RT Offset 0.005 min. Peak Name 13441 CAS Number None Quan Ions 122.0+136.0+150.0 Area >=2 248287 Pass Height 120057 Amount >= 0.000 mg/L 17.113 mg/L Pass Reverse Match N-R >= 900 999 Pass Calibration Equation Linear, Include, None y = +1.4601e+4x -1580.2803 Baseline Code BB Status Pass Error None

Match Types: N-R : Normal-Reverse

12.25 12.50 12.75 13.00 minutes 0 25 50 75 100 kCounts Quan

Start Search End Search Start Integration End Integration 75 100 125 150 175 200 225 250 m/ z 0% 25% 50% 75% 100%

77 929

82 3191

98 1197 122 6987

123 1875 150 2458

166 719

178 2506 220 2698

221 750 Sample Spectrum for Scan: 1166 RT: 12.733 min. BC BP 122 (6987=100%) dai13441_0,1_vf1_1001.sms

75 100 125 150 175 200 225 250 m/ z 0% 25% 50% 75% 100%

82 456 97 371 122 999

123 270 150 350 192 229 220 389 Reference Spectrum for 13441 Scan: 1169 RT: 12.793 min. 13441_mtbe_std_10,0_1.sms

75 100 125 150 175 200 225 250 m/z -100% -50%

0% 50% 100% Sample Spectrum for Scan: 1166 RT: 12.733 min. BC minus

Seg 2, Scan, Time: 5.00-18.00, EI-Auto-Full, 75-250 m/z

Figure 8: Chromatogram & Spectrum of the Test Item (Old Medium, 24 h) 0.1 mg/L, enrichment factor 500 (dated 2010-05-22)DR.U.NOACK-LABORATORIEN

Report Page 36 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441

Target Compound Report for #1 from dai13441_ktr_vf1_1001.sms

Sample ID: DAI13441_KTR_VF1 Operator: Da Instrument ID: GCMSGC1 Last Calibration: 27.05.2010 12:11 Measurement Type: Area Calibration Type: External Standard Acquisition Date: 22.05.2010 10:16 Data File: ...441_ktr_vf1_1001.sms Calculation Date: 27.05.2010 12:11 Method: ...e0_promth_100520.mth Sample Type: Analysis Inj. Sample Notes: [090916AB] DAI 134412 E0 in MTBE 500:1 Divisor: 1.000000 Multiplier: 1.000000

Parameter Specification Actual Status Compound Number 1 Retention Time 12.702 +/- 0.300 12.702 min. Fail RT Offset 0.000 min. Peak Name 13441 CAS Number None Quan Ions 122.0+136.0+150.0 Area >=2 0 Fail Height 0 Amount >= 0.000 mg/L 0.000 mg/L Reverse Match N-R >= 900 795 Fail Calibration Equation Linear, Include, None y = +1.4601e+4x -1580.2803 Baseline Code Status X MY Error Error Y

Match Types: N-R : Normal-Reverse Status and Errors: X : Error M : Missing Peak. Y : Peak not detected, < Size Threshold, or not in Search Window.

12.25 12.50 12.75 13.00 minutes 50 75 100 125 150 175 Counts Quan

Start Search End Search Start Integration End Integration 75 100 125 150 175 200 225 m/z 0% 25% 50% 75% 100%

81 53 85 115

86 22 97

70

106 33 122 44 136 72

137 16

145 44

159 12 174 25 192 13 210 31 226 8 242 14 Sample Spectrum for Scan: 1172 RT: 12.781 min. BC BP 85 (115=100%) dai13441_ktr_vf1_1001.sms

75 100 125 150 175 200 225 m/z 0% 25% 50% 75% 100%

82 456 97 371 122 999

123 270 150 350 192 229 220 389 Reference Spectrum for 13441 Scan: 1169 RT: 12.793 min. 13441_mtbe_std_10,0_1.sms

75 100 125 150 175 200 225 m/z -100% -50%

0% 50% 100% Sample Spectrum for Scan: 1172 RT: 12.781 min. BC minus

Seg 2, Scan, Time: 5.00-18.00, EI-Auto-Full, 75-250 m/z

Figure 9: Chromatogram & Spectrum of the Control (Old Medium, 24 h) < LOQ, enrichment factor 500 (dated 2010-05-22)DR.U.NOACK-LABORATORIEN

Report Page 37 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## 11 Certificate of Test Item AnalysisDR.U.NOACK-LABORATORIEN

Report Page 38 of 38 Oleylnitrile (CAS No. 112-91-4) Acute Immobilization Test to Daphnia magna Project-No. 090916AB with Natural River Water, semi-static, 48 h, acc. to OECD 202 (2004) Study-No. DAI13441



## 12 GLP-Certificate of D R 

## .U.N OACK 

## -L ABORATORIEN