### FINA L REPOR T

AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T

O F

R-901 0

STUD Y KS-3 B

Author : Mi a Clinckspoo r

OW S N V •Do k

Noor d 5 •B-900 0 Gen t •Belgiu m www.ows.b o •tel . +3

2 9 233020

4 •fax +3

2 9 2332825

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### Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 

### TABL E O F CONTENT S 1 . IDENTIFICATIO N O F TES T 3 1.1 . GENERA L INFORMATION 3 1.2 . STUDY PERSONNEL 4 1.3 . STUDY SCHEDUL E 4 1.4 . ARCHIVIN G 4 2 . CONFIDENTIALIT Y STATEMEN T 5 3. GL PCOMPLIANC E STATEMEN T 5 4 . QUALIT Y ASSURANC E AUDI T STATEMEN T 5 5 . SUMMAR Y AN D CONCLUSION S 6 6 . INTRODUCTIO N 7 6.1 . PRINCIPL

E O F TES T METHO D 7 6.2 . GUIDELINE S USED 7 7 . MATERIAL S AND METHOD S 8 7.1. TES T ITE

M AND REFERENC E ITE M 8 7.2 . GENERA L PROCEDUR E 8 7.2.1. Preparation of the inoculum 8 7.2.2. Preparation of Control, Reference and Test Reactors 5 7.2.3. Performance of the Test 9 7.3 . ANALYTICA L METHOD S 1 0 8 . RESULT S 1 2 8.1 . ANALYSE S INOCULUM AN D TES T ITE M 1 2 8.2 . TES T CONDITION S AND SET-U

P1 3 8.3 . 0 2CONSUMPTION 1 3 8.4 . CORRECTION O

F 0 2UPTAK E FORINTERFERENC E B Y NITRIFICATIO N 1

5 8.5 . ANALYSE S A T TH E EN D O F TH E TES T 1 6 8.6 . BIODEGRADATIO N PERCENTAGE S 1 7 8.6.1. Evolution of

the biodegradation percentages (based on oxygen consumption without correction for NOD or

biomass) / 7 8.6.2. Biodegradation percentages (after correction for

NOD but

without correction for

biomass) ... 19 8.6.3. Biodegradation percentages (after correction for

NOD and

for biomass) 20Date : Oct-26-1 1 ISUIUZI^SZI Page : 3 o f 2 0 

### Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 

### 1 . IDENTIFICATIO N O F TES T 1.1 . GENERA L INFORMATIO N Project Number KS-3 B Sponsor

Monitoring Scientist

Testing Facility ORGANI C WAST E SYSTEM S N.V . D o k Noor d5 B-900 0 Gen t BELGIU M Contact in the United

States M r . Richar dTillinge r rtillinger@worldnet. att. net Mrs . Norm aMcDonal d norma. mcdonald@ows. be Test Item u .->.v - R-901 0 Re ference Item Cellulos e Duration Phone : + " Fax : +
Phone : +32/9/233.02.0 4 Fax : +32/9/233.28.2 5 bruno. dewilde@ows. be mia. clinckspoor@ows. be

Phone : +1/937/829.688 8
Phone:+1/513/535.676 0

2 8 day sCode : R-KS-3 B WM^ ^ 0 0 Date : Oct-26-1 1 Mlij^*** w 7 Page : 4 o f 2 0 Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 1.2 . STUD Y PERSONNE L Stud y Director : Replacemen t

Stud y Director : Qualit y Assuranc e Manager : Technician : 1.3 . STUD Y SCHEDUL E Study initiatio n date : Study completio n date : Experimenta l startin g date : Starting dat

e o f incubation : Completio n dat

e o f incubation : Duratio n o f

incubation : Experimenta l completio n date : Mi aCLINCKSPOO R Brun o D E WILD E Johan VERMEULE N Lyn n SERBRUYN S Sep-14-1 1 Oct-26-1 1 Sep-15-1 1 Sep-20-1 1 Oct-18-1 1 2 8 day s Oct-21-1 1 1.4 . ARCHIVIN G A l l ra w dat aan drecord s necessai y t o reconstruc t the study an ddemonstrate adherenc e to th e stud y pla n wil l b e maintaine di n th e archive s o f O.W.S . Thes e record s includ e notebooks , stud y plan , study report , sample s o f tes t item s an dspecimens . The y wil l b e store di n afil e coded : KS- 3 Th e trainin g record s o f personne l ar

e store di n the map s 'Organisatio n an dPersonnel' . Thes e file s ar e store dper

perso n an dadministere db y th e La b Qualit y Mange r an dth e Assistan t

La b Qualit y Manager . Afte r seve n (7 ) years , al l dat aan drecord s wil l b e destroye do r returne dt o th e sponso r afte r agreement

i n writin g b y th e involve dSponso r an dth e Stud y Director . I n cas e n o writte n agreement

o f th e sponso r

ca n b e obtaine dafte r 7 years , th e dat aan drecord s wil l b e destroyed .Code : R-KS-3 B Date : Oct-26-1 1 

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AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 

### 2 . CONFIDENTIALIT Y STATEMEN T Th e Testin g Facilit y wil l trea t strictl y confidentia l al l relevan t informatio n o n the Tes t item s disclose db y the Sponso r as wel l a

s al l result s obtaine di n executin g the Test .



### 3 . GL PCOMPLIANC E STATEMEN T Th e tes t i s performe di n accordanc e wit h the OEC D principle s o f Goo dLaborator y Practice s



### 4 . QUALIT Y ASSURANC E AUDI T STATEMEN T Th e result s reporte dare i n accordanc e wit h the Stud y Plan , SOP' s (standardoperatin g procedures ) an dRa w Data , A qualit y contro l i s execute don.. , Thi s quality contro l ensure s tha t the fina l repor

t i s complete an daccuratel y reflect s the conduc t

Brun o D E WILD E La b Manage r

(GLP) .

Mi aCLTNCKSPOO R Study Directo r

an dra w datao f

th e study . Joha n VERMEULE N Q A Manage rCode : R-KS-3 B 

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### Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 

### 5 . SUMMAR Y AN D CONCLUSION S Th e biodegradatio n a

t ambien t temperature o f

tes t ite mi R-901 0 wa s teste dtogethe r wit h cellulos e a s positiv e referenc e ite mi n an aquatic , aerobi c biodegradatio n tes t usin g sludg e withou t

any pre-adaptatio n to th e tes t ite mdurin g aperio do f 2 8 days . Th e biodegradatio n percentage s ar e base do n oxyge n consumption , determinatio n o f minera l nitroge n level s an dcalculatio n o f biomas s yield . Th e tes t wa s execute daccordin g to the standar dprocedur e IS O 1485 1 an dAST M D5271-0 2 a t atemperatur e o f

21° C (ambien t

temperature) . Accordin g t o th e IS O 1485 1 guideline , th e tes t i s considere dvali di f

a) th e degre e o f

biodegradatio n o f

th e referenc e materia l i s > 6 0 % a t th e en do f

th e test , an db ) the oxyge n consumptio n o f the blan k

i s no t exceedin g an uppe r limi t valu e obtaine db y experienc e (thi s valu e depend s o n the amount o f inoculu man dis , fo r exampl e abou t

6 0 m g 0 2 

### / l i n the cas e o f 3 0 m g TSS/ 1 o f

sludge) . Afte r

2 8 day s o f

incubatio n adefinit e biodegradatio n o f 88.7 % + 8.9 % wa s measure dfo r the referenc e ite mcellulose . Th e averag e oxyge n consumptio n o f the blan k

wa s wit h avalu e o f

39. 8 m g 0 2 

### / l (fo r

28. 4 m g TSS/1) . A s suc h bot

h requirement s wer

e clearl y fulfilled . Fo r th e tes t

ite m. \R-901 0 afina l absolut e biodegradatio n percentag e o f 99.2% ± 3.1% o o r 111.8 % relativ e t o th e suitabl e referenc e materia l cellulos e wa s obtaine dafte r 28 day s o f incubation . Fro mthes e result s i t ca n b e conclude dtha

t th e tes t ite mI •

• • • R-901 0 wa s completel y biodegrade dwithi n 2 8 days . Th e tes t ite mi R-901 0 ma y therefor e b e regarde das completel y biodegradabl e a t ambien t temperature withi n 2 8 day s unde r aerobi c condition s considerin g that : a ) accordin g t o th e America n standardAST M D6400-0 4 'Standard Specification for Compostable Plastics'

arelativ e biodegradatio n percentag e o f 60 % fo r homopolymers , whil e arelativ e biodegradatio n percentag e o f 90 % i s require dfo r

product s consistin g o f mor e than on e polymer . b ) accordin g t o th e Europea n StandardE N 1343 2 (2000 ) 'Requirements for packaging recoverable through composting and biodegradation - Test

scheme and

evaluation criteria for

the final acceptance ofpackaging' an dIS O 1708 8 (2008 ) 'Specifications for compostable plastics' amateria l ca n onl y b e calle dbiodegradabl e whe n th e percentag e o f

biodegradatio n i s a

t leas t 90 % i n tota l o r

90 % o f

th e maximu m degradatio n o f

asuitabl e referenc e ite mafte r aplatea u ha s been reache dfo r bot h referenc e andtes t

item , andthi s withi n aperio do f maximu m18 0 days .Code : R-KS-3 B OTjT^/^r) Revision : 0 0 Date : Oct-26-1 1 

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### 6 . INTRODUCTIO N 6.1 . PRINCIPL E O F TES T METHO D Th e aqueou s biodegradatio n tes t determine s th e biodegradatio n o f ates t ite munder

laboratoi y condition s b y aconsortiu mo f bacteri afro mdifferen t source s o r

aconditione dsludg e pre expose dt o th e tes t

ite mo r structure-relate dcomponent . Th e tes t materia l i s brought

int o a chemicall y defined(mineral ) liqui dmedium , essentiall y fre e o f other organi c carbo n sources , an dspike dwit h micro-organisms. Durin g the aerobi c biodegradatio n o f organi c material s i n a n aqueou s medium , oxyge n i s consume dandcarbo n i s converte dto gaseous , minera l C (under the for mo f

carbo n dioxide , C0 2 

### ) . Par

t o f the organi c materia l i s assimilate dfo r cel l growth . NaO H pellet s tra p the C0 2 

### release dan dth e induce dpressure-dro p i s directl y relate dt o the consume doxyge n andhenc e to the biodegradatio n o f

th e tes t item . Th e amoun

t o f biodegradatio n i s expresse da s the rati o o f the BO D (correcte dfo r the blank ) t o the Theoretica l Oxyge n Deman d(ThOD ) o r Chemica l Oxyge n Deman d(COD ) o f the use dtes t item . 6.2. GUIDELINE S USE D Th e tes t i s performedaccordin g to : IS O 14851:2004 : Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium - Method by measuring the oxygen demand in a closed

respirometer (ISO 14851:1999). An d AST M D5271-02 : Standard Test Method for Determining the Aerobic Biodegradation of Plastic Materials in an activated-Sludge-Wastewater-Treatment System. Thi s tes t metho di s equivalen t

t o IS O 14851 .Code : R-KS-3 B I^TtT ^rZ^ZTt Revision : 0 0 Date : Oct-26-1 1 

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# I Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 

### 7 . MATERIAL S AN D METHOD S 7.1 . TES T ITE M AN D REFERENC E ITE M 

### TEST ITEM Name : Manufacturer' s name : Description : Colour : Batc h number : Productio n date : Storag e conditions : ThOD : R-901 0 Liqui d Transparent , ligh t

yello w UBS07151 1 7/201 1 Roo mtemperature s i n th e dar k 2.79 2 g/g (calculate dfro melemental analysis ) 

### REFERENCE ITEM Name : Cellulos e Physica l form : Powde r Color : Whit e Storag e conditions : Roo mtemperatur e i n the dar

k COD : 1.14 1 g/g ( ± 0.02 7 g/g ) (determine db y OWS ) 7.2. GENERA L PROCEDUR E 7.2.1 . Preparatio n o

f th e inoculum Th e sourc e o f

microbia l flor awa s amixtur e o f activate dsludg e fro mdifferen t sewage treatmen t

plant s i n Gen t

(Ossemeersen) , Lokeren , Zele , Brech t an dLandegem . Thes e site s treat wastewater

fro mdomesti c and/o r industria l origin . Th e differen t sludge s wer e sieve d ove r a n 80u msiev e an dth e mixtur e mad e o f equa l portion s o f

th e differen t

sludge s wa s aerate dfo r

4 hours . Th e fina l inoculu mwa s obtaine dafte r remova l o f

supernatant

liqui dan d replacemen t

wit h minera l mediu muse dfo r th e remova l o f

solubl e components . 7.2.2 . Preparatio n o

f Control , Reference and Tes tReactor s A t th e star

t o f th e experiment , eac h reacto r wa s fille dwit h th e sam e amoun t o f minera l medium . A precis e amoun

t o f inoculu m(1-5% ) i s adde dt o eac h reacto r t o obtai n ates t

mediu mwit h a concentratio n o f

approximatel y 3 0 m g suspende dsolids/1 . Th e referenc e an dtes t ite mare adde d directl y t o the reactors . Th e vessel s ar

e late r o n aerate dfo r

anothe r 1 5 minute s i n th e incubato r befor e closin g an d initiatin g th e actual incubatio n perio dfo r biodegradation . Thi s fina l aeratio n perio di s neede dt o equilibrat e the fina l mixtur e andt o stabilis e th e temperature .Code : R-KS-3 B 

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### 7.2.3 . Performanc e o f th e Tes t Fo r the set-up ofth e tes t

system , see Figur e 1 . Afte r filling th e reactors , the y ar e put o n an inductiv e stirrer . NaO H pellet s ar e adde dt o th e rubbe r carrie r an dth e OXITOP-O C head s ar e connected . A magneti c ro di s present t o kee p th e referenc e item , tes t

ite man dth e growin g biomas s int o suspensio n throughou t the test . Durin g th e test , th e NaO H pellet s absor b th e C0 2

### produced . Th e reactor s ar e incubate da

t a constan t temperature (21° C + 1°C ) i n the dar k fo r aperio do f

minimu m2 8 days .

Figure J. Respirometric test set-upCode : R-KS-3 B 

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### Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 

### 7.3 . ANALYTICA L METHOD S Weight Determination Durin g th e tes t

2 type s o f

balance s were used . A Sartoriu s A C 21 0 S wit h interna l calibratio n (max . 20 0 g ; d= 0. 1 mg ) fo r th e determinatio n o f

dr y an dvolatil e matter an dfo r th e weigh t o f tes t an dreferenc e items . A Sartoriu s E D 3202S-C W (max . 320 0 g ; d= 0.0 1 g ) o r a Accula b ATL-420 2 (max . 420 0 g ; d= 0.0 1 g ) wa s use dfo r th e weigh t

o f inoculu man d minera l medium . Dry MatterorTotal

Solids Th e dry matte

r i s determine db y dryin g a t 105° C unti l constant

weigh t i s reache dandweighing , a s describe di n MET H L.00 9 "Determinatio n o f moistur e content" . Th e dry matte r i s give n i n percent o n we t weight . Volatile Solids -Ash Th e volatil e solid s andas h content i s determine db y heatin g the drie dsampl e a t 550° C fo r

a fe w hour s an dweighing , a s describe di n MET H L.Ol O "Determinatio n o f organi c matte

r an d carbo n content" . Th e result s ar e give n i n percent

o n dry matter . pH Th e p H i s determine da s describe di n MET H L.00 6 "Determinatio n o f p H andelectrica l conductivity" . COD (chemical

oxygen demand) Th e chemica l oxyge n deman di s determine da s describe di n MET H L.10 3 "Detenninatio n o f COD" . Th e result s ar

e give n i n mg/l . ThOD (Theoretica

l Oxyge n Demand) Th e ThO D i s calculate dfro mthe chemica l formul ao r base do n the elemental analysi s (whic h i s determine di n anothe r lab ) o f th e tes t material , accordin g to the formul adescribe di n IS O 14851 , anne x A . Th e result s ar e give n i n g/g . Elemental

analysis CHN Th e elemental analysi s i s determine di n anothe r la b an danalyse da

s describe di n AST M D529 1

- 1 0 "Standar dTes t

Method s fo r

Instrumental Determinatio n o f Carbon , Hydrogen , an d Nitroge n i n Petroleu mProduct s andLubricants" . Th e result s ar e expresse di n % . TSS& VSS Th e tota l suspende dsolid s wer e determine dusin g centrifugation . A well-know n sampl e o f th e sludg e (50-ml ) wa s brought i n atub e an dcentrifuge dfo r 1 5 mi n a

t 400 0 rpm . Afte r decantin g th e supernatant the pelle t

wa s drie d(105° C overnight ) as describe di n "SO P o n determinatio n o f

suspende dsolid s (TSS ) an dvolatil e suspende dsolid s (VSS) , Editio n 3" .Code : R-KS-3 B 

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### Th e volatil e solid s an das h conten t wa s determine db y heatin g th e drie dsampl e a t 55 0 ° C fo r 8 hour s an dweighin g a s describe di n "SO P o n determinatio n o f suspende dsolid s (TSS ) an d volatil e suspende dsolid s (VSS) , Editio n 3" . Th e result s ar e give n i n g/1 . Ammonium- and

Nitrate-N

analyses Th e minera l N analyse s ar e determine da

t OWS . Th e amount

o f ammoniu mi s determine db y Fiastar 500 0 a s describe di n MET H L.01 6 "Detenninatio n o f ammoni anitrogen-b y FI A an d spectrometri c detection" . Th e sampl e containin g ammoniu mion s i s injecte dint o acontinuou s carrie r strea mb y mean s o f

an injectio n valv e andi s mixe dwit h acontinuousl y streamin g flo w o f a n alkalin e solution . Th e gaseou s ammoni aforme di s separate dthroug h adiffusio n cel l fro mth e solutio n ove r ahydrophobi c sem i permeabl e membran e an dtake n u p b y a streamin g recipien t

flo w containin g ap H indicator . Du e t o th e resultin g p H shift , th e indicato r solutio n wil l chang e it s colou r whic h i s measure dcontinuousl y i n the flo w photomete r

a t 59 0 nm . Th e amount

o f nitrate i s als o measure db y Fiastar 500 0 a s describe di n MET H L.01 7 "Detenninatio n o f

tota l oxidize dnitrogen-b y FI A an dspectrometri c detection" . Th e sampl e containin g nitrite/nitrat e ion s i s fe dint o acontinuousl y flowin g buffe r solutio n (carrie r stream ) b y mean s o f

a n injectio n valve . Nitrat e i n th e sampl e i s reduce dt o nitrite i n a cadmiu mreductor . O n th e additio n o f a n acidi c sulphanilamid e solution , nitrite initiall y presen t an dnitrite forme dfro mreductio n o f nitrate wil l for madiaz o compound . Thi s compoun di s couple dwit h N-(l-naphtyl)-ethylen e diamin e dihydrochlorid e (NED ) to for ma purpl e az o dye . Thi s az o dy e i s measure da t 54 0 nm . Kjeldahl Nitrogen (N) Thi s analysi s i s don e as describe di n "Metho dL.204 . "Determinatio n o f Kjeldah l nitrogen" . I n th e presenc e o f

acatalysin g agen

t (K 2 

### S0 4 

### -Se-mixture ) an dunde r boilin g condition s (380°C ) wit h amixtur e o f sulphuri c aci dboun dnitroge n i s converte dint o the sal t (NFLO2SO4 . Afterward s the ammoni ai s liberate dusin g stron g alkal i an ddistille dfo r subsequent determinatio n b y titration . Th e ammoni ai s capture di n abori c acid/indicato r solution . Determinatio n o f

ammoniu mio n i n the distillat e i s don e b y titratio n wit h standardacid . Th e result s ar e give n i n g per

k g tota l solids .Code : R-KS-3 B 

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### Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 

### 8 . RESULT S 8.1 . ANALYSE S INOCULU M AN D TES T ITE M Th e inoculu mi s amixtur e o f activate dsludg e fro mdifferen t sewage-treatment

plant s (Gent , Lokeren , Zele , Brech t

an dLandegem ) treatin g domesti c and/o r industria l wastewater . Afte r filtration ove r

a n 80u msieve , decantatio n o f the supernatant , an dreplacemen t wit h minera l medium , th e final inoculu mwa s obtained . Th e TSS , VS S an dp H value s o f the inoculu m itsel f ar e give n i n Tabl e 1 . Th e final suspende dsolid s conten t (afte r dilutio n wit h minera l medium ) wa s 28. 4 m g TSS/ 1 an dth e final valu e fo r VS S wa s 20. 1 mg/l . Table 1. Analyses of

the inoculum. Inoculu m TS S (g/1 ) 1.41 8 VS S (g/1 ) 1.00 6 VS S ( % o n TS ) 71. 0 p H 7. 2 Th e referenc e ite mcellulos e wa s analyse dfo r Kjeldahl- N an dfo r

CO D (Chemica l Oxyge n Demand) . Th e ThO D (Theoretica l Oxyge n Demand ) o f tes t ite mHallGree n R-901 0 wa s calculate dfro mthe elementa l analysi s CHN . Th e result s ar e summarise di n Tabl e 2 . Table 2. Elemental

analysis CHN, Kjeldahl-N, COD and ThOD of

the test and reference item. Tes tite m C H N Kjeldahl- N CO D (g/g ) ThO D (g/g

) (% ) (% ) (% ) (%

) HallGree n R-901 0 77. 2 10. 7 0.0 9 - - 2.79 2 Cellulos e - 

### - - <0. 1 1.14 1 ±0.02 7 1.142Code : R-KS-3 B IfW^f^ l °° Date : Oct-26-1 1 

### lUJVVjJj-^^l Page : 1 3 of2 0 Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 8.2. TES T CONDITION S AN D SET-U P A set o f 9 equa l reacto r

vessel s wit h atota l volum e o f 50 0 m l eac h wa s used . Th e tota l volum e o f

the tes t solutio n wa s 25 0 ml . T o 24 5 m l minera l medium , 5 m l inoculu mwa s added . A s aresul t

atota l suspende dsoli dcontent o f 28. 4 mg/ l an datota l volatil e suspende d soli dcontent

o f 20. 1 mg/ l wer e obtaine di n the final tes t medium . Both , referenc e ite m cellulos e an dtes t

ite m" R-9010 , wer e adde ddirectl y t o th e vessels . Th e tes t set-up i s give n i n Tabl e 3 . Afte r the additio n o f

the referenc e andtes t

item , the reactor s were put o n a n inductiv e stirrer . A magneti c ro dkep t the referenc e items , tes t item s an dth e growin g biomas s int o suspensio n throughou t the test . Th e reactor s wer e incubate da t aconstant

temperatur

e o f 2 1 ° C ( + 1°C ) i n the dar k fo r aperio d o f 2 8 days . Table 3. Test

set-up. Reacto r Tes tserie

s Minera l Inoculu m Cellulos e Tes tite m Numbe r mediu m (ml/reactor) (mg/reactor ) (mg/reactor) (ml/reactor) 1 Blan k

1 24 5 5 / / 2 Blan k

2 24 5 5 / / 3 Blan k

3 24 5 5 / / 4 Cellulos e 1 24 5 5 25. 4 / 5 Cellulos e 2 24 5 5 25. 2 / 6 Cellulos e 3 24 5 5 25. 0 / 7 R-901 0 1 24 5 5 / 9. 9 8 R-9010 2 24 5 5 / 10. 5 9 a-.-.. - R-9010 3 24 5 5 / 10. 3
8.3 . 0 2CONSUMPTIO N Th e evolutio n o f

the cumulativ e 0 2 -consumptio n o f th e inoculu m(blank ) i s represente di n Figur e 2 . Th e tota l 0 2 -consumptio n fo r 3 replicate s afte r

2 8 day s o f

testin g wa s 39. 8 + 2. 9 m g 0 2 / l medium . Figur e 3 show s th e evolutio n o f th e 0 2 -consumptio n o f the positiv e referenc e ite m (Cellulose) . Th e final 0 2-consumptio n fo r 3 replicate s o f

th e referenc e ite mi s 128. 7 ± 15. 5 m g 0 2 / l . Th e final ne

t Q 2 -consumptio n fo r

th e referenc e ite mi s 88. 9 ± 15. 5 m g 0 2 / l . I n Figur e 4 the evolutio n o f th e 0 2 -consumptio n o f the tes t ite mHallGree n R-901 0 i s seen . Th e final 0 2-consumptio n fo r th e 2 replicate s (one replicat e wa s omitte ddu e t o aleakage ) i s 143. 0 ± 8. 5 m g 0 2 / l . Th e final ne t O2 -consumptio n afte r

i s 103. 2 ± 8. 5 m g 0 2 / l . Th e evolutio n o f

th e net 0 2 -consumptio n o f

the referenc e (averag e 3 replicates ) an dthe tes t ite m(averag e 2 replicates ) i s represente di n Figur e 5 .Code : R-KS-3 B IrlM^ ^Revision : 0 0 Date : Oct-26-1 1 lUJWJJJm ^l Page : 1 4 o f 2 0 Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T

15 0

0 7 1 4 2 1 2 8 Tim e (days ) -—•Blan k 1 —Blan

k 2 —-Blankj j Figure 2. Evolution of

the cumulative 02-consumption ofthe inoculum.

15 0

Tim e (days ) —-Cellulose 1 Cellulos e 2 Cellulos

e 3 Figure 3. Evolution of

the cumulative 0 2 -consumption ofthe reference item.Code : R-KS-3 B 

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### Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T

Tim e (days ) ~«-HallGree n 1 —HallGree n 2 

### Figure 4. Evolution of

the cumulative C>2-consumption ofthe test item.

15 0

Tim e (days ) Cellulos e —HallGree n R-901

0 

### Figure 5. Evolution of

the net average 0 2 

### -consumption ofthe reference and the test item.Code : R-KS-3 B l/WMCp^^Revision : 0 0 Date : Oct-26-1 1 

## MWSSSU Page : 1 6 o f 2 0 Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 8.4 . CORRECTIO N FO R0 2UPTAK E B Y NITRIFICATIO N Th e aerobi c biodegradatio n o f a n organi c compoun dresult s i n productio n o f C0 2 , water

an d minera l salt s o f

an y other

element s present

(mineralisation ) an dthe productio n o f ne w biomass . I n cas e o f

nitrogen-containin g compound s th e nitroge n ma y b e eliminate das ammonia , nitrite , o r

nitrate . Nitrificatio n i s amicrobia l proces s whic h consume s oxyge n i n a two-ste p process . a) nitrit e productio n NH 3 + 0 2—»N0 2 b ) nitrat e productio n N0 2+ 0 2— > N0 3 NO D i s th e nitroge n oxyge n demand , whic h i s the oxyge n consumptio n relate dt o nitrification . Nitrificatio n itsel f i s the biologica l oxidatio n o f ammonia-nitroge n (NHJ-N ) t o nitrat e nitroge n (N O 3 -N ) wit h nitrit e nitroge n (NO~-N ) a s a n intermediat e step . A s such , a hig h leve l o f

nitrat e i s asig n o f

nitrificatio n andoxyge n consumptio n no t relate dt o carbo n conversion . NO D i s calculate da s (Nitrate- N x 4.57 + Nitrite- N x 3.43) . I f nitrificatio n ha s occurre dbu t i s no t complete , th e observe doxyge n uptake b y th e reactio n mixtur e (se e 8.3 . 0 2-consumption ) ma y b e correcte dfo r th e amount

o f oxyge n use di n oxidizin g ammoniu mt o nitrite , andnitrite t o nitrate . T o calculat e thi s amount th e initia l an d final reactio n mixtur e mus t b e analyze dfo r

nitrit e an dnitrate . 8.5 . ANALYSE S A T TH E EN D O F TH E TES T A t th e endo f

th e tes t perio d(afte r

2 8 days) , the p H wa s measure dan dthe vessel s wer e checke db y mean s o f

nitrite/nitrat e strip s o n th e presenc e o f nitrite . A s can b e seen fro m Tabl e 4 , th e p H ofth e blan k reactor s wa s almos t

equa l t o th e reference/tes t ite mreactors . Al l blan k reactors , cellulos e an d7 > - R-901 0 reactor s wer e afterward s analyse dfo r N H + - N an dNO3

- N (a s nitrit e seeme dabsen t i n al l vessels) . Almos t complet e nitrificatio n wa s observe dfo r

blank , cellulos e andtes t item . Th e result s ar

e summarise di n Tabl e 5 an dals o th e calculate dNO D (nitroge n oxyge n demand ) i s mentioned . Table 4. pHat

the end

ofthe test

(after 28days). Reacto r numbe r Tes tserie s P H 1 Blan k 1 6. 7 2 Blan k 2 6. 8 3 Blan k 3 6. 8 4 Cellulos e 1 6. 8 5 Cellulos e 2 6. 8 6 Cellulos e 3 6. 8 7 1 R-901 0 1 6. 7 8 R-9010 2 6. 7 9 R-9010 3 6. 7Code : R-KS-3 B KjfDYW^^l Revision : 0 0 Date : Oct-26-1 1 Page : 1 7 o f 2 0 Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T Table 5. Ammonium-N

and Nitrate-N

(precise analyses) and calculated

NOD after

28days. NH;- N N03- N NO D Reacto r number (mg/l ) (mg/l) (mg/l) 1 < 0.0 1 6.0 0 27. 4 2 0.1 4 5.9 3 27. 1 3 < 0.0 1 5.9 4 27. 1 4 0.2 3 5.2 6 24. 0 5 < 0.0 1 4.8 4 22. 1 6 0.5 4 5.0 0 22. 9 7 0.0 7 5.3 6 24. 5 8 < 0.0 1 5.4 3 24. 8 9 < 0.0 1 5.4 5 24. 9 Notes: NOD is calculated as (Nitrate-Nx

4.57) 8.6. BIODEGRADATIO N PERCENTAGE S 8.6.1 . Evolutio n of

th e biodegradation percentage s (base d o n oxyge n consumption withoutcorrectio n fo rNO D o rbiomass ) Figur e 6 show s th e evolutio n o f th e averag e percentag e o f biodegradatio n fo r

th e referenc e ite mCellulos e an dth e tes t ite m1 R-9010 . Figure s 7 an d8 represent th e biodegradatio n o f

the separat

e replicates . I

t shoul db e note dtha t th e calculatio n o f

th e intermediar y biodegradatio n percentage s i s base do n th e ne t oxyge n consumptio n (afte r subtractio n ofth e oxyge n consume di n th e blan k reactors ) i n th e tes t

o r referenc e reacto r

an d o n th e CO D adde dt o eac h reactor . N o intermediat e correctio n i s made fo r th e oxyge n consumptio n fo r

nitrification . S o Figure s 6 up t o 8 giv e agoo dindicatio n ofth e evolutio n o f th e biodegradatio n but

no t ofth e absolut e valu e o f biodegradation . Th e biodegradabilit y o f th e referenc e ite mcellulos e starte dafte r approximatel y 2 days . I t too k 2. 4 day s t o reac h 11.4% . Afte r 1 1 day s a

n averag e biodegradatio n percentag e o f mor e tha n 60 % wa s measured . Afte r 2 8 day s abiodegradatio n valu e o f 77.3 % ± 13.1 % wa s measured . Th e tes t

i s considere dvalid , i f a t the en do f

th e tes t th e biodegradatio n percentag e ofth e referenc e ite mi s more than 60% . Th e result s sho w clearl y tha t thi s requiremen t

wa s fulfilled . Th e biodegradatio n ofth e tes t ite m: - R-901 0 starte dafte r 1 day an dth e leve l o f

10 % wa s passe dafte r

1. 6 days . Afte r

5 day s an averag e biodegradatio n o f mor e tha n 60 % wa s observed . A t

th e en dofth e tes t

(afte r 2 8 days ) a

n absolut e biodegradatio n o f 90.6 % ± 3.7 % wa s obtained . A plateau i n biodegradatio n wa s observe dfo r both th e referenc e an dtes t item .Code : R-KS-3 B 

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### Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T
10 0 T

Tim e (days )

—Cellulos

e -» - HallGree

n R-901 0 

### Figure 6. Evolution of the average biodegradation percentage of cellulose and

test item

. ' R-9010(based

on COD/ThOD values).
10 0

Tim e (Days ) 

### Figure 7. Evolution of

the biodegradation percentage of the replicates of

cellulose.Code : Date : R-KS-3 B

Oct-26-1 1 Organic Waste Systems Revision : 0 0 1 9 o f 2 0
AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T

Tim e (Days ) Figure 8. Evolution of

the biodegradation percentage of the replicates of: R-9010. 8.6.2. Biodegradatio n percentage s (afte r correctio n fo rNO D bu twithou tcorrectio n fo rbiomass) Tabl e 6 give s th e tota l amoun t o f oxyge n consume dpe r reactor , th e oxyge n consumptio n fo r oxidatio n o f

carbo n (afte r

subtractio n o f

th e NOD ) an dthe net oxyge n consumptio n o f th e tes t an dreferenc e ite m(afte r subtractio n o f

th e oxyge n consumptio n i n th e blank) . Th e biodegradatio n percentag e base do n the oxidatio n o f carbo n onl y i s als o reporte di n Tabl e 6 . Table 6. Biodegradation percentages after correction for

NOD, based

on BODc. Tes tite m Tes tite

m Tota l 0 2 Total 0 2 Nett o Biodegradation c ThOD/CO D Consumptio n Consumptio n minus NO D BOD c (m g / l tes t (mg/

l tes t (mg/

l tes t (mg/

l tes t (%

) medium ) medium) medium) medium) Blank 1 - 36. 6 9. 2 Blan k 2
- 40. 7 13. 6 Blan k 3 - 42. 1 15. 0 Cellulos e 1 115. 9 144. 0 120. 0 107. 4 92. 7 Cellulos e 2 115. 0 113. 0 90. 9 78. 3 68. 1 Cellulos e 3 114. 0 129. 0 106. 1 93. 6 82. 0

" : " . J1 110. 5 137. 0 112. 5 99. 9 90. 4 r '..--'^l 2 117. 2 149. 0 124. 2 111. 6 95. 2Code : R-KS-3 B 

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### Organic Waste Systems AQUEOU S AEROBI C RESPIROMETRI C BIODEGRADATIO N TES T 

### 8.6.3 . Biodegradatio n percentage s (afte r correctio n fo rNO D and forbiomass ) Durin g biodegradatio n o f organi c materia l par t o f i t i s incorporate dint o ne w biomass . Thu s th e rea l percentag e o f biodegradatio n i s base do n th e amoun t o f tes t o r referenc e ite m oxidise dt o carbo n dioxid e afte r subtractin g NO D (=BOD ) an dafte r addin g th e amoun t

o f tes t o r referenc e ite mtransforme dt o biomas s ( = biomass-COD) . Fo r determinatio n o f

th e biomass-CO D th e followin g assumption s ar e made : biomas s contain s 5 0 % carbo n o n V S basi s an d5 0 % o f

th e V S ar e protein . C/ N rati o o f protei n i s equa l t o 6,2 5 an dl g o f biomas s V S i s equa l t o 1 g o f

COD . Tabl e 7 give s th e result s o f th e biodegradatio n percentage s base do n BO D an dbiomass-COD . Th e nitroge n fixe di n biomas s i s obtaine db y subtractin g th e tota l amoun t o f minera l nitroge n i n th e tes t o r referenc e reactor s fromth e tota l amoun t o f minera l nitroge n i n th e blan k

reactors . Th e tota l amoun t o f minera l nitroge n ca n b e foun di n Tabl e 5 an di s th e su m o f NTLt +
- N an dN0 3 "-N . Whe n th e minera l N i n th e tes t an dth e referenc e reactor s i s lowe r tha n i n th e blan k

reactor s (whic h i s actuall y th e case) , par t o f th e N ha s bee n assimilate db y micro

organism s an dextr abiomas s ha s bee n forme di n thes e reactors . Table 7. Definite biodegradation percentages based on BODc and

biomass yield. Tes tite m Tes tite

m BOD c Ni n Biomas s - Definit e Biodegradatio n COD/ThO D biomass CO D (% ) (m g / I tes t (mg/

l tes t (mg/l) (mg/l) Av g St d Re l medium) medium) Cellulos e 1 115, 9 107. 4 0.5 2 6. 5 98. 3 88 . 7 8 . 9 100. 0 Cellulos e 2 115. 0 78. 3 1.1 6 14. 5 80. 7 Cellulos e 3 114. 0 93. 6 0.4 6 5. 7 87. 1

1 110. 5 90. 4 0.5 8 7. 2 97. 0 99. 2 3. 1 111. 8

. . -i 2 117. 2 95. 2 0.5 8 7. 2 101. 4 With Avg = average; Std = standard

deviation and

Rel = relative biodegradation Fo r th e positiv e referenc e ite mCellulos e a n averag e biodegradatio n resul t o f 88.7 % + 8.9 % wa s obtaine dat

th e en do f

th e tes t

(afte r 2 8 days) . Fo r

th e tes t

ite miR-901 0 a n averag e absolut e biodegradatio n percentag e o f 99.2 % + 3.1 % wa s obtaine dafte r 2 8 day s o f incubation . O n arelativ e basi s wit h cellulos e a s th e suitabl e referenc e materia l 111.8 % wa s calculated . Fro mthes e result s i t ca n b e conclude dtha t th e tes t ite m. R-901 0 wa s completel y biodegrade dan dca n therefor e b e regarde da s completel y biodegradabl e a t ambien t temperatur e withi n 2 8 day s unde r aerobi c condition s considerin g that : b ) accordin g t o th e America n standar dAST M D6400-0 4 'Standard Specification for Compostable Plastics'

arelativ e biodegradatio n percentag e o f 60 % fo r

homopolymers , whil e arelativ e biodegradatio n percentag e o f 90 % i s require dfo r

product s consistin g o f mor e tha n on e polymer , b ) accordin g t o th e Europea n Standar dE N 1343 2 (2000 ) 'Requirements for packaging recoverable through composting and biodegradation - Test scheme and

evaluation criteria for

the final

acceptance of

packaging'

an dIS O 1708 8 (2008 ) 'Specifications for compostable plastics' amateria l ca n onl y b e calle dbiodegradabl e whe n th e percentag e o f

biodegradatio n i s a t leas t 90 % i n tota l o r

90 % o f

th e maximu m degradatio n o f

asuitabl e referenc e ite mafte r aplatea u ha s bee n reache dfo r bot h referenc e an dtes t

item , an dthi s withi n aperio do f maximu m18 0 days .