Technology&EngineeringB.V.EnvironmentalChemistryTitleAuthorDateDocumentnumberStudynumberPOBox93006800SBArnhemtheNetherlandsReportCONFIDENTIALAl<zoNobelTomorrow'sAnswersTodayBIODEGRADABILITYOFOLEYLNITRILE(CAS112-91-4)INTHECLOSEDBOTTLETESTe.G.vanGinkel2009-10-192.389.780-T09034CBIODEGRADABILITY

OF OLEYLNITRILE (CAS 112-91-4)

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# Al<zoNobel Tomorrow's Answers Today 

### ABSTRACT In order to assess the biotic degradation,

a ready biodegradability
test was performed which allows the biodegradability

to be measured

in an aerobic aqueous medium. The ready biodegradability
was determined

in the Closed Bottle test performed according

to slightly modified OECD, EU and ISO Test Guidelines, and in 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.

2BIODEGRADABILITYOFOLEYLNITRILE(CAS112-91-4)INTHECLOSEDBOTTLETEST2.389.780-STUDYORGANISATIONSponsorStudymonitorStudylocationStudydirectorQualityAssuranceUnitManagementExperimentalstartingdateExperimentalcompletiondateARCHIVINGANDSTORAGEAl<zoNobelTomorrow'sAnswersTodayAkzoNobelSurfaceChemistryABStenungsund,S-44485SwedenactingastheleadcompanyforthefattynitrileconsortiumMGJGeurtsAkzoNobelTechnology&EngineeringVelperweg766824BMArnhemTheNetherlandsAkzoNobelTechnology&EngineeringVelperweg766824BMArnhemTheNetherlandse.G.vanGinkelH.vanDaalenR.Borgmann-Strahsen2009-07-222009-08-26Theprojectfileincludingthefinalreport,amendmentstothefinalreport,thestudyplan,amendmentstothestudyplan,recordsofqualityassuranceinspections,allletters,memosandnotesandrawdatapertainingtothestudywillberetainedinthearchivesofAkzoNobelTechnology&EngineeringArnhemforaperiodoftenyears.Otherrecordsincludingmasterschedulesheet,laboratorynotebooks,logbooks,recordsofthemaintenanceandcalibrationofequipment,summaryoftraining,curriculavitaeandjobdescriptionsofthepersonnelinvolvedinthestudy,recordsrelatedtolocationandstorageofthetestsubstancewillalsobekeptintheAkzoNobelTechnology&EngineeringArnhemarchivesforaperiodoftenyears.TestmaterialwillbestoreddeepfrozenunderthesamplecodeT09034fortenyearsoronlyaslongasthequalityofthetestsubstancepermitsevaluation.3BIODEGRADABILITY

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r s T oday GLP COMPLIANCE STATEMENT The study reported here was carried out according

to the study plan in compliance with the OECD Principles
of Good Laboratory Practice excluding test substance data submitted
by the sponsor. The report contains
an accurate description

of the results. Study director C.G. van Ginkel 

# .. . . **~?.** 

# ...........

. .. . Management R. Borgmann-Strahsen 

## I< 

## r-
, - ()))._

, - '- .......

; .... **~** .............

. .....

.

4BIODEGRADABILITY

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w

' s Ans we rs Toda
y QUALITY ASSURANCE STATEMENT This report was audited by the Quality Assurance Unit contracted
by AkzoNobel Technology

& Engineering Arnhem.

It is considered

to be an accurate presentation

of the methods and procedures applied

in the course of the study and an accurate reproduction

of the data recorded. Listed below are the phases of the study, the dates of inspection

of this study by the Quality Assurance Unit and the dates on which its findings were reported
to Study Director and Management. Phase of the study 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

5BIODEGRADABILITY

OF OLEYLNITRILE (CAS 112-91-4)

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. 780- CONTENTS ABSTRACT STUDY ORGANISATION ARCHIVING AND STORAGE GLP COMPLIANCE STATEMENT QUALITY ASSURANCE STATEMENT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION AMENDMENTS AND DEVIATIONS REFERENCES TABLES FIGURE ANNEXES Al<zoNobel Tomorrow's Answers Today 2 3 3 4 5 7 8 11 12 12 13 14 15 6BIODEGRADABILITY

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y 

## INTRODUCTION Biodegradation

of an organic chemical refers to the reduction

in complexity

of the chemical through metabolic activity of microorganisms. Under aerobic conditions, microorganisms convert organic substances

in carbon dioxide, water and biomass. The objective
of this study

is to assess the biodegradability

of oleylnitrile using

a 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

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# MATERIALS AND METHODS Test material A sample of oleylnitrile was received from Akzo Nobel Surface Chemistry
on 06- 07-2009. The following test substance data were submitted
by the sponsor, who 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. Thesubstance 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.

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### Nutrients and stock solutions The water used
in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2P04, 21.75 mg K2HP04, 33.3 mg Na 2 HP04·2H

2 0, 22.5 mg MgS0

4 ·7H 2 0, 27.5 mg CaCb, 0.25 mg FeCI

3 ·6H 2 0. Ammonium chloride was omitted from the medium
to prevent nitrification. Sodium acetate was added to the bottles using stock solutions
of 1.0 g/L. Oleylnitrile was administered using

a stock solution
of 1.0 g/L in dichloromethane. 

### 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 bottlescontaining 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)


## = M e (day 0)

- M e (day 28) M e is the mean oxygen level in the control bottle inoculated

w

i th 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 )+3 S + 2t P+ tN a-O) ThOD N H 3 

### (m g 0 2 

### I mg
)

, _.:.....____.:__:__ ___ __:_ ___ =-------='-----'- 

### MW

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# Calculation
of the biochemical oxygen demand (BOD) Provided that the oxygen concentrations

in all bottles at the start of the test were equal, the amounts
of oxygen consumed

in test and reference compound bottles were calculated
as follows: Oxygen consumptionn (mg/L) by test substance



# = Mes- Mt 

# Oxygen consumptionn (mg/L) by reference compound



# = Me- Ma Me ores 

# = the mean oxygen level in the control bottles inoculated with activated 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).

10BIODEGRADABILITY

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## RESULTS AND DISCUSSION Theoretical oxygen demand {ThOD) The calculated theoretical oxygen demand
of oleylnitrile

is 3.1 mg/mg. The theoretical oxygen demand
of sodium acetate

is 0.8 mg/mg. 

### 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 oleyln

i trile 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 Oleyln

i trile 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 apply

i ng the time window (Richterich and Steber

, 2001). Oleylnitrile should therefore
be classified
as readily biodegradable.

11

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# AMENDMENTS AND DEVIATIONS Guidelines One minor deviation from the guidelines
of the Closed Bottle test was introduced; a) ammonium chloride was omitted from the medium
to prevent oxygen consumption due to nitrification (omission does not result
in nitrogen limitation
as 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.

12BIODEGRADABILITY

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I Dissolved oxygen concentrations (mg/L)
in the closed bottles. Time (days) Oxygen concentration

( mg/L) Ocs

Ot

Oc

Oa 0

8.9

8.9
8.9

8.9 8.9
8.9
8.9
8.9 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 O c s 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. O a 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

13BIODEGRADABILITY

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# 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.

14BIODEGRADABILITYOFOLEYLNITRILE(CAS112-91-4)INTHECLOSEDBOTTLETEST2.389.780-Annex1CompliancestatementmvoedselenWarenautoriteitENDORSEMENTOFCOMPUANCEWITHTHEOECDPRINCIPLESOFGOODLABORATORYPRACTICEPursuanttotheNetherlandsGLPComplianceMonitoringProgrammeandaccordingtoDirective2004/9/ECtheconformitywiththeOECDPrinciplesofGLPwasassessedon29-31January2008atAkzoNobelTechnology&EngineeringBVEnvironmentalChemistry&RegulatoryAffairsDepartmentVelperweg766824BMAmhemItisherewithconfirmedthattheafore-mentionedtestfacilityiscurrentlyoperatingincompliancewiththeOECDPrinciplesofGoodLaboratoryPracticeinthefollowingareasofexpertise:Physical-chemicaltesting,environmentaltoxicitystudiesonaquaticandterrestrialorganismsandtestsonbehaviourinwater,soilandair;bioaccumulation.DenHaag,2008.DrTh.HelderManagerGLPComplianceMonitoringProgramFoodandConsumerProductSafetyAuthority0/'N/\JPnnsesBeatriJdaan2,2595ALDenHaagPostbus19506,2500CMDenHaagTheNetherlandsAl<zoNobelTomorrow'sAnswersToday15BIODEGRADABILITYOFOLEYLNITRILE(CAS112-91-4)INTHECLOSEDBOTTLETEST2.389.780-Annex2Certificateofanalysis(suppliedbythesponsor)Al<zoNcbelSurfateCherr.slrfA3CertificateofAnalysisAkzoNobelSurfaceChemistryAB,444 85 Stenungsund,SwedenDa!;.2009-09-21F;;g;Cr.Je-•NLrn';:e.·AU:<J!-:ote:l:P:tdutlllamArneel0cwr.!ltrc•s112-91-4LO',Eat:!l5001149COHctPm<uT.on;200801305;stt>H::cH>a:.:Alleastto20180130AnalysisofActivecontentNftrilesC-chaindistributionofactivecontentC14partCl6partCl7partC18part•ClO.C18issat3ndunsatUnitw/w"!0101.1w/w"k410WiVJ%2w/w%64Al<zoNobelTomorrow'sAnswersTodayAkzoNobelTodDyMethodNMRSMACG/1.005SMACG/1.005SMACGI1.005SMACGII.OC516