Document number 

Study number 

2.389.780-

T09034 C 
compliance with the OECD principles of Good Laboratory Practice. 

Oleylnitrile did not cause a reduction in the endogenous respiration. The test 

substance is therefore considered to be non-inhibitory to the inoculum. Oleylnitrile 

was biodegraded 85% at day 28 in the Closed Bottle test. Hence this compound 

should be classified as readily biodegradable. 

The test is valid as shown by an endogenous respiration of 0.9 mg/L arid by the 

total mineralization of the reference compound, sodium acetate. Sodium acetate 

was degraded 81% of its theoretical oxygen demand after 14 days. Finally, the 

most important criterion was met by oxygen concentrations >0.5 mg/L in all 

bottles during the test period. 

2 
Study monitor 

Study location 

Study director 

Quality Assurance Unit 

Management 

Experimental starting date Experimental completion date 

ARCHIVING AND STORAGE 

acting as the lead company for the 

fatty nitrile consortium 

MGJ Geurts 

Akzo Nobel Technology & Engineering 

Velperweg 76 

6824 BM Arnhem 

The Netherlands 

Akzo Nobel Technology & Engineering 

Velperweg 76 

6824 BM Arnhem 

The Netherlands 

e.G. van Ginkel 

H. van Daalen 

R. Borgmann-Strahsen 

2009-07-22 2009-08-26 

The project file including the final report, amendments to the final report, the 

study plan, amendments to the study plan, records of quality assurance 

inspections, all letters, memos and notes and raw data pertaining to the study will 

be retained in the archives of Akzo Nobel Technology & Engineering Arnhem for a 

period of ten years. Other records including master schedule sheet, laboratory 

notebooks, logbooks, records of the maintenance and calibration of equipment, 

summary of training, curricula vitae and job descriptions of the personnel 

involved in the study, records related to location and storage of the test 

substance will also be kept in the Akzo Nobel Technology & Engineering Arnhem 

archives for a period of ten years. Test material will be stored deepfrozen under 

the sample code T09034 for ten years or only as long as the quality of the test 

substance permits evaluation. 

3 
Study director 

C.G. van Ginkel 

.... ~?. .............. . 

Management 

R. Borgmann-Strahsen 

I< r- , -()))._ ,-'-

....... ; .... ~ ................... . 

4 
p

y

the Quality Assurance Unit and the dates on which its findings were reported to 

Study Director and Management. 

Phase of the study 

Study plan 

Test (experimental conditions) 

Report 

Quality Assurance Unit 

H. van Daalen 

Date 3 .. : ... .. ~ Dates of inspection 31-07-2009 03-08-2009 27-08-2009 Dates of reporting 31-07-2009 03-08-2009 27-08-2009 

5 
INTRODUCTION 

MATERIALS AND METHODS 

RESULTS AND DISCUSSION 

AMENDMENTS AND DEVIATIONS 

REFERENCES 

TABLES 

FIGURE 

ANNEXES 

7 

8 

11 

12 

12 

13 

14 

15 

6 
ready biodegradability test. In ready biodegradability tests, microorganisms are 

inoculated into a chemically defined liquid medium containing the test substance 

as sole carbon and energy source. The test substance is exposed to the inoculum, 

consisting of relatively low numbers of microorganisms from an activated sludge 

plant, under aerobic conditions for a period of at least 28 days. 

The Closed Bottle test is chosen as the most appropriate ready biodegradability 

test available. This test has been performed according to slightly modified EEC, 

OECD and ISO Test Guidelines (OECD, 1992; EU 1992; ISO, 1994) in compliance 

with the OECD principles of Good Laboratory Practice (Annex 1). 

7 
accepted full responsibility for the validity thereof. 

• chemical name (active ingredient) oleylnitrile 

• CAS reg. No. (active ingredient) 112-91-4 

• Purity (see annex 2) 100% 

• product name Arneel 0 

• batch/lot No. S001194 

• 

• 

• 

appearance 

stability 

yellowish liquid 

not relevant 

soluble at test concentrations 

• 

solubility in water 

storage at ambient temperature in the dark 

under nitrogen gas 

The concentrations cited in this report refer to the as-received sample of 

oleyl nitrile. 

Reference substances and chemicals 

Sodium acetate anhydrous was used as a reference substance in the Closed Bottle 

test. This compound was purchased from Acros Organics, Geel, Belgium. The substance data were submitted by the supplier. 

• Reference compound acetic acid, sodium salt 

• CAS reg. No. 127-09-3 

• purity >99% 

• batch/lot No. A0206783001 

• appearance white crystals 

• solubility in water soluble in water 

• storage at ambient temperature in the dark 

All other chemicals used were of reagent grade quality. 

Deionized water 

Deionized water containing no more than 0.01 mg/L Cu (Sterlab certified; nonGLP analysis) was prepared in a water purification system. 

Inoculum 

Secondary activated sludge (25-07-2009) was obtained from the wastewater 

treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated 

sludge plant treating predominantly domestic wastewater. The activated sludge 

was preconditioned to reduce the endogenous respiration rates. To this end, 400 

mg dry weight (DW)/L of activated sludge was aerated for one week. The sludge 

was diluted in the BOD bottles (van Ginkel and Stroo, 1992). 

Test bottles 

The test was performed in 0.30 L BOD (biological oxygen demand) bottles with 

glass stoppers. 

8 
g

Test procedures 

The Closed Bottle test was performed according to the study plan. The study plan 

was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 10 bottles to which dichloromethane was first added and 

subsequently evaporated containing only inoculum, 10 bottles containing 

inoculum and test substance, and 6 bottles containing sodium acetate and 

inoculum. Accurate administration of water-insoluble oleylnitrile at a 

concentration of 2.0 mg/L was accomplished by dissolving the test substance in 

dichloromethane (Nyholm and Seiero, 1989). The test substance in 

dichloromethane was directly added to bottles. Before the start of the test, the 

solvent was allowed to evaporate by placing the bottles on a roller bank in a 

ventilated hood for 24 hours to obtain an even distribution of the test substance 

on the walls of the bottles. The concentration of sodium acetate in the bottles was 

6. 7 mg/L. Each of the prepared solutions was dispensed into the respective group 

of BOD bottles so that all bottles were completely filled without air bubbles. The 

zero time bottles were immediately analyzed for dissolved oxygen using an 

oxygen electrode. The remaining bottles were closed and incubated in the dark. 

Two duplicate bottles of all series were withdrawn for analyses of the dissolved 

oxygen concentration at day 7, 14, 21, and 28. 

Analyses 

The dissolved oxygen concentrations were determined electrochemically using an 

oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (Retsch, 

Ochten, The Netherlands). The pH was measured using a Knick 765 calimatic pH 

meter (Eiektronische Messgerate GmbH, Berlin, Germany). The temperature was 

measured and recorded with a sensor connected to a data logger. 

Calculation of the results 

Calculation of endogenous respiration 

The endogenous respiration (oxygen depletion in the control) was 

calculated as follows; 

Oxygen depletion (endogenous respiration) (mg/L) = Me (day 0) - Me (day 28) 

Me is the mean oxygen level in the control bottle inoculated with activated sludge. 

Calculation of the theoretical oxygen demand (ThOD) 

The ThODs of oleylnitrile, and sodium acetate were calculated from their 

molecular formulae and molecular weights as follows. 

16(2C+t (H -Cl- 3N)+3S +2t P+ tNa-O) ThODNH3 (mg0 2 I mg) ,_.:.....____.:__:__ ___ __:_ ___ =-------='-----'- MW 

9 
yg

sludge, n days after the start of the test (c). One control was carried out in bottles 

to which dichloromethane was first added and subsequently evaporated (cs). 

Mt or a = the mean oxygen concentration in the bottles containing the test 

substance (t) or the reference compound, sodium acetate (a), and inoculated with 

activated sludge n-days after the start of the test. 

The biological oxygen demand (BOD) mg/mg of the test substance and sodium 

acetate was calculated by dividing the oxygen consumption by the concentration 

of the test substance and sodium acetate in the closed bottle, respectively. 

Calculation of the biodegradation percentages 

The biodegradation was calculated as the ratio of the biochemical oxygen demand 

(BOD) to the theoretical oxygen demand (ThOD). 

10 
Toxicity 

Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate 

by the test compound in the Closed Bottle test was not determined because 

possible toxicity of oleylnitrile to microorganisms degrading acetate is not 

relevant. Inhibition of the endogenous respiration of the inoculum by the test 

substance was not detected. Therefore, no inhibition of the biodegradation due to 

the "high" initial concentration of the test substance is expected. 

Test conditions 

The pH of the media was 7.1 at the start of the test. The pH of the medium at day 

28 was 7.1 (control of test) and 7.0 (control; no dichlorometane) and 6.8 (test). 

Temperatures were within the prescribed temperature range of 22 to 24°C. 

Validity of the test 

The validity of the test is demonstrated by an endogenous respiration of 0.9 mg/L 

at day 28 (Table I). Furthermore, the differences of the replicate values at day 28 

were less than 20%. The biodegradation percentage of the reference compound, 

sodium acetate, at day 14 was 81. Finally, the validity of the test is shown by 

oxygen concentrations >0.5 mg/L in all bottles during the test period. 

The Closed Bottle test 

Oleylnitrile is biodegraded 85% at day 28 in the Closed Bottle test (Figure and 

Tables I-II), Over 60% biodegradation was not achieved within a period of 10 

days (14 days for the Closed Bottle test) immediately following the attainment of 

10% biodegradation. Oleylnitrile is a poorly water soluble substance. Poor water 

solubility is expected to affect biodegradation kinetics because slow desorption 

and dissolution rates of the test substance present at high concentrations in 

ready biodegradability tests. It should be noted that under environmental 

conditions substances will be present in the environment at very low 

concentrations. Poor water solubility of fatty amine derivatives is therefore an 

important reason for not applying the time window (Richterich and Steber, 

2001). 

Oleylnitrile should therefore be classified as readily biodegradable. 

11 
consumption due to nitrification (omission does not result in nitrogen limitat

o

shown by the biodegradation of the reference compound). 

Study plan 

There are no amendments to the study plan. There are no deviations from the 

study plan. 

REFERENCES 

OECD (1992) Guideline for testing of chemicals, Degradation and accumulation, 

No. 301: Ready biodegradability Paris Cedex France. 

EU (1992) Official Journal of the European Communities L383, Part C. Methods for 

the determination of ecotoxicity, method C.4. Degradation-biotic degradation: 

Closed Bottle test. 

ISO 10707 (1994) Water quality - Evaluation in an aqueous medium of the 

ultimate aero.bic biodegradability of organic compounds - Method by analysis of 

biochemical oxygen demand (Closed Bottle test). 

C.G. van Ginkel, C.A. Stroo (1992) Simple method to prolong the Closed Bottle 

test for the determination of the inherent biodegradability. Ecotoxicology and 

Environmental Safety. 24 319-327. 

N. Nyholm, C. Seiero (1989) Biodegradability testing of poorly soluble compounds 

by means of manometric respirometry. ISO/TC 147SC5/WG4. 

K. Richterich and J. Steber (2001) The time-window - an inadequate criterion 

for the ready biodegradability assessment of technical su rfactants. 

Chemosphere 44 1649-1654. 

12 
Mean (M) 8.9 8.9 8.9 8.9 

7 8.2 5.6 8.2 4.1 

8.2 5.5 8.2 4.2 

Mean (M) 8.2 5.6 8.2 4.2 

14 8.1 3.8 8.2 3.7 

8.2 3.8 8.2 3.8 

Mean (M) 8.0 3.8 8.2 3.8 

21 8.1 3.4 8.1 

8.1 3.3 8.1 

Mean (M) 8.1 3.4 8.1 

28 8.0 2.8 7.9 

8.0 2.6 8.0 

Mean (M) 8.0 2.7 8.0 

Ocs Mineral nutrient solution without test material but with inoculum. Before 

filling the bottles, dichoromethane (0.6 ml) was added to these bottles and 

subsequently allowed to evaporate. 

Ot Mineral nutrient solution with test material (2.0 mg/L) administered using a 

stock solution in dichloromethane solution, and inoculum. 

Oc Mineral nutrient solution with only inoculum. 

Oa Mineral nutrient solution with sodium acetate (6. 7 mg/L) and with inoculum. 

Table II Oxygen consumption (mg/L) and the percentages biodegradation of 

oleylnitrile (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle 

test. 

Time (days) Oxygen consumption (mg/L) Biodegradation (%) 

Test Acetate Test Acetate 

substance substance 

0 0.0 0.0 0 0 

7 2.6 4.0 42 71 

14 4.2 4.4 68 81 

21 4.6 74 

28 5.3 85 

13 
100 

.....--.. ~ 80 0 

............. 

c 

0 60 

......, 

ro 

""C 

ro 

1... 40 0"1 

<V 

""C 

0 

ca 20 

0--------'--------'--------' 

0 10 20 30 

Time (days) 

Figure Percentage of biodegradation of oleyl nitrile ( • ) and sodium acetate (o) 

versus time in a Closed Bottle test. 

14 
ENDORSEMENTOFCOMPUANCE 

WITH THE OECD PRINCIPLES OF 

GOOD LABORATORY PRACTICE 

Pursuant to the Netherlands GLP Compliance Monitoring Programme and 

according to Directive 2004/9/EC the conformity with the OECD Principles of GLP 

was assessed on 29-31 January 2008 at 

Akzo Nobel Technology & Engineering BV 

Environmental Chemistry & Regulatory Affairs Department 

Velperweg 76 

6824 BM Am hem 

It is herewith confirmed that the afore-mentioned test facility is currently operating 

in compliance with the OECD Principles of Good Laboratory Practice in the 

following areas of expertise: Physical-chemical testing, environmental toxicity studies 

on aquatic and terrestrial organisms and tests on behaviour in water, soil and air; 

bioaccumulation. 

Den Haag, 2008 . 

Dr Th. Helder 

Manager GLP Compliance Monitoring Program 

Food and Consumer Product Safety Authority 0/'N/\J 

Pnnses BeatriJdaan 2, 2595 AL Den Haag 

Postbus 19506, 2500 CM Den Haag 

The Netherlands 

15 
Akzo Nobel Surface Chemistry AB, 444 85 Stenungsund, Sweden 

Da!; . 2009-09-21 

F;;g; 

Cr.J e-• NLrn';:e.· AU:<J!-:ote:l: 

P:tdutlllam Arneel 0 

cwr.!ltr 

c•s 112-9 1-4 

LO',Eat:!l 5001149 

COH ct Pm<uT.on; 200801 30 

5;st t>H::cH>a:.: All east to 20180130 

Analysis of 

Active content Nftriles 

C-chain distribution of 

active content 

C14 part 

C l 6 part 

C l 7 part 

C18 part 

• C l O. C 18 is sat 3nd unsat 

Unit 

w/w "!0 101.1 

w/w "k 4 

10 

WiVJ% 2 

w/w% 64 

AkzoNobel 

TodDy 

Method 

NMR 

SMA CG/1.005 

SMA CG/1 .005 

SMA CGI 1.005 

SMA CGII .OC5 

16