FINAL REPORT 

AQUEOUS AEROBIC RESPIROMETRIC 

BIODEGRADATION TEST 

OF 

R-9010 

STUDY KS-3B 

Author: Mia Clinckspoor 

OWS NV • Dok Noord 5 • B-9000 Gent • Belgium 

www.ows.bo • tel. +32 9 2330204 • fax +32 9 2332825 
fAlu/JFf^l Revision : 0 0 

lUfVVlJ I page; 2 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

TABLE OF CONTENTS 

1. IDENTIFICATION OF TEST 3 

1.1. GENERAL INFORMATION 3 

1.2. STUDY PERSONNEL 4 

1.3. STUDY SCHEDULE 4 

1.4. ARCHIVING 4 

2. CONFIDENTIALITY STATEMENT 5 

3. GLP COMPLIANCE STATEMENT 5 

4. QUALITY ASSURANCE AUDIT STATEMENT 5 

5. SUMMARY AND CONCLUSIONS 6 

6. INTRODUCTION 7 

6.1. PRINCIPLE OF TEST METHOD 7 

6.2. GUIDELINES USED 7 

7. MATERIALS AND METHODS 8 

7.1. TEST ITEM AND REFERENCE ITEM 8 

7.2. GENERAL PROCEDURE 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. ANALYTICAL METHODS 10 

8. RESULTS 12 

8.1. ANALYSES INOCULUM AND TEST ITEM 12 

8.2. TEST CONDITIONS AND SET-UP 13 

8.3. 02 CONSUMPTION 13 

8.4. CORRECTION OF 02 UPTAKE FOR INTERFERENCE BY NITRIFICATION 15 

8.5. ANALYSES AT THE END OF THE TEST 16 

8.6. BIODEGRADATION PERCENTAGES 17 

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) 20 
Date: Oct-26-11 ISUIUZI^SZI Page: 3 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

1. IDENTIFICATION OF TEST 

1.1. GENERAL INFORMATION 

Project Number 

KS-3B 

Sponsor 

Monitoring Scientist 

Testing Facility 

ORGANI C WAST E SYSTEM S N.V . 

D o k Noord 5 

B-9000 Gent 

BELGIU M 

Contact in the United States 

M r. Richard Tillinger 

rtillinger@worldnet. att. net 

Mrs. Norma McDonald 

norma. mcdonald@ows. be 

Test Item 

u .->.v- R-9010 

Re ference Item 

Cellulose 

Duration 

Phone: +" 

Fax: + 

Phone: +32/9/233.02.04 

Fax: +32/9/233.28.25 

bruno. dewilde@ows. be 

mia. clinckspoor@ows. be 

Phone: +1/937/829.6888 

Phone:+1/513/535.6760 

28 days 
Code: R-KS-3B WM^ ^ 00 

Date: Oct-26-11 Mlij^***w7 Page: 4 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

1.2. STUDY PERSONNEL 

Study Director: 

Replacement Study Director: 

Quality Assurance Manager: 

Technician: 

1.3. STUDY SCHEDULE 

Study initiation date: 

Study completion date: 

Experimental starting date: 

Starting date of incubation: 

Completion date of incubation: 

Duration of incubation: 

Experimental completion date: 

Mia CLINCKSPOOR 

Bruno DE WILDE 

Johan VERMEULE N 

Lynn SERBRUYNS 

Sep-14-11 

Oct-26-11 

Sep-15-11 

Sep-20-11 

Oct-18-11 

28 days 

Oct-21-11 

1.4. ARCHIVING 

A l l raw data and records necessaiy to reconstruct the study and demonstrate adherence to the 

study plan will be maintained in the archives of O.W.S. These records include notebooks, 

study plan, study report, samples of test items and specimens. They will be stored in a file 

coded: 

KS-3 

The training records of personnel are stored in the maps 'Organisation and Personnel'. These 

files are stored per person and administered by the Lab Quality Manger and the Assistant Lab 

Quality Manager. 

After seven (7) years, all data and records will be destroyed or returned to the sponsor after 

agreement in writing by the involved Sponsor and the Study Director. In case no written 

agreement of the sponsor can be obtained after 7 years, the data and records will be 

destroyed. 
Code: R-KS-3B 

Date: Oct-26-11 

Organic Waste Systems 

Revision: 00 

Page: 5 of 20 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

2. CONFIDENTIALITY STATEMENT 

The Testing Facility will treat strictly confidential all relevant information on the Test items 

disclosed by the Sponsor as well as all results obtained in executing the Test. 

3. GLP COMPLIANCE STATEMENT 

The test is performed in accordance with the OECD principles of Good Laboratory Practices 

4. QUALITY ASSURANCE AUDIT STATEMENT 

The results reported are in accordance with the Study Plan, SOP's (standard operating 

procedures) and Raw Data, 

A quality control is executed on.., 

This quality control ensures that the final report is complete and accurately reflects the conduct 

Bruno DE WILDE 

Lab Manager 

(GLP). 

Mi a CLTNCKSPOOR 

Study Director 

and raw data of the study. 

Johan VERMEULE N 

QA Manager 
Code: R-KS-3B IfWliW**^ Revision: 00 

Date: Oct-26-11 Page: 6 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

5. SUMMARY AND CONCLUSIONS 

The biodegradation at ambient temperature of test item i R-9010 was tested together 

with cellulose as positive reference item in an aquatic, aerobic biodegradation test using 

sludge without any pre-adaptation to the test item during a period of 28 days. The 

biodegradation percentages are based on oxygen consumption, determination of mineral 

nitrogen levels and calculation of biomass yield. 

The test was executed according to the standard procedure ISO 14851 and AST M D5271-02 

at a temperature of 21°C (ambient temperature). According to the ISO 14851 guideline, the 

test is considered valid if a) the degree of biodegradation of the reference material is > 60 % 

at the end of the test, and b) the oxygen consumption of the blank is not exceeding an upper 

limit value obtained by experience (this value depends on the amount of inoculum and is, for 

example about 60 mg 0 2/l in the case of 30 mg TSS/1 of sludge). After 28 days of incubation 

a definite biodegradation of 88.7% + 8.9% was measured for the reference item cellulose. 

The average oxygen consumption of the blank was with a value of 39.8 mg 0 2/l (for 28.4 mg 

TSS/1). As such both requirements were clearly fulfilled. 

For the test item . \ R-9010 a final absolute biodegradation percentage of 99.2%± 

3.1%o or 111.8% relative to the suitable reference material cellulose was obtained after 28 

days of incubation. From these results it can be concluded that the test item I •• • • R-9010 

was completely biodegraded within 28 days. 

The test item i R-9010 may therefore be regarded as completely biodegradable at 

ambient temperature within 28 days under aerobic conditions considering that: 

a) according to the American standard AST M D6400-04 'Standard Specification for 

Compostable Plastics' a relative biodegradation percentage of 60% for homopolymers, 

while a relative biodegradation percentage of 90% is required for products consisting 

of more than one polymer. 

b) according to the European Standard E N 13432 (2000) 'Requirements for packaging 

recoverable through composting and biodegradation - Test scheme and evaluation 

criteria for the final acceptance ofpackaging' and ISO 17088 (2008) 'Specifications 

for compostable plastics' a material can only be called biodegradable when the 

percentage of biodegradation is at least 90% in total or 90% of the maximum 

degradation of a suitable reference item after a plateau has been reached for both 

reference and test item, and this within a period of maximum 180 days. 
Code: R-KS-3B OTjT^/^r) Revision: 00 

Date: Oct-26-11 I^EIa'^"! Page: 7 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

6. INTRODUCTION 

6.1. PRINCIPLE OF TEST METHOD 

The aqueous biodegradation test determines the biodegradation of a test item under laboratoiy 

conditions by a consortium of bacteria from different sources or a conditioned sludge preexposed to the test item or structure-related component. The test material is brought into a 

chemically defined (mineral) liquid medium, essentially free of other organic carbon sources, 

and spiked with micro-organisms. 

During the aerobic biodegradation of organic materials in an aqueous medium, oxygen is 

consumed and carbon is converted to gaseous, mineral C (under the form of carbon dioxide, 

C02 ). Part of the organic material is assimilated for cell growth. NaOH pellets trap the C0 2 

released and the induced pressure-drop is directly related to the consumed oxygen and hence to 

the biodegradation of the test item. 

The amount of biodegradation is expressed as the ratio of the BOD (corrected for the blank) to 

the Theoretical Oxygen Demand (ThOD) or Chemical Oxygen Demand (COD) of the used test 

item. 

6.2. GUIDELINES USED 

The test is performed according to: 

ISO 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). 

And 

AST M D5271-02: Standard Test Method for Determining the Aerobic Biodegradation of 

Plastic Materials in an activated-Sludge-Wastewater-Treatment System. 

This test method is equivalent to ISO 14851. 
Code: R-KS-3B I^TtT^rZ^ZTt Revision: 00 

Date: Oct-26-11 ^HUd^^d I Page: 8 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

7. MATERIALS AND METHODS 

7.1. TEST ITEM AND REFERENCE ITEM 

TEST ITEM 

Name: 

Manufacturer's name: 

Description: 

Colour: 

Batch number: 

Production date: 

Storage conditions: 

ThOD: 

R-9010 

Liquid 

Transparent, light yellow 

UBS071511 

7/2011 

Room temperatures in the dark 

2.792 g/g (calculated from elemental analysis) 

REFERENCE ITEM 

Name: Cellulose 

Physical form: Powder 

Color: White 

Storage conditions: Room temperature in the dark 

COD: 1.141 g/g (± 0.027 g/g) (determined by OWS) 

7.2. GENERAL PROCEDURE 

7.2.1. Preparation of the inoculum 

The source of microbial flora was a mixture of activated sludge from different sewagetreatment plants in Gent (Ossemeersen), Lokeren, Zele, Brecht and Landegem. These sites 

treat wastewater from domestic and/or industrial origin. The different sludges were sieved 

over an 80um sieve and the mixture made of equal portions of the different sludges was 

aerated for 4 hours. The final inoculum was obtained after removal of supernatant liquid and 

replacement with mineral medium used for the removal of soluble components. 

7.2.2. Preparation of Control, Reference and Test Reactors 

At the start of the experiment, each reactor was filled with the same amount of mineral 

medium. 

A precise amount of inoculum (1-5%) is added to each reactor to obtain a test medium with a 

concentration of approximately 30 mg suspended solids/1. The reference and test item are added 

directly to the reactors. 

The vessels are later on aerated for another 15 minutes in the incubator before closing and 

initiating the actual incubation period for biodegradation. This final aeration period is needed to 

equilibrate the final mixture and to stabilise the temperature. 
Code: R-KS-3B [fiWufffii^l Revision: 00 

Date: Oct-26-11 if^WJjJ-^^J Page: 9 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

7.2.3. Performance of the Test 

For the set-up ofthe test system, see Figure 1. 

After filling the reactors, they are put on an inductive stirrer. NaOH pellets are added to the 

rubber carrier and the OXITOP-OC heads are connected. A magnetic rod is present to keep 

the reference item, test item and the growing biomass into suspension throughout the test. 

During the test, the NaOH pellets absorb the C0 2 produced. The reactors are incubated at a 

constant temperature (21°C + 1°C) in the dark for a period of minimum 28 days. 

Figure J. Respirometric test set-up 
Code: R-KS-3B M^(7*^7 Revision: 00 

Date: Oct-26-11 I^WJjjmp^f Page: 10 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

7.3. ANALYTICAL METHODS 

Weight Determination 

During the test 2 types of balances were used. A Sartorius A C 210 S with internal calibration 

(max. 200 g; d = 0.1 mg) for the determination of dry and volatile matter and for the weight 

of test and reference items. A Sartorius ED 3202S-CW (max. 3200 g; d = 0.01 g) or a 

Acculab ATL-4202 (max. 4200 g; d = 0.01 g) was used for the weight of inoculum and 

mineral medium. 

Dry Matter or Total Solids 

The dry matter is determined by drying at 105°C until constant weight is reached and weighing, 

as described in MET H L.009 "Determination of moisture content". The dry matter is given in 

percent on wet weight. 

Volatile Solids -Ash 

The volatile solids and ash content is determined by heating the dried sample at 550°C for a 

few hours and weighing, as described in MET H L.OlO "Determination of organic matter and 

carbon content". The results are given in percent on dry matter. 

pH 

The pH is determined as described in METH L.006 "Determination of pH and electrical 

conductivity". 

COD (chemical oxygen demand) 

The chemical oxygen demand is determined as described in MET H L.103 "Detennination of 

COD". The results are given in mg/l. 

ThOD (Theoretical Oxygen Demand) 

The ThOD is calculated from the chemical formula or based on the elemental analysis (which 

is determined in another lab) of the test material, according to the formula described in ISO 

14851, annex A. The results are given in g/g. 

Elemental analysis CHN 

The elemental analysis is determined in another lab and analysed as described in ASTM D5291 

- 10 "Standard Test Methods for Instrumental Determination of Carbon, Hydrogen, and 

Nitrogen in Petroleum Products and Lubricants". The results are expressed in %. 

TSS& VSS 

The total suspended solids were determined using centrifugation. A well-known sample of 

the sludge (50-ml) was brought in a tube and centrifuged for 15 min at 4000 rpm. After 

decanting the supernatant the pellet was dried (105°C overnight) as described in "SOP on 

determination of suspended solids (TSS) and volatile suspended solids (VSS), Edition 3". 
Code: R-KS-3B fMWFjf^^l Revision : 0 0 

Date: Oct-26-11 MIlM ^ S / Page: 11 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

The volatile solids and ash content was determined by heating the dried sample at 550 °C for 

8 hours and weighing as described in "SOP on determination of suspended solids (TSS) and 

volatile suspended solids (VSS), Edition 3". The results are given in g/1. 

Ammonium- and Nitrate-N analyses 

The mineral N analyses are determined at OWS. The amount of ammonium is determined by 

Fiastar 5000 as described in MET H L.016 "Detennination of ammonia nitrogen-by FIA and 

spectrometric detection". The sample containing ammonium ions is injected into a continuous 

carrier stream by means of an injection valve and is mixed with a continuously streaming 

flow of an alkaline solution. The gaseous ammonia formed is separated through a diffusion 

cell from the solution over a hydrophobic semi permeable membrane and taken up by a 

streaming recipient flow containing a pH indicator. Due to the resulting pH shift, the 

indicator solution will change its colour which is measured continuously in the flow 

photometer at 590 nm. 

The amount of nitrate is also measured by Fiastar 5000 as described in METH L.017 

"Detennination of total oxidized nitrogen-by FIA and spectrometric detection". The sample 

containing nitrite/nitrate ions is fed into a continuously flowing buffer solution (carrier 

stream) by means of an injection valve. Nitrate in the sample is reduced to nitrite in a 

cadmium reductor. On the addition of an acidic sulphanilamide solution, nitrite initially 

present and nitrite formed from reduction of nitrate will form a diazo compound. This 

compound is coupled with N-(l-naphtyl)-ethylene diamine dihydrochloride (NED) to form a 

purple azo dye. This azo dye is measured at 540 nm. 

Kjeldahl Nitrogen (N) 

This analysis is done as described in "Method L.204. "Determination of Kjeldahl nitrogen". 

In the presence of a catalysing agent (K2S04 -Se-mixture) and under boiling conditions 

(380°C) with a mixture of sulphuric acid bound nitrogen is converted into the salt (NFLO2SO4. 

Afterwards the ammonia is liberated using strong alkali and distilled for subsequent 

determination by titration. The ammonia is captured in a boric acid/indicator solution. 

Determination of ammonium ion in the distillate is done by titration with standard acid. The 

results are given in g per kg total solids. 
Code: R-KS-3B IfWfCf***! °° 

Date: Oct-26-11 MEJd **""/ Page: 12 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

8. RESULTS 

8.1. ANALYSES INOCULUM AND TEST ITEM 

The inoculum is a mixture of activated sludge from different sewage-treatment plants (Gent, 

Lokeren, Zele, Brecht and Landegem) treating domestic and/or industrial wastewater. After 

filtration over an 80um sieve, decantation of the supernatant, and replacement with mineral 

medium, the final inoculum was obtained. The TSS, VSS and pH values of the inoculum 

itself are given in Table 1. The final suspended solids content (after dilution with mineral 

medium) was 28.4 mg TSS/1 and the final value for VSS was 20.1 mg/l. 

Table 1. Analyses of the inoculum. 

Inoculum 

TSS (g/1) 1.418 

VSS (g/1) 1.006 

VSS (% on TS) 71.0 

pH 7.2 

The reference item cellulose was analysed for Kjeldahl-N and for COD (Chemical Oxygen 

Demand). The ThOD (Theoretical Oxygen Demand) of test item HallGreen R-9010 was 

calculated from the elemental analysis CHN. The results are summarised in Table 2. 

Table 2. Elemental analysis CHN, Kjeldahl-N, COD and ThOD of the test and reference item. 

Test item C H N Kjeldahl-N COD (g/g) ThOD (g/g) 

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

HallGreen R-9010 77.2 10.7 0.09 - - 2.792 

Cellulose - - - <0.1 1.141 ±0.027 1.142 
Code: R-KS-3B IfW^f^ l °° 

Date: Oct-26-11 lUJVVjJj-^^l Page: 13 of20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

8.2. TEST CONDITIONS AND SET-UP 

A set of 9 equal reactor vessels with a total volume of 500 ml each was used. The total 

volume of the test solution was 250 ml. To 245 ml mineral medium, 5 ml inoculum was 

added. As a result a total suspended solid content of 28.4 mg/l and a total volatile suspended 

solid content of 20.1 mg/l were obtained in the final test medium. Both, reference item 

cellulose and test item " R-9010, were added directly to the vessels. The test set-up 

is given in Table 3. After the addition of the reference and test item, the reactors were put on an 

inductive stirrer. A magnetic rod kept the reference items, test items and the growing biomass 

into suspension throughout the test. 

The reactors were incubated at a constant temperature of 21 °C (+ 1°C) in the dark for a period 

of 28 days. 

Table 3. Test set-up. 

Reactor Test series Mineral Inoculum Cellulose Test item 

Number medium (ml/reactor) (mg/reactor) (mg/reactor) 

(ml/reactor) 

1 Blank 1 245 5 / / 

2 Blank 2 245 5 / / 

3 Blank 3 245 5 / / 

4 Cellulose 1 245 5 25.4 / 

5 Cellulose 2 245 5 25.2 / 

6 Cellulose 3 245 5 25.0 / 

7 R-9010 1 245 5 / 9.9 

8 R-90102 245 5 / 10.5 

9 a-.-.. - R-90103 245 5 / 10.3 

8.3. 02 CONSUMPTION 

The evolution of the cumulative 02-consumption of the inoculum (blank) is represented in 

Figure 2. The total 02-consumption for 3 replicates after 28 days of testing was 39.8 + 2.9 mg 

0 2/ l medium. 

Figure 3 shows the evolution of the 02-consumption of the positive reference item 

(Cellulose). The final 0 2 -consumption for 3 replicates of the reference item is 128.7 ± 15.5 

mg 02/ l. The final net Q2 -consumption for the reference item is 88.9 ± 15.5 mg 0 2/ l. 

In Figure 4 the evolution of the 02-consumption of the test item HallGreen R-9010 is seen. 

The final 0 2 -consumption for the 2 replicates (one replicate was omitted due to a leakage) is 

143.0 ± 8.5 mg 0 2/ l. The final net O2-consumption after is 103.2 ± 8.5 mg 0 2/ l. 

The evolution of the net 02-consumption of the reference (average 3 replicates) and the test 

item (average 2 replicates) is represented in Figure 5. 
Code: R-KS-3B IrlM^ ^ Revision: 00 

Date: Oct-26-11 lUJWJJJm^l Page: 14 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

150 

0 7 14 21 28 

Time (days) 

-—•Blank 1 — Blank 2 —-Blankjj 

Figure 2. Evolution of the cumulative 02-consumption ofthe inoculum. 

150 

Time (days) 

—-Cellulose 1 Cellulose 2 Cellulose 3 

Figure 3. Evolution of the cumulative 02-consumption ofthe reference item. 
Code: R-KS-3B IfWRf^l °° 

Date: Oct-26-11 01!j2J^n Page: 15 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

Time (days) 

~«-HallGree n 1 —HallGreen 2 

Figure 4. Evolution of the cumulative C>2-consumption ofthe test item. 

150 

Time (days) 

Cellulose —HallGreen R-9010 

Figure 5. Evolution of the net average 02-consumption ofthe reference and the test item. 
Code: R-KS-3B l/WMCp^^ Revision: 00 

Date: Oct-26-11 MWSSSU Page: 16 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

8.4. CORRECTION FOR 0 2 UPTAKE BY NITRIFICATION 

The aerobic biodegradation of an organic compound results in production of C0 2, water and 

mineral salts of any other elements present (mineralisation) and the production of new 

biomass. In case of nitrogen-containing compounds the nitrogen may be eliminated as 

ammonia, nitrite, or nitrate. Nitrification is a microbial process which consumes oxygen in a 

two-step process. 

a) nitrite production NH3 + 0 2 —»N02 

b) nitrate production N0 2 + 0 2 —> N0 3 

NOD is the nitrogen oxygen demand, which is the oxygen consumption related to 

nitrification. Nitrification itself is the biological oxidation of ammonia-nitrogen (NHJ-N) to 

nitrate nitrogen (NO 3 -N) with nitrite nitrogen (NO~-N) as an intermediate step. As such, a 

high level of nitrate is a sign of nitrification and oxygen consumption not related to carbon 

conversion. NOD is calculated as (Nitrate-N x 4.57+ Nitrite-N x 3.43). 

If nitrification has occurred but is not complete, the observed oxygen uptake by the reaction 

mixture (see 8.3. 0 2 -consumption) may be corrected for the amount of oxygen used in 

oxidizing ammonium to nitrite, and nitrite to nitrate. To calculate this amount the initial and 

final reaction mixture must be analyzed for nitrite and nitrate. 

8.5. ANALYSES AT THE END OF THE TEST 

At the end of the test period (after 28 days), the pH was measured and the vessels were 

checked by means of nitrite/nitrate strips on the presence of nitrite. As can be seen from 

Table 4, the pH ofthe blank reactors was almost equal to the reference/test item reactors. Al l 

blank reactors, cellulose and 7 > - R-9010 reactors were afterwards analysed for N H + -N 

and NO3-N (as nitrite seemed absent in all vessels). Almost complete nitrification was 

observed for blank, cellulose and test item. The results are summarised in Table 5 and also 

the calculated NOD (nitrogen oxygen demand) is mentioned. 

Table 4. pHat the end ofthe test (after 28 days). 

Reactor number Test series PH 

1 Blank 1 6.7 

2 Blank 2 6.8 

3 Blank 3 6.8 

4 Cellulose 1 6.8 

5 Cellulose 2 6.8 

6 Cellulose 3 6.8 

7

 1 R-9010 1 6.7 

8 R-90102 6.7 

9 R-90103 6.7 
Code: R-KS-3B KjfDYW^^l Revision: 00 

Date: Oct-26-11 Page: 17 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

Table 5. Ammonium-N and Nitrate-N (precise analyses) and calculated NOD after 28 days. 

NH;- N N03-N NOD 

Reactor number (mg/l) (mg/l) (mg/l) 

1 < 0.01 6.00 27.4 

2 0.14 5.93 27.1 

3 < 0.01 5.94 27.1 

4 0.23 5.26 24.0 

5 < 0.01 4.84 22.1 

6 0.54 5.00 22.9 

7 0.07 5.36 24.5 

8 < 0.01 5.43 24.8 

9 < 0.01 5.45 24.9 

Notes: NOD is calculated as (Nitrate-Nx 4.57) 

8.6. BIODEGRADATION PERCENTAGES 

8.6.1. Evolution of the biodegradation percentages 

(based on oxygen consumption without correction for NOD or biomass) 

Figure 6 shows the evolution of the average percentage of biodegradation for the reference 

item Cellulose and the test item

 1 R-9010. Figures 7 and 8 represent the 

biodegradation of the separate replicates. It should be noted that the calculation of the 

intermediary biodegradation percentages is based on the net oxygen consumption (after 

subtraction ofthe oxygen consumed in the blank reactors) in the test or reference reactor and 

on the COD added to each reactor. No intermediate correction is made for the oxygen 

consumption for nitrification. So Figures 6 up to 8 give a good indication ofthe evolution of 

the biodegradation but not ofthe absolute value of biodegradation. 

The biodegradability of the reference item cellulose started after approximately 2 days. It 

took 2.4 days to reach 11.4%. After 11 days an average biodegradation percentage of more 

than 60% was measured. After 28 days a biodegradation value of 77.3% ± 13.1% was 

measured. The test is considered valid, if at the end of the test the biodegradation percentage 

ofthe reference item is more than 60%. The results show clearly that this requirement was 

fulfilled. 

The biodegradation ofthe test item : - R-9010 started after 1 day and the level of 10% 

was passed after 1.6 days. After 5 days an average biodegradation of more than 60% was 

observed. At the end ofthe test (after 28 days) an absolute biodegradation of 90.6% ± 3.7% 

was obtained. 

A plateau in biodegradation was observed for both the reference and test item. 
Code: R-KS-3B I*K>jm^T) Revision: 00 

Date: Oct-26-11 ^r^^-w^i Page : 18of2 0 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

100 T 

Time (days) 

— Cellulose -» - HallGreen R-9010 

Figure 6. Evolution of the average biodegradation percentage of cellulose and test item 

. ' R-9010 (based on COD/ThOD values). 

100 

Time (Days) 

Figure 7. Evolution of the biodegradation percentage of the replicates of cellulose. 
Code: 

Date: 

R-KS-3B 

Oct-26-11 

Organic Waste Systems 

Revision: 00 

19 of 20 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

Time (Days) 

Figure 8. Evolution of the biodegradation percentage of the replicates of: R-9010. 

8.6.2. Biodegradation percentages 

(after correction for NOD but without correction for biomass) 

Table 6 gives the total amount of oxygen consumed per reactor, the oxygen consumption for 

oxidation of carbon (after subtraction of the NOD) and the net oxygen consumption of the 

test and reference item (after subtraction of the oxygen consumption in the blank). The 

biodegradation percentage based on the oxidation of carbon only is also reported in Table 6. 

Table 6. Biodegradation percentages after correction for NOD, based on BODc. 

Test item Test item Total 0 2 Total 02 Netto Biodegradationc 

ThOD/COD Consumption Consumption 

minus NOD 

BODc 

(mg /l test (mg/l test (mg/l test (mg/l test (%) 

medium) medium) medium) medium) 

Blank 1 - 36.6 9.2 

Blank 2 - 40.7 13.6 

Blank 3 - 42.1 15.0 

Cellulose 1 115.9 144.0 120.0 107.4 92.7 

Cellulose 2 115.0 113.0 90.9 78.3 68.1 

Cellulose 3 114.0 129.0 106.1 93.6 82.0 

"

:

" . J 1 110.5 137.0 112.5 99.9 90.4 

r '..--'^l 2 117.2 149.0 124.2 111.6 95.2 
Code: R-KS-3B fflWufff^^l Revision: 00 

Date: Oct-26-11 MEIzlf^l Page: 20 of 20 

Organic Waste Systems 

AQUEOUS AEROBIC RESPIROMETRIC BIODEGRADATION TEST 

8.6.3. Biodegradation percentages 

(after correction for NOD and for biomass) 

During biodegradation of organic material part of it is incorporated into new biomass. Thus 

the real percentage of biodegradation is based on the amount of test or reference item 

oxidised to carbon dioxide after subtracting NO D (=BOD) and after adding the amount of test 

or reference item transformed to biomass (= biomass-COD). For determination of the 

biomass-COD the following assumptions are made: biomass contains 50 % carbon on V S basis 

and 50 % of the V S are protein. C/ N ratio of protein is equal to 6,25 and l g of biomass V S is 

equal to 1 g of COD . Table 7 gives the results of the biodegradation percentages based on BO D 

and biomass-COD. The nitrogen fixed in biomass is obtained by subtracting the total amount of 

mineral nitrogen in the test or reference reactors from the total amount of mineral nitrogen in 

the blank reactors. The total amount of mineral nitrogen can be found in Table 5 and is the sum 

of NTLt +-N and N0 3 "-N . When the mineral N in the test and the reference reactors is lower than 

in the blank reactors (which is actually the case), part of the N has been assimilated by microorganisms and extra biomass has been formed in these reactors. 

Table 7. Definite biodegradation percentages based on BODc and biomass yield. 

Test item Test item BODc Nin Biomass - Definite Biodegradation 

COD/ThOD biomass COD (%) 

(mg /I test (mg/l test (mg/l) (mg/l) Avg Std Rel 

medium) medium) 

Cellulose 1 115,9 107.4 0.52 6.5 98.3 88.7 8.9 100.0 

Cellulose 2 115.0 78.3 1.16 14.5 80.7 

Cellulose 3 114.0 93.6 0.46 5.7 87.1 

1 110.5 90.4 0.58 7.2 97.0 99.2 3.1 111.8 

.. -i2 117.2 95.2 0.58 7.2 101.4 

With Avg = average; Std = standard deviation and Rel = relative biodegradation 

For the positive reference item Cellulose an average biodegradation result of 88.7% + 8.9% 

was obtained at the end of the test (after 28 days). For the test item i R-9010 an 

average absolute biodegradation percentage of 99.2% + 3.1% was obtained after 28 days of 

incubation. On a relative basis with cellulose as the suitable reference material 111.8% was 

calculated. From these results it can be concluded that the test item . R-9010 was 

completely biodegraded and can therefore be regarded as completely biodegradable at 

ambient temperature within 28 days under aerobic conditions considering that: 

b) according to the American standard AST M D6400-04 'Standard Specification for 

Compostable Plastics' a relative biodegradation percentage of 60% for homopolymers, 

while a relative biodegradation percentage of 90% is required for products consisting 

of more than one polymer, 

b) according to the European Standard E N 13432 (2000) 'Requirements for packaging 

recoverable through composting and biodegradation - Test scheme and evaluation 

criteria for the final acceptance of packaging' and ISO 17088 (2008) 'Specifications 

for compostable plastics' a material can only be called biodegradable when the 

percentage of biodegradation is at least 90% in total or 90% of the maximum 

degradation of a suitable reference item after a plateau has been reached for both 

reference and test item, and this within a period of maximum 180 days.