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 Netherlands 
DR.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 Oleylnitrile (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 DR.U.NOACK-LABORATORIEN 38
DR.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: EC10-, EC50- (with Confidence Interval), EC100 - 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 Oleylnitrile (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 Oleylnitrile (CAS No. 112-91-4) after 24 h 21

Figure 2: Concentration-Effect Relationship of Oleylnitrile (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) 36
DR.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 DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany. 

The study was conducted with natural river water 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 organisms 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 soluble substances in general but long chain nitriles 

in particular rank among the most difficult substances to test in environmental toxicology. 

Standard guideline studies are inappropriate to test 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 

PECaquatic,bulk/PNECaquatic,bulk approach and is considered to be conservative but more 

environmentally realistic than the standard method. This approach is based on PEC estimations 

representing ‘total aquatic concentrations’. To characterize the risk to the aquatic compartment 

the PECaquatic,bulk is compared with the PNECaquatic,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 are 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-effect 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 substances 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 tests are therefore much easier to interpret, more environmental 

realistic, and if compared to PECbulk clearly provide a more appropriate 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 Oleylnitrile 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) 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 concentration procedure (s. Preparation of Adsorption sample) was determined 

to be below LOQS 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 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 dissolved 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: EC10-, EC50- (with Confidence Interval), EC100 - Values, NOEC and LOEC

(based on the geometric mean initially 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] 

EC10 24 0.0160 --- 

 48 0.0160 --- 

EC50 24 0.542 -- 

 48 0.0302 0.0262 – 0.0348 

EC100 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 temperature, 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 the 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 EC10-, EC50- and EC100-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 DR.U.NOACK-LABORATORIEN, 

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 ELENDT (1990), modified to a total 

hardness of 160 to 180 mg CaCO3/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 

> 106 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 validity 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. (SIGMA)

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 < EC50 (24 h) < 2.5 mg/L, according to AQS P 9/2 (clone 5) 

 0.6 mg/L < EC50 (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 mL 
DR.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, 

MACHEREY-NAGEL) 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 O2/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 CO3/L] 154 

Storage conditions 6 ± 2 °C 

Number of study organisms 20 animals, divided into 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 (1st 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 total 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 (pH 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 O2 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 immobile, if it is not able to swim 

within 15 seconds after gentle agitation of the test vessel. 

 

Equipment Oximeter, HQ 40d multi (HACH LANGE) 

 pH-Meter, inoLab pH Level 1 (WTW) 

 Thermohygrograph (LUFFT) 

 Conductometer, Cond 340i (WTW) 

 Spectrophotometer, DR 5000 (HACH LANGE) 

 Analytical balance (SARTORIUS) and balances (METTLER and KERN) 

 Rotating shaker, 3040 (GFL) 

 Incubator, Rumed (RUBARTH APPARATE GMBH) with Timer
DR.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 EC10- and EC100- values after 24 and 48 h were deduced directly 

OF EVALUATION from the observation data without mathematical calculation. 

EC-values and The EC50-values for the reference test and for the definitive test 

statistical analyses after 48 h were calculated by using sigmoidal dose-response 

regression. The confidence intervals for both EC50-values were 

calculated from the best-fit values, the standard error and the tdistribution with the software GraphPad prism. 

The EC50-value for the definitive test after 24 h was deduced 

directly from the observation data without mathematical 

calculation. The concentration effect relationships are presented 

graphically. 

Software All data were computer processed and rounded for presentation 

from the full derived data. Consequently 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, GRAPHPAD SOFTWARE, INC. 

 - Microsoft® Office Excel, MICROSOFT CORPORATION

DEFINITIONS 

NOEC (No observed The highest tested concentration level at which the test item is 

effect concentration) observed to have no biologically 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 biologically 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 data 
DR.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 O2 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 conducted under semi-static conditions with the 

water soluble fraction (WSF) with a nominal loading of 10 mg/L and further two dilution levels of 

1 and 0.1 mg/L. The percentage immobility was determined in all test item and control groups 

after 24 and 48 h of exposure. The immobilization rates are given in Table 2. 

Table 2: Immobilization Rates of the Preliminary 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 value 
DR.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 test 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 EC50-value after 48 h was calculated by sigmoidal dose-response 

regression (Table 1). The EC50-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, determined 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 samples of the fresh media (0 h and 24 h) showed 

recoveries in the range of 44 to 62 % of the nominal 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 concentration 1.00 mg/L was after a process of concentrating 

determined below the LOQS. 

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 

Nominal Loading Oleylnitrile (CAS No. 112-91-4) 

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 -- -- < LOQS -- -- -- -- 

Control < LOQM < LOQM < LOQM < LOQM

Meas. conc. = Measured concentration of test item, enrichment and dilution taken into 

 account; mean value of 2 injections 

Geometric Mean Meas. Concentration = Geometric Mean Measured 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 

LOQS = Limit of quantification of the analytical system (1.0 mg/L)

LOQM = Limit of quantification of the analytical method (2.0 μg/L, enrichment 

 factor taken into account) 

* = Re-injected on 31.05.2010 

-- = Not analysed 
DR.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 EC50-value with 95 % confidence limits (CI) was calculated by sigmoidal 

dose-response regression to be: 

 EC50 : 1.70 mg/L (CI 1.51 - 1.97 mg/L) 

The EC50-value of the reference item potassium dichromate 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 EC50-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 media (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 measured 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 Guideline 202 (2004). The validity criteria were 

fulfilled: 

• In the control group no Daphnia were immobilized or showed other signs of disease or 

stress, e.g. discoloration or unusual behaviour such as trapping on the surface of the water, 

during the 48 h test period. 

• The dissolved O2 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 Oleylnitrile was found to be toxic to Daphnia magna after 48 h at 

concentrations of the test item of 0.0569 mg/L (LOEC48 h) and higher. 

Based on the geometric mean of the initially measured test item concentrations the 48-hour 

EC50 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 concentration 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 concentration procedure (s. Preparation of 

Adsorption sample) was determined to be below LOQS 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 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 %. 

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): Bestimmung der Wirkung von Wasserinhaltsstoffen 

auf Kleinkrebse 

(3) ELENDT B. P. (1990): Selenium Deficiency in Crustacea, Protoplasma 154, 25 - 30 

(4) ICH Harmonised Tripartite Guideline, Validation 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: Guidance for generating and reporting methods of 

analysis in support of pre-registration data requirements 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 measured 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 measured 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 replicate per loading level and control) 

0 h 24 h 

Oleylnitrile 

Nominal Loading 

Level of the 

Water Soluble 

Fraction (WSF) 

[mg/L] 

pH-Value 

Dissolved 

O2-Concentration 

[mg/L] 

pH-Value 

Dissolved 

O2-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 O2 -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 8.75 
DR.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 O2 -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 

O2-Concentration

[mg/L] 

Temperature

[°C] 

Conductivity 

[μS/cm] 

Total Hardness 

[mg CaCO3/L] 

2010-05-19 7.85 9.63 19.2 266 92 

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

Report Page 24 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 GC-MS Analysis of Oleylnitrile (CAS No. 112-91-4) 

10.1 Method 

METHOD OF Various concentrations of Oleylnitrile (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, VARIAN

 Autosampler : CTC Combi PAL, CTC ANALYTICS 

 Detector : MS, Saturn 2000, VARIAN

 Software : Saturn GC/MS Workstation 6.8 (SP1), 

 VARIAN

 

Additional equipment Chromabond cartridges HR-X 3mL/200mg, 

 batch 60.042 and 59.328, MACHEREY&NAGEL 

 

 Evaporator, vapotherm mobil s, BARKEY

 Rotary evaporator, BÜCHI

Reagents tert-Butylmethylether (MTBE), Rotisolv, HPLC grade, ROTH 

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, VARIAN

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 3 
DR.U.NOACK-LABORATORIEN 

Report Page 25 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 

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 

Report Page 26 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 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 

Report Page 27 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 

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 response 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 analytical system (LOQS) was fixed at 

1 mg test item/L. With enrichment factor taken into account, the 

limit auf quantification of the analytical method (LOQM) 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 LOQM) and 40 μg test item/L (20 x LOQM) as 

well as two blank replicates containing no test item were prepared 

as described before for the test samples (enrichment factor 500 

for 1 x LOQM and blanks and factor 50 for 20x LOQM) 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 LOQM values. 

Precision Relative standard deviations lower than 20 % were necessary to 

demonstrate sufficient precision of the analytical method. 

Specificity Two dilution water blank samples were used to prove specificity 

and blank values being < 30 % of the LOQM. 

*) 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 (R2

) 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 variation 
DR.U.NOACK-LABORATORIEN 

Report Page 29 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 

10.2.3 Limit of Quantification (LOQ) 

The limit of quantification (LOQM) of the analytical method (including enrichment steps) was 

fixed at 2 μg Oleylnitrile (CAS no. 112-91-4)/L. The 1 x LOQM and 20 x LOQM 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 LOQM) and 40 μg/L (20 x LOQM) 

Oleylnitrile (CAS no. 112-91-4) 

1 x LOQM 20 x LOQM

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 concentration, 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 LOQM level indicating sufficient accuracy and precision according 

to SANCO 3029/99 rev.4 (2000). 

Furthermore validation series spiked on 20 x LOQM level fulfilled requirements of the 

corresponding guideline with a mean recovery of 90% and a CV of 7.0%. 

Specificity of the analytical method was demonstrated 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%

y = +1.498791e+4x -3802.787109 Coeff. Det.(r2): 0.999938

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 1
DR.U.NOACK-LABORATORIEN 

Report Page 31 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 

10.4 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 13.00minutes

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 factor 500 (dated 2010-05-31) 
DR.U.NOACK-LABORATORIEN 

Report Page 33 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_0,1_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 

Report Page 35 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_0,1_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 Analysis 
DR.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 DR.U.NOACK-LABORATORIEN