ADHESIVE ST6093G + ST6097 · OWS NV • Dok Noord 5 • B-9000 Gent • Belgium • tel. +32 9...

FINAL REPORT

COMPOSTABILITY TESTING PROGRAM

ON

ADHESIVE ST6093G + ST6097

• EXECUTIVE SUMMARY

• HEAVY METALS

• AEROBIC BIODEGRADATION TEST

• ECOTOXICITY TESTS

BARLEY PLANT GROWTH TEST CRESS TEST

EXECUTIVE SUMMARY

OWS NV • Dok Noord 5 • B-9000 Gent • Belgium www.ows.be • tel. +32 9 2330204 • fax +32 9 2332825

EXECUTIVE SUMMARY

COMPOSTABILITY TESTING PROGRAM

ON

ADHESIVE

ST6093G + ST6097

ACCORDING TO

EN 13432 (2000), ASTM D 6868-11 & ISO 17088 (2008)

STUDY TJO-1

Scitech Adhesive Systems Ltd. Flint CH6 5AX UNITED KINGDOM

Code:

Date:

R-TJO-1-ex

Jul-28-11

Revision:

Page:

00

2 of 3

EXECUTIVE SUMMARY

A compostability testing program was performed on adhesive ST6093G + ST6097 in line with CEN norm EN 13432 of September 2000 ‘Requirements for packaging recoverable through composting and biodegradation - Test scheme and evaluation criteria for the final acceptance of packaging’, ASTM D 6868-11 ‘Standard Specification for Labeling of End Items that Incorporate Plastics and Polymers as Coatings or Additives with Paper and Other Substrates Designed to be Aerobically Composted in Municipal or Industrial Facilities’ and ISO 17088 (2008) ‘Specifications for compostable plastics’. Adhesive ST6093G + ST6097 is obtained by mixing ST6093G and ST6097 in a concentration of 97.8% and 2.2%, respectively. Following basic characteristics were evaluated: material characteristics, biodegradation and plant ecotoxicity tests. The detailed test results are given in the various reports. The study plans are given in Appendix 1. Material characteristics Test item ST6093G + ST6097 showed a volatile solids content of 99.7% on total solids content (TS), which is well above the minimum volatile solids content of 50% on TS as prescribed by EN 13432 (2000) and ISO 17088 (2008). The heavy metals content of ST6093G + ST6097 is reported in report R-TJO-1/1. Adhesive ST6093G + ST6097 does fulfill the heavy metal requirements of EN 13432 (2000), ASTM D 6868-11 and ISO 17088 (2008). Biodegradation under aerobic conditions Adhesive ST6093G + ST6097 was tested on biodegradation in a controlled composting test at 58°C (see report R-TJO-1/2). After 137 days of incubation an absolute biodegradation of 84.8% was measured for ST6093G + ST6097, or 90.4% on a relative basis compared to the suitable reference material cellulose. Material ST6093G + ST6097 fulfills the 90% biodegradability requirement of EN 13432 (2000), ASTM D 6868-11 and ISO 17088 (2008) within 180 days, and can be called biodegradable under controlled composting conditions. Pilot-scale composting test As material ST6093G + ST6097 will only be used as an additive in a compostable product, no disintegration needed to be evaluated on the adhesive itself. However, the final product, containing adhesive ST6093G + ST6097, will have to demonstrate that it fulfills the 90% pass level for disintegration as stipulated by EN 13432 (2000), ASTM D 6400-04, ASTM D 6868-11 and ISO 17088 (2008). Compost quality - Ecotoxicity tests The addition of ST6093G + ST6097 in a maximum concentration of 10.0% on dry weight to a compostable material, exerted no negative effect on the emergence and growth of barley plants (see report R-TJO-1/4) and cress plants (see report R-TJO-1/5). Adhesive ST6093G + ST6097 fulfills the ecotoxicity requirements of EN 13432 (2000), ASTM D 6868-11 and ISO 17088 (2008) till a maximum concentration on dry weight of 10.0% in a compostable material.

HEAVY METALS

Report R-TJO-1/1


FINAL REPORT

HEAVY METAL ANALYSES

OF

ST6093G +ST6097

AND

ST6103 + ST6104

STUDY TJO-1/1 Scitech Adhesive Systems Ltd. Author: Steven VERSTICHEL Casle Park Flint CH6 5AX UNITED KINGDOM

AEROBIC BIODEGRADATION TEST

Report R-TJO-1/2


FINAL REPORT

AEROBIC BIODEGRADATION UNDER CONTROLLED COMPOSTING CONDITIONS

OF

ST6093G + ST6097

AND

ST6103 + ST6104

STUDY TJO-1/2

Scitech Adhesive Systems Ltd. Author: Mia CLINCKSPOOR Casle Park Flint CH6 5AX UNITED KINGDOM

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

2 of 23


TABLE OF CONTENTS

1. IDENTIFICATION OF THE 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 AND REFERENCE ITEM ....................................................................................................................8 7.2. GENERAL PROCEDURE ..............................................................................................................................9

7.2.1. Composting under dynamic conditions.......................................................................................................9 7.2.2. Composting under static conditions ......................................................................................................... 10

7.3. ANALYTICAL METHODS ........................................................................................................................ 12

8. TEST SET-UP .................................................................................................................................................. 14

8.1. TEST CONDITIONS AND SET-UP............................................................................................................ 14 8.2. ANALYSES INOCULUM, TEST ITEMS AND REFERENCE ITEM ...................................................... 15 8.3. CO2 PRODUCTION ..................................................................................................................................... 16 8.4. BIODEGRADATION PERCENTAGES ..................................................................................................... 19 8.5. VISUAL PERCEPTIONS AND ANALYSES AT END OF TEST ............................................................. 23

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

3 of 23


1. IDENTIFICATION OF THE TEST

1.1. GENERAL INFORMATION

Project Number TJO-1/2 Sponsor

Scitech Adhesive Systems Ltd. Casle Park Flint CH6 5AX UNITED KINGDOM Monitoring Scientist Mr. Trevor JONES Phone: +44/1352.732007 [email protected] Fax: +44/1352.732045 Testing Facility ORGANIC WASTE SYSTEMS N.V. Phone: +32/9/233.02.04 Dok Noord 5 Fax: +32/9/233.28.25 B-9000 Gent [email protected] BELGIUM [email protected]

Test Items

ST6093G + ST6097 ST6103 + ST6104 Reference Item

Cellulose

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

4 of 23


1.2. STUDY PERSONNEL

Study Director: Mia CLINCKSPOOR Replacement Study Director: Bruno DE WILDE Quality Assurance Manager: Steven VERSTICHEL

1.3. STUDY SCHEDULE

After 45 days of incubation the test was stopped for test item ST6103 + ST6104. On Dec-14-10, after 47 days of testing, the test was changed from dynamic composting conditions to static conditions. At the same time a re-inoculation with 10% fresh VGF (Vegetable, Garden and Fruit waste) was executed in order to renew the microbial population and to supply extra nutrients, in an attempt to increase the biodegradation rate of the test items. The total test duration was 137 days. Study initiation date: Oct-27-10 Study completion date: Mar-17-11 Experimental starting date: Oct-27-10 Starting date of incubation under dynamic conditions: Oct-28-10 Completion date of incubation under dynamic conditions: Dec-14-10 Duration incubation under dynamic conditions: 47 days Starting date of incubation under static conditions: Dec-14-10 Completion date of incubation under static conditions: Mar-14-11 Duration incubation under static conditions: 90 days Total test duration: 137 days Experimental completion date: May-20-11

1.4. ARCHIVING

All raw data and records necessary 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, training records of personnel, samples of test and reference item and specimens. They will be stored in a file coded:

TJO-1/2

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:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

6 of 23


5. SUMMARY AND CONCLUSIONS

Test items ST6093G + ST6097 and ST6103 + ST6104 were tested on biodegradability under dry, aerobic conditions in a controlled composting test according to ISO 14855. The incubation temperature was continuously kept at 58°C. After 45 days the test was stopped for ST6103 + ST6104. After 47 days the test was converted from dynamic to static conditions and at the same time a re-inoculation with 10% fresh VGF (Vegetable, Garden and Fruit waste) was executed in attempt to increase the biodegradation rate of ST6093G + ST6097. The test was ended after 137 days. According to the standard ISO 14855 a CO2-production between 50 mg and 150 mg CO2/g VS should be measured for the blank during the first 10 days of the test. After 10 days a background activity of 106 mg CO2/g VS was measured, which indicates the good quality of the inoculum. The biodegradation of the reference item cellulose started almost immediately at a high rate. After 9 days cellulose was already degraded for 71.6%. The biodegradation went on and after 45 days a biodegradation of 96.0% was obtained, while at the end of test (after 137 days) a plateau in biodegradation at a level of 93.8%+9.7% was measured. The results of third replicate of cellulose (RN 10) were omitted due to a leak during the beginning of the test. The test is considered valid if after 45 days the biodegradation percentage of the reference item is more than 70% and if the standard deviation of the biodegradation percentage of the reference item is less than 20% at the end of the test. Both requirements were easily fulfilled. The biodegradation of test items ST6093G + ST6097 and ST6103 + ST6104 started also immediately at a good rate and after 17 days an absolute biodegradation of 51.8% and 55.5% was observed, respectively. From then the biodegradation rate decreased considerably and the biodegradation proceeded further at a moderate rate. After 45 days test item ST6103 + ST6104 reached an absolute biodegradation of 67.9%+2.7% or 70.7% on a relative basis compared to the suitable reference substrate cellulose. However, the biodegradation of this material was still slowly going on. At the end of test (after 137 days) a biodegradation of 84.8%+4.0% was reached for test item ST6093G + ST6097, or 90.4% on a relative basis compared to the suitable reference substrate cellulose. According to the European standard EN 13432 (2000) ‘Requirements for packaging recoverable through composting and biodegradation – Test scheme and evaluation criteria for the final acceptance of packaging’ and the international standard 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. The American standard ASTM D 6400-04 ‘Standard Specification for Compostable Plastics’ stipulates 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. The maximum allowed test duration by the standards is 180 days. From the results it can be concluded that test item ST6093G + ST6097 fulfills the biodegradation requirement of these standards within 137 days. Test item ST6103 + ST6104 does not fulfill the biodegradation requirement of the standards within 45 days, but the biodegradation of this material was still slowly going on. The results, obtained in this test, are valid for solid aerobic conditions only and cannot be directly used for aqueous or for anaerobic conditions. Other tests are more suited to simulate and examine the degradation under these circumstances.

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

7 of 23


6. INTRODUCTION

6.1. PRINCIPLE OF TEST METHOD

The controlled composting biodegradation test is an optimized simulation of an intensive aerobic composting process where the biodegradability of a test item under dry aerobic conditions is determined. The inoculum consists of stabilized and mature compost derived from the organic fraction of municipal solid waste. The test item is mixed with the inoculum and introduced into static reactor vessels where it is intensively composted under optimum oxygen, temperature and moisture conditions. During the aerobic biodegradation of organic materials, a mixture of gases, principally carbon dioxide and water, are the final decomposition products while part of the organic material will be assimilated for cell growth. The carbon dioxide production is continuously monitored and integrated to determine the carbon dioxide production rate and the cumulative carbon dioxide production. After determining the carbon content of the test item, the percentage of biodegradation can be calculated as the percentage of solid carbon of the test item, which has been converted to gaseous, mineral C under the form of CO2. Also the kinetics of the biodegradation can be established.

6.2. GUIDELINES USED

The test is performed according to ISO 14855 (2005) method: "Determination of the Ultimate Aerobic Biodegradability and Disintegration of Plastics under Controlled Composting Conditions", which is part of the CEN standard EN 13432 (2000): "Requirements for packaging recoverable through composting and biodegradation - Test scheme and evaluation criteria for the final acceptance of packaging" and the international standard ISO 17088 (2008) ‘Specifications for compostable plastics’. The standard is identical to CEN norm EN 14046 (2003): “Packaging. Evaluation of the ultimate aerobic biodegradability and disintegration of packaging materials under controlled composting conditions. Method by analysis of released carbon dioxide”.

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

8 of 23


7. MATERIALS AND METHODS

7.1. TEST AND REFERENCE ITEM

TEST ITEM 1

Name: ST6093G + ST6097 Sample preparation: Mixing ST6093G + ST6097 in a ratio 500 g + 11 g Name: ST6093G Batch number: 1671L/1 Description: Liquid Colour: Whitish Name: ST6097 Batch number: Sample 13/10/10 Description: Viscous liquid (Cross-linker) Colour: Transparent

TEST ITEM 2

Name: ST6103 + ST6104 Sample preparation: Mixing ST6103 + ST6104 in a ratio 500 g + 16.4 g Name: ST6103 Batch number: 1671L/2 Description: Liquid Colour: Whitish Name: ST6104 Description: Liquid (Cross-linker) Colour: Whitish

REFERENCE ITEM

Name: Cellulose Purity: Native cellulose powder for thin layer chromatography (Avicel) Physical form: Powder Colour: White Batch number: K39658431930 Expiration date: October 2014 Brand: Merck Art. Nr. 2331

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

9 of 23


7.2. GENERAL PROCEDURE

7.2.1. Composting under dynamic conditions The inoculum is derived from the organic fraction of municipal solid waste, which is stabilized further and matured in a composting bin at the laboratory under controlled aeration conditions (see Figure 1). Before use the mature compost is sieved on a screen of 5 mm. The fine fraction is the inoculum. It is recommended that it has a total solids content of roughly 50-55% and a volatile solids concentration of 30% (±5%) on dry solids. The test and reference item are mixed with the inoculum in a ratio of roughly 1 to 1.5 parts of total solids to 6 parts of total solids and introduced into the reactors. These reactors are closed airtight and put into the incubators (see Figure 2). The temperature of the reactors is continuously controlled and kept at 58°C. Pressurized dry air is sent over a gas flow controller, which regulates very precisely the flow rate and blown into the composting vessel at the bottom through a porous plate. Through biodegradation solid carbon of the test compound is converted and CO2 is produced. The gas leaving each individual reactor is continuously analyzed on regular intervals (about every 6 hours) for CO2 and O2 concentration. Also the flow rate is measured regularly. Likewise the cumulative CO2 production can be determined. The percentage of biodegradation is determined as the percentage of solid carbon of the test compound that is converted to gaseous, mineral C under the form of CO2. More details on the procedure for the particular test reported, are given in the study plan.

Figure 1. Set-up composting bin for compost maturation.

GAS SAMPLING

COMPOST DRUM

GRIDGAS METER

FLOW CONTROL

SURPRESSOR

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

10 of 23


Figure 2. Set-up controlled composting test.

7.2.2. Composting under static conditions After 47 days of composting under dynamic conditions a homogeneous compost sample (about 100 g) of each reactor mixture was further composted under static conditions. The sample is put in a beaker and placed in a reactor, which can be closed airtight (Figure 3). The reactor also contains a beaker with KOH to absorb the carbon dioxide, released during the composting process. The two beakers are put on a perforated plate. A same amount of water is put on the bottom of the reactors in order to prevent a drying out of the compost sample. Three technical controls are included in the test set-up. The technical control has the same composition as the other reactors, except that no beaker with compost sample is added. The exact test set-up can be found in study plan amendment 1. The reactors are stored in an incubator at 58°C. The CO2 production is determined by titration. The percentage of biodegradation is calculated as the percentage of solid carbon of the test item, which has been converted to gaseous, mineral C under the form of CO2. After each titration, a new beaker with KOH is put into the reactor. At the same time the compost sample is stirred and moistened if needed.

1.

2.

3.

4.

5.

Air

Flow Control

Humidifier

Composting Vessel

Condensate

PRINTER

DATA

PROCESSOR

GAS

CHROMATOGRAPH

1

23

45

6

7 8

9

10

6.

7.

8.

9.

10.

Multiport Valve

Test Gas

Carrier Gas

Sample Loop

Flow MeterINCUBATOR COOLING UNIT

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

11 of 23


Figure 3. Set-up controlled composting test under static conditions.

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

12 of 23


7.3. ANALYTICAL METHODS

Weight Determination During the test 3 types of balances are used. A Sartorius AC 210 S with internal calibration (max. 200 g; d = 0.1 mg) for the determination of dry and volatile matter. A Mettler PJ12 (max. 12000 g, d = 0,1 g), Sartorius BL6100 (max. 6100 g, d = 0.1 g) or Sartorius CP 12001 S (max. 12100 g, d = 0,1 g) are used for weighing of the inoculum and test items and weighing of the water added throughout the composting process. A Toledo G3 balance (max. 150 kg; d = 50 g) is used for weighing of the biowaste and the composting bins. Dry Matter or Total Solids

The dry matter is determined by drying at 105°C for at least 16 hours and weighing, as described in "METH L.009. Compost – 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 "METH L.010. Compost – Determination of organic matter and carbon content". The results are given in percent on dry matter. pH

The pH is measured with a pH meter after calibration with standard buffer solutions (pH = 4.00, pH = 7.00 and pH = 10.00), as described in "METH L.006. Compost - Determination of pH and electrical conductivity". Before inserting the electrode the sample is diluted with distilled water at a ratio of 5 to 1 (5 parts of demineralised water versus 1 part of sample) and thoroughly mixed. Salt Content (Electrical Conductivity, E.C.) The salt content is measured with a conductivity meter after calibration in a 0.01 M KCl solution, as described in "METH L.006. Compost - Determination of pH and electrical conductivity". Before inserting the electrode the sample is diluted with distilled water at a ratio of 5 to 1 (5 parts of distilled water versus 1 part of sample) and thoroughly mixed, as described in "METH L.012. Compost – Preparation of extracts and analysis solutions". Ammonium - Nitrogen (NH 4

+ -N)

This analysis is done as described in "METH L.016. Compost - Determination of ammonia-nitrogen by FIA and spectrometric detection". The ammonium-N of compost is determined in an aqueous extract (5 parts of demineralised water versus 1 part of sample; see METH L.012). 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 results are given in g per l wet weight.

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

13 of 23


Nitrate and Nitrite - Nitrogen (NO x

− -N) This analysis is done as described in "METH L.017. Compost-Determination of nitrite and nitrate nitrogen and the sum of both by FIA and spectrometric detection". The determination is performed on an aqueous extract (5 parts of demineralised water versus 1 part of sample; see METH L.012). 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 tot 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-(1-naphtyl)-ethylene diamine dihydrochloride (NED) to form a purple azo dye. This azo dye is measured at 540 nm. The results are given in g per l wet weight. Total Nitrogen (N)

This analysis is done as described in "METH L.005. Compost-Determination of total nitrogen". In the presence of a catalysing agent (K2SO4-mixture) and under boiling conditions (380°C) with a mixture of sulphuric acid- salicylic acid bound nitrogen is converted into the salt (NH4)2SO4. 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. Volatile Fatty Acids (VFA)

The volatile fatty acids are determined as described in "METH L.203 Determination of volatile fatty acids". The sample is diluted with water and centrifuged to remove the suspended solids. Afterwards ether is added and the acids are extracted by centrifugation. The actual analysis is done by gas chromatography. The gas chromatograph is a Mega 8130 DPFC (8000 Top Serie). The column used is a Stabilwax of 30 m. The carrier gas is He. A mixture with precise concentrations of eight reference volatile fatty acids is used for calibration while 2-methyl-caproic acid is used as an internal standard. The results are given in g per l wet weight. Gas Composition The gas analyses are performed on a PerkinElmer AutoSystem XL gas chromatograph with CTRI column as described in "SOP on general procedure of the composting unit 1, Edition 3". The gas chromatograph is calibrated with a standard gas mixture consisting out of 15% O2, 6% CO2 and 79% N2. The calibration is done just before the start of each cycle of analyses. Total Organic Carbon (TOC)

The TOC, total organic carbon, is determined in another laboratory. The sample is pretreated with acid to remove the inorganic carbon. The used method is an element CHN analysis. The sample is burned in a small excess of oxygen at 900°C. Due to the exothermic combustion of the tin capsule the temperature raises to about 1800°C. The sample is at that temperature decomposed to NOx, N2, CO, CO2, H2O, SO2, SO3, salts and metals. The gas mixture is led to oxidators and catalysts and converted to NOx, N2, CO2 and H2O. After reduction, the end products CO2 and H2O are separated on different absorption columns, while the N2 gas is directly measured by means of a katharometer. Thereafter CO2 and H2O are measured. The carbon content is given in percent on wet weight. The apparatus used is an Elementar Vario EL III. Reagents used are oxygen gas (as burning gas), helium gas (carrier stream), and standards for calibration. The analysis is executed according to EN 13137 procedure B, Direct method.

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

14 of 23


8. TEST SET-UP

8.1. TEST CONDITIONS AND SET-UP

A set of 12 equal composting vessels with a total volume of 4 l each was used, incubated at a constant temperature of 58°C + 2°C. The test set-up is given in Table 1. Reference material cellulose was added as powder, while the test items were prepared just before start of the test. ST6093G and ST6097 were mixed in a ratio of 500 g + 11 g, while ST6103 and ST6104 were blended in a ratio of 500 g + 16.4 g. Extra water was added to the cellulose reactors to maintain optimal moisture conditions. After 45 days the test was stopped for ST6103 + ST6104. After 47 days the test was converted from dynamic to static conditions and at the same time a re-inoculation with 10% fresh VGF (Vegetable, Garden and Fruit waste) was executed. The total test duration was 137 days. Table 1. Test set-up controlled composting test.

RN Test Series Inoculum (g) Item (g) Water (g)

1

2 3

4

5

6 7

8

9

10 11

12

Control

Cellulose ST6093G + ST6097

ST6103 + ST6104

Control


ST6103 + ST6104

Control


ST6103 + ST6104

1201

1199 1198

1198

1199

1199 1200

1199

1200

1200 1200

1209

0

70 179

179

0

70 180

180

0

70 180

181

0

45 0

0

0

43 0

0

0

46 0

0

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

15 of 23


8.2. ANALYSES INOCULUM, TEST ITEMS AND REFERENCE ITEM

The inoculum was derived from the organic fraction of municipal solid waste. The waste was stabilized and aerated in a composting bin at the laboratory under controlled conditions for more than 20 weeks. Before use the compost was sieved through 5 mm. The characteristics of the inoculum used at start are given in Table 2. The inoculum should have a total dry solids (TS) content between 50% and 55% and a volatile solids content (VS) on TS of 30%(±5%). Moreover the pH should be between 7.0 and 9.0. As can be seen from Table 2 these requirements were largely fulfilled. The inoculum showed just a little higher total solids content of 56.3%. However, as the test materials were added as liquids it was aimed for a little higher dry matter content in order to obtain optimal moisture conditions in these reactors. The mixtures must feel somewhat sticky and have some free water available when pressed by hand. Some extra water was added to the cellulose reactors in order to obtain optimal moisture conditions. Moreover a volatile solids content of 32.3% on TS and a pH of 8.6 was measured. According to the norm ISO 14855 a CO2-production between 50 mg and 150 mg CO2/g VS should be measured for the blank during the first 10 days of the test. After 10 days a background activity of 106 mg CO2/g VS was measured, which indicates the good quality of the inoculum. Table 2. Characteristics of the inoculum.

Characteristics Inoculum

Total solids (TS, %) Moisture content (%) Volatile solids (VS, % on TS) Ash content (% on TS) pH E.C. (µS/cm) Volatile Fatty Acids (VFA, g/l) Total N (g/kg TS) NH4

+-N (mg/l) NOx

--N (mg/l) C/N ratio

56.3 43.7 32.3 67.7 8.6

2190 b.r. 17.2 23 316 9

b.r. = below reporting limit: VFA = 0.3 g/l The reference item and test items were analyzed for total solids (TS), volatile solids (VS) and total organic carbon content (TOC) (see Table 3). Table 3. Total solids (TS), volatile solids (VS) and total organic carbon (TOC) content of the

reference and test items.

Test item TS (%) VS (% on TS) TOC (%)

Cellulose

ST6093G + ST6097

ST6103 + ST6104

96.4

39.0

39.3

100.0

99.7

99.5

42.5

24.1

26.3

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

16 of 23


8.3. CO2 PRODUCTION

The total cumulative CO2 production for each reactor after 45 days (end of test for ST6103 + ST6104) and after 137 days (end of test for ST6093G + ST6097) is given in Table 4. Also the net cumulative CO2 production of the reference and test items is given in g absolute and in mg per g of test item. The results of third replicate of cellulose (RN 10) were omitted due to a leak during the beginning of the test. Therefore these results were reported between brackets and shown as dashed lines in the figures. Cellulose replicate RN10 was not taken into account for the calculation of the final biodegradation percentages. Figures 4 up to 7 show the evolution of the total cumulative CO2 production (g CO2/kg inoculum). Table 4. CO2 production after 45 days (end of test for ST6103 + ST6104) and at end of test

(after 137 days).

RN Test Series Total CO2

(g) Net CO2

(g) (mg/g test item)

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 5 6 7 9 10 11

After 45 days Control Cellulose ST6093G + ST6097 ST6103 + ST6104 Control Cellulose ST6093G + ST6097 ST6103 + ST6104 Control Cellulose ST6093G + ST6097 ST6103 + ST6104 After 137 days Control Cellulose ST6093G + ST6097 Control Cellulose ST6093G + ST6097 Control Cellulose ST6093G + ST6097

86.3 183.9 194.4 184.1

64.0 169.2 183.5 193.0

65.5

(182.1) 190.1 192.5

161.4 271.1 297.0

151.4 265.0 289.4

148.0

(261.0) 301.9

-

112.1 122.6 112.2

-

97.4 111.6 121.2

-

(110.2) 118.2 120.0

-

109.9 135.6

-

94.8 127.5

-

(100.1) 139.9

-

1601 684 626

-

1392 621 674

-

(1576) 658 664

-

1569 756

-

1355 709

-

(1432) 779

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

17 of 23


Figure 4. Total CO2 production of the control.

Figure 5. Total CO2 production of cellulose.

0

20

40

60

80

100

120

140

160

180

200

220

240

260

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Tot

al C

O2

prod

ucti

on (

g C

O2

/ kg

inoc

ulum

)

Time (Days)

RN 1 RN 5 RN 9

after 47 days re-inoculation with 10% VGF

0

20

40

60

80

100

120

140

160

180

200

220

240

260

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Tot

al C

O2

prod

ucti

on (

g C

O2

/kg

inoc

ulum

)

Time (Days)

RN 2 RN 6 RN 10



Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

18 of 23


Figure 6. Total CO2 production of ST6093G + ST6097.

Figure 7. Total CO2 production of ST6103 + ST6104.

0

20

40

60

80

100

120

140

160

180

200

220

240

260

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Tot

al C

O2

prod

ucti

on (

g C

O2

/kg

inoc

ulum

)

Time (Days)

RN 3 RN 7 RN 11


0

20

40

60

80

100

120

140

160

180

200

220

240

260

0 5 10 15 20 25 30 35 40 45

Tot

al C

O2

prod

ucti

on (

g C

O2

/kg

inoc

ulum

)

Time (Days)

RN 4 RN 8 RN 12

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

19 of 23


8.4. BIODEGRADATION PERCENTAGES

The results on the calculation of the biodegradation percentages after 45 days (end of test for ST6103 + ST6104) and after 137 days (end of test for ST6093G + ST6097) is given in Table 5. The percentages are determined by the ratio of gaseous carbon, which is found back under the form of carbon dioxide at the end of the incubation period, to the original amount of carbon input. Figure 8 shows the evolution of the average biodegradation percentages, while Figures 9 up to 11 represent the biodegradation of the separate replicates. Table 5. Biodegradation percentages after 45 days (end of test ST6103 + ST6104) and after

137 days (end of test).

Item

Average Cinput

Average Cgaseous

Biodegradation (%)

95% CL

(g) (g) AVG STD REL

After 45 days Cellulose

ST6093G + ST6097

ST6103 + ST6104

After 137 days Cellulose ST6093G + ST6097

29.7

43.2

47.3

29.7 43.2

28.6

32.0

32.1

27.9 36.6

96.0

74.2

67.9

93.8 84.8

9.5

3.6

2.7

9.7 4.0

100.0

77.2

70.7

100.0 90.4

30.0

13.8

12.4

30.3 14.2

With AVG = average, STD = standard deviation, REL = relative biodegradation and CL = confidence limits. The biodegradation of the reference item cellulose started almost immediately at a high rate. After 9 days cellulose was already degraded for 71.6%. The biodegradation went on and after 45 days a biodegradation of 96.0% was obtained, while at the end of test (after 137 days) a plateau in biodegradation at a level of 93.8%+9.7% was measured. The results of third replicate of cellulose (RN 10) were omitted due to a leak during the beginning of the test. The test is considered valid if after 45 days the biodegradation percentage of the reference item is more than 70% and if the standard deviation of the biodegradation percentage of the reference item is less than 20% at the end of the test. Both requirements were easily fulfilled. The biodegradation of test item ST6103 + ST6104 started also immediately at a good rate and after 17 days an absolute biodegradation of 55.5% was observed. From that moment the biodegradation rate decreased and the biodegradation proceeded only at a low rate. After 45 days (end of test) an absolute biodegradation of 67.9%+2.7% was reached, or 70.7% on a relative basis compared to the suitable reference substrate cellulose. However, the biodegradation of this material was still slowly going on. Also the biodegradation of test item ST6093G + ST6097 started at a high rate and after 17 days an absolute biodegradation of 51.8% was observed. From then on the rate decreased and after 47 days an absolute biodegradation of 74.8% was observed. At that time a re-inoculation with 10% fresh VGF (Vegetable, Garden and Fruit waste) was executed in order to renew the microbial population and to supply extra nutrients in an attempt to increase the biodegradation rate of the test item. At the end of test (after 137 days) a biodegradation at a level of 84.8%+4.0% was reached, or 90.4% on a relative basis compared to the suitable reference substrate cellulose.

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

20 of 23


According to the European standard EN 13432 (2000) ‘Requirements for packaging recoverable through composting and biodegradation – Test scheme and evaluation criteria for the final acceptance of packaging’ and the international standard 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. The American standard ASTM D 6400-04 ‘Standard Specification for Compostable Plastics’ stipulates 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. The maximum allowed test duration by the standards is 180 days. From the results it can be concluded that test item ST6093G + ST6097 fulfills the biodegradation requirement of these standards within 137 days. Test item ST6103 + ST6104 does not fulfill the biodegradation requirement of the standards within 45 days, but the biodegradation of this material was still slowly going on.

Figure 8. Evolution of the biodegradation percentage of reference item and test items.

0

10

20

30

40

50

60

70

80

90

100

110

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Bio

degr

adat

ion

(%)

Time (Days)

Cellulose ST6093G+ST6097 ST6103+ST6104


Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

21 of 23


Figure 9. Evolution of the biodegradation percentage of the replicates of cellulose.

Figure 10. Evolution of the biodegradation percentage of the replicates of ST6093G + ST6097.

0

10

20

30

40

50

60

70

80

90

100

110

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Bio

degr

adat

ion

(%)

Time (Days)

RN 2 RN 6 RN 10

0

10

20

30

40

50

60

70

80

90

100

110

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Bio

degr

adat

ion

(%)

Time (Days)

RN 3 RN 7 RN 11

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

22 of 23


Figure 11. Evolution of the biodegradation percentage of the replicates of ST6103 + ST6104.

0

10

20

30

40

50

60

70

80

90

100

110

0 5 10 15 20 25 30 35 40 45

Bio

degr

adat

ion

(%)

Time (Days)

RN 4 RN 8 RN 12

Code:

Date:

R-TJO-1/2

May-20-11

Revision:

Page:

00

23 of 23


8.5. VISUAL PERCEPTIONS AND ANALYSES AT END OF TEST

Once every week during the incubation, at each time of shaking, the reactors were inspected visually for several aspects such as moisture content, structure of the mixture, development of fungi and visual appearance of the test items. Control and cellulose showed a good structure and moisture content during the test. No fungi growth was observed in the cellulose reactors. After 41 days no reference material could be detected anymore. Also a good structure and moisture conditions were observed in the different test reactors during the test. After 2 weeks of incubation some fungi growth was seen for both test items. At the different stop time of the materials the test series were analyzed for TS and VS content and pH (see Table 6). Table 6. pH, TS and VS content after 45 days (end of test for ST6103 + ST6104), and at end of

test (after 137 days ).

Test item TS (%) VS (% on TS) pH

After 45 days Control Cellulose

ST6093G + ST6097 ST6103 + ST6104

After 137 days Control Cellulose

ST6093G + ST6097

58.4 56.3

52.9 53.4

64.0 60.6

61.5

29.9 29.3

30.6 31.2

26.1 26.8

27.9

8.4 8.7

8.5 8.3

8.5 8.7

8.6

ECOTOXICITY TESTS

Barley plant growth test on compost residuals of ST6093G + ST6097

Report R-TJO-1/4


FINAL REPORT

ECOTOXICITY TEST

BARLEY PLANT GROWTH TEST

ON

COMPOST RESIDUALS

OF

ST6093G + ST6097

STUDY TJO-1/4

Scitech Adhesive Systems Ltd. Author: Nike MORTIER Casle Park Flint CH6 5AX UNITED KINGDOM

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

2 of 18


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 ................................................................................................................................................ 8 7.2. TEST COMPOSTS .................................................................................................................................... 8 7.3. GENERAL PROCEDURE ........................................................................................................................ 8 7.4. ANALYTICAL METHODS ................................................................................................................... 10

8. RESULTS .......................................................................................................................................................... 12

8.1. TEST CONDITIONS AND SET-UP....................................................................................................... 12 8.2. RESULTS AND DISCUSSION .............................................................................................................. 14

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

3 of 18


1. IDENTIFICATION OF TEST


Project Number

TJO-1/4 Sponsor

Scitech Adhesive Systems Ltd. Casle Park Flint CH6 5AX UNITED KINGDOM Monitoring Scientist

Mr. Trevor JONES Phone: +44/1352 732007 [email protected] Fax: +44/1352 732045

Testing Facility ORGANIC WASTE SYSTEMS N.V. Phone: +32/9/233.02.04 Dok Noord 5 Fax: +32/9/233.28.25 B-9000 Gent [email protected] BELGIUM [email protected] Test Item

Standard compost with a test mixture containing 1 test item: a mixture of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis), added in a 2.710% concentration. This concentration is equivalent to a maximum concentration in a compostable material on dry weight basis of 10.0%. Reference Item Standard compost without any addition (= control compost).

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

4 of 18



Study Director: Nike MORTIER Replacement Study Director: Bruno DE WILDE Quality Assurance Manager: Mia CLINCKSPOOR

1.3. STUDY SCHEDULE

Before the actual start-up of the barley plant growth test the test item was added to mature compost in a defined concentration and composted for 50 days at 58°C ± 2°C and 2 days at ambient temperature in the dark. Study initiation date: Apr-15-11 Study completion date: Jun-30-11 Experimental starting date: Apr-19-11 Starting date of incubation: Jun-10-11 Completion date of incubation: Jun-21-11 Duration of incubation: 11 days Experimental completion date: Jun-23-11

1.4. ARCHIVING

All raw data and records necessary 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:

TJO-1/4

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:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

6 of 18



A barley plant growth test, which is representative for monocotyledonous plants, was performed on a test mixture consisting of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis) in a specific concentration. A mixture of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis) was tested for a maximum concentration on dry weight in the compostable material of 10.0%. This maximum concentration and the assumption that the compostable material is added to the raw biowaste in a 10% concentration at start of the composting test and is degraded for 90% at the end of the composting test, results in a concentration in the final compost on dry weight of 2.710%. To obtain this concentration, the test item was mixed in an exact amount with mature compost (= test compost). Previous to the plant test a stabilization process was performed during 50 days in the dark at 58°C ± 2°C and 2 days at lab conditions (± 20°C) in order to prevent possible toxic effects because of biological activity and immaturity of the compost due to the addition of the test item. According to EN 13432 (2000), ASTM D 6400-04 and ISO 17088 (2008) such a stabilization could be continued for 12 weeks. The shortening of the stabilization period does make the ecotoxicity test more stringent. The test is executed according to the following standards: the European norm EN 13432 (2000) “Requirements for packaging recoverable through composting and biodegradation - Test scheme and evaluation criteria for the final acceptance of packaging”, the American standard ASTM D 6400-04 “Standard Specification for Compostable Plastics” and the international standard ISO 17088 (2008) “Specifications for compostable plastics”. The test was stopped after 11 days, which is within the prescribed time interval of “Methodenbuch 1998, Kapitel II: 5. Pflanzen-verträglichkeit – Bundesgütegemeinschaft Kompost e.V.” . According to EN 13432 (2000), ASTM D 6400-04 and ISO 17088 (2008) the germination rate and the plant biomass of the test compost should be more than 90% of those from the corresponding blank compost. The test compost easily fulfilled these criteria for barley plants. In conclusion, it can be stated that the addition of a mixture of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis), in a maximum concentration of 10.0% to a compostable material (on dry weight basis), exerts no negative effect on the emergence and growth of barley plants.

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

7 of 18


6. INTRODUCTION


The barley plant growth test is applied after a preceding composting test. The compost produced at the end of the composting test may contain residuals of the original test item such as metabolites, undegraded components and inorganic components. The purpose of the barley plant growth test is to evaluate any toxic effect of the test compost containing the test item residuals in comparison to blank compost to which no reference or test item was added at the start of the preceding composting test. The barley plant is chosen as representative of monocotyledonous plants. The test includes germination and growth of barley in mixtures of reference substrate and compost. At the end of the test the fresh and dry weight of the plants is determined for each test series and compared. Also the germination rate is measured.


The test is executed in line with the European norm EN 13432 (2000) “Requirements for packaging recoverable through composting and biodegradation - Test scheme and evaluation criteria for the final acceptance of packaging”, the American standard ASTM D 6400-04 “Standard Specification for Compostable Plastics” and the international standard ISO 17088 (2008) “Specifications for compostable plastics”. The time duration was based on “Methodenbuch 1998, Kapitel II: 5. Pflanzenverträglichkeit – Bundesgütegemeinschaft Kompost e.V.”.

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

8 of 18



7.1. TEST ITEM

Name: ST6093G + ST6097 Sample preparation: Mixing ST6093G + ST6097 in a ratio 500 g + 11 g Total solids: 39.1% Name: ST6093G Batch number: 1840L/1 Description: Liquid (Adhesive) Colour: Whitish Name: ST6097 Batch number: 100809/10/4 Description: Viscous liquid (Cross-linker) Colour: Transparent

7.2. TEST COMPOSTS

Following types of compost were tested for their ecotoxicological effect: Control compost: < 10 mm fraction of mature compost. Test compost: < 10 mm fraction of the control compost + 2.710% of a mixture of

97.8% ST6093G and 2.2% ST6097 (on wet weight basis). Table 1. Composition of control and test compost.

Component Control compost (g)

Test compost (g wet weight)

Test compost (g dry weight)

% test item in test compost

(%) Mature compost

ST6093G & ST6097

3500.00

-

3405.20

242.82

1511.57

94.85

-

2.710

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

9 of 18



The barley plant growth test is done in flower pots of 500 ml, containing a mixture of compost and reference substrate. Each compost is tested in 2 mixing ratios of compost and reference substrate: 1/3 and 1/1 on a volumetric basis. Each mixture is tested in 4 replicates. At the start of the test, each flower pot is filled with 500 ml of compost/reference substrate mixture and 100 ml demi water is added. Subsequently, 50 barley seeds are put on top of the mixture and covered with a thin layer of siliceous sand. Finally, an extra amount of demineralised water can be added to assure optimal moisture content. After the flower pots have been completely prepared, they are covered with a glass plate and incubated at a constant temperature of 20 ± 2 °C in the dark. After germination, the plate is removed and the pots are exposed to a light intensity of at least 3000 lux during at least 12 hours per day. During the test, extra water is added if needed, and visual perceptions are noted. In order to avoid side effects, the position of each pot is changed during the testing period, according to a logical rotation scheme. The test is finished after 11 (± 1) days. At the end of the test the total fresh and dry weight of the above-soil plant material is determined for each flower pot separately. Also the germination rate is measured. The toxicity of possible residuals of the test item is evaluated by comparing the results on germination and plant yield of test compost to blank compost. More details on the procedure for the particular test reported, are given in the Study Plan.

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

10 of 18



Weight Determination During the test two balances were used. A Sartorius AC 210 S with internal calibration (max. 200 g, d = 0.1 mg) for the determination of dry and volatile matter and weighing of the plants. A Mettler PJ12 (max. 12000 g, d = 0,1 g) was used for weighing of the composts and reference substrate and the determination of the density. Dry Matter or Total Solids

The dry matter is determined by drying at 105°C during at least 16 hours and weighing, as described in “METH L.009 Compost - Determination of moisture content”. The dry matter is given in percent on wet weight. Volumetric Density The volumetric density is determined by filling a 1 l cylinder and measuring the weight after compression with a 650 g plunger for 180 s. The exact procedure is described in “METH L.011 Compost - Determination of volumetric density”. Germinative Capacity Five ml of demineralised water are added to a petri dish with a filter paper on top of a layer of cotton. Twenty seeds of barley are put on top of the filter paper. A second wet filter paper is put on top of the seeds. The petri dish is sealed with parafilm and left in the dark and at room temperature. After 4 days the number of germinated seeds is counted. The germination is given in % on the amount of seeds at start. The germinative capacity is tested in 5 replicates. 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 “METH L.010 Compost - Determination of organic matter and carbon content”. The results are given in percent on dry matter. pH

The pH is measured with a pH meter after calibration with standard buffer solutions (pH = 4.00, pH = 7.00 and pH = 10.00), as described in “METH L.006. Compost - Determination of pH and electrical conductivity”. Before inserting the electrode the sample is diluted with distilled water at a ratio of 5 to 1 (5 parts of demineralised water versus 1 part of sample) and thoroughly mixed.

Salt Content (Electrical Conductivity, E.C.) The salt content is measured with a conductivity meter after calibration in a 0.01 M KCl solution, as described in “METH L.006. Compost - Determination of pH and electrical conductivity”. Before inserting the electrode the sample is diluted with distilled water at a ratio of 5 to 1 (5 parts of distilled water versus 1 part of sample) and thoroughly mixed, as described in “METH L.012. Compost – Preparation of extracts and analysis solutions”.

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

11 of 18


Ammonium - Nitrogen (NH 4

+ -N)

This analysis is done as described in "METH L.016. Compost - Determination of ammonia-nitrogen by FIA and spectrometric detection". The ammonium-N of compost is determined in an aqueous extract (5 parts of demineralised water versus 1 part of sample; see METH L.012). 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 results are given in g per l wet weight. Nitrate and Nitrite - Nitrogen (NO x

− -N) This analysis is done as described in "METH L.017. Compost-Determination of nitrite and nitrate nitrogen and the sum of both by FIA and spectrometric detection". The determination is performed on an aqueous extract (5 parts of demineralised water versus 1 part of sample; see METH L.012). 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-(1-naphtyl)-ethylene diamine dihydrochloride (NED) to form a purple azo dye. This azo dye is measured at 540 nm. The results are given in g per l wet weight. Total Nitrogen (N) This analysis is done as described in “METH L.005. Compost - Determination of total nitrogen”. In the presence of a catalysing agent (K2SO4-mixture) and under boiling conditions (380°C) with a mixture of sulphuric acid- salicylic acid bound nitrogen is converted into the salt (NH4)2SO4. 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:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

12 of 18


8. RESULTS


The test compost was prepared by mixing an exact amount of the test item with mature compost to obtain a concentration of 2.710% for a mixture of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis). This concentration is based on a maximum concentration of 10.0% on dry weight basis in the compostable material and the assumption that the compostable material is added to the raw biowaste in a 10% concentration at start of the composting test and is degraded for 90% at the end of the composting test. During composting only part of the raw waste and the compostable material will end up in the final compost (fraction < 10 mm), while the test item, in the worst case scenario, will end up completely in the final compost (for more details see Study Plan TJO-1/4). Because in normal conditions the test item undergoes a composting process before it comes in contact with plants, a stabilization process of 50 days in the dark at 58°C ± 2°C and 2 days at lab conditions (± 20°C) was performed. Attention is paid to aeration and moisture content. During the composting process the content of the reactors is stirred regularly. During this process the test item can be partially or completely biodegraded. Without the composting, the test item may have a toxic effect to the plants because of biological activity and immaturity of the compost, which is not representative for reality. According to EN 13432 (2000), ASTM D 6400-04 and ISO 17088 (2008) such a stabilization could be continued for 12 weeks. It must be noted that the shortening of the stabilization period does make the ecotoxicity test more stringent. The compost is the < 10 mm fraction of fresh, pretreated municipal solid waste (biowaste) that has been aerobically composted for more than 12 weeks. The characteristics of the compost are given in Table 2. The control and test compost were thoroughly mixed, prior to use. Table 2. Characteristics of the mature compost.

Characteristics Compost Total solids (TS, %) Volatile solids (VS, % on TS) Ash content (% on TS) pH E.C. (µS/cm) NOx

--N (mg/l) NH4

+-N (mg/l) Total N (g/kg TS) C/N

44.4 43.5 56.5 8.4

1650 268 b.r. 19.2 11

b.r. = below reporting limit reporting limit: NH4

+-N = 5.5 mg/l

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

13 of 18


In total 16 flower pots were used. The mixtures of reference substrate and compost are given on volumetric basis. In practice, the amounts of compost and reference substrate are added on weight basis, based on measurements of the volumetric density of the compost and reference substrate. Table 3 describes the test set-up. Table 3. Test set-up barley plant growth test.

Treatment Volume of Weight of Ref. Sub.

(ml/pot) Compost(a)

(ml/pot) Ref. Sub.

(g/pot) Compost(a)

(g/pot)

4 x Blank compost (1:3) 375 125 144 72

4 x Blank compost (1:1) 250 250 96 144

4 x Test compost (1:3) 375 125 144 77

4 x Test compost (1:1) 250 250 96 155 (a) Volumetric density of Reference substrate = 0.385 kg/l Blank compost = 0.575 kg/l Test compost = 0.619 kg/l The used reference substrate is “Einheitserde O” (EEO), which is produced by Einheitserdewerk Hameln A. Stangenberg GmbH, Kiebitzweg 3, D-31789 Hameln in Germany. The seeds are barley seeds “Barke non treated” and are derived from AVEVE, Tiensestraat 300, B-3400 Landen, Belgium. The seeds were examined for their germinative capacity. The germinative capacity was 100%, which is above the recommended value of 90%.

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

14 of 18


8.2. RESULTS AND DISCUSSION

The test was stopped after 11 days. Table 4 represents the average germination rate as a percentage of the total amount of seeds added at start. The relative germination rate is also shown in Figure 1. Table 5 shows the average fresh and dry weight yield (of above-soil plant parts) for each test series, as well as the standard deviation. The results are also shown in Figure 2. The comparison is made on dry weight basis and not on fresh weight because differences in fresh plant yield are not only caused by differences in compost mixtures, but also differences in watering (quantity and timing) and evaporation can have an influence on the fresh weight yield. Dry weight plant yield differences are more directly related to compost and reference substrate conditions. Therefore, dry weight yield is used for further comparison. For statistical analysis of the results, the ‘Anova single factor test’ at P≤ 0.05 was used. The results are compared with the lowest significant difference procedure of Fisher. Figure 1 and 2 give a visual presentation of the statistical analyses. Bars, which have at least one letter in common, are not significantly different from each other at the P≤ 0.05 level of significance. Table 4. Germination rate of barley (%).

Test series Germination rate (%) AVG STD Blank compost 1/3 98.5 1.9 Blank compost 1/1 98.5 1.0 Test compost 1/3 99.0 1.2 Test compost 1/1 98.0 1.6

With AVG = average, STD = standard deviation.

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

15 of 18


Figure 1. Average germination rate (as a percentage to the total amount of seeds added at

start). Bars, which have at least one letter in common, are not significantly different from each other at the P≤�0.05 level of significance (Anova single factor Test).

Figure 2. Absolute plant dry weight (g/pot). Bars, which have at least one letter in common,

are not significantly different from each other at the P ≤ �0.05 level of significance (Anova single factor Test).

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

16 of 18


Table 5. Absolute fresh and dry weight yield of barley plants.

Test series Fresh Weight Yield (g) AVG STD Blank compost 1/3 13.65 0.65 Blank compost 1/1 10.01 0.09 Test compost 1/3 13.70 0.36 Test compost 1/1 9.60 0.52 Test series Dry Weight Yield (g) AVG STD Blank compost 1/3 1.28 0.04 Blank compost 1/1 1.12 0.02 Test compost 1/3 1.22 0.01 Test compost 1/1 1.03 0.06 With AVG = average, STD = standard deviation.

According to EN 13432 (2000) “Requirements for packaging recoverable through composting and biodegradation - Test scheme and evaluation criteria for the final acceptance of packaging”, ASTM D 6400-04 “Standard Specification for Compostable Plastics” and ISO 17088 (2008) “Specifications for compostable plastics” the germination rate and the plant biomass in the test compost should be more than 90% of those in the corresponding blank compost. From Table 6 it is seen that these criteria are easily reached for both the germination rate and the plant biomass of both mixtures of the test compost. Therefore, it can be stated that the requirements of EN 13432 (2000), ASTM D 6400-04 and ISO 17088 (2008) on ecotoxicity are fulfilled for barley plants.

Table 6. Germination and dry weight plant yield of both mixtures of the test compost as a percentage of the corresponding mixture of blank compost.

Test series Germination Dry weight plant yield (as % of blank compost) (as % of blank compost) Test compost 1/3 100.5 95.7 Test compost 1/1 99.5 91.6 Figures 3 to 6 give a visual presentation of the plant growth of the barley plants in the 1/3 mixtures (Figures 3 & 4) and the 1/1 mixtures (Figures 5 & 6) of the compost/reference substrate mixtures. The visual observations during and at the end of the test showed no significant difference in growth for the 1/3 mixture of the test compost when compared to the corresponding mixture of the blank compost, while the plants in the 1/1 mixture of the test compost were somewhat smaller when compared to the plants in the corresponding mixture of the blank compost. Furthermore, no signs of chlorosis and necrosis were seen for the different compost mixtures.

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

17 of 18


As a general conclusion it can be stated that the addition of a mixture of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis), in a maximum concentration of 10.0% to a compostable material (on dry weight basis), exerts no negative effect on the emergence and growth of barley plants.

Figure 3. Overview of the barley plant growth after an incubation period of 6 days (from

bottom to top): 1/3 mixture of blank compost and 1/3 mixture of test compost.

Figure 4. Detailed barley plant growth after an incubation period of 6 days (from left to

right): 1/3 mixture of blank compost and 1/3 mixture of test compost.

Code:

Date:

R-TJO-1/4

Jun-30-11

Revision:

Page:

00

18 of 18


Figure 5. Overview of the barley plant growth after an incubation period of 6 days (from


Figure 6. Detailed barley plant growth after an incubation period of 6 days (from left to


ECOTOXICITY TESTS

Cress test on compost residuals of ST6093G + ST6097

Report R-TJO-1/5


FINAL REPORT

ECOTOXICITY TEST

CRESS TEST

ON

COMPOST RESIDUALS

OF

ST6093G + ST6097

STUDY TJO-1/5

Scitech Adhesive Systems Ltd. Author: Nike MORTIER Casle Park Flint CH6 5AX UNITED KINGDOM

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

2 of 18

CRESS 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 ................................................................................................................................................ 8 7.2. TEST COMPOSTS .................................................................................................................................... 8 7.3. GENERAL PROCEDURE ........................................................................................................................ 9 7.4. ANALYTICAL METHODS ................................................................................................................... 10

8. RESULTS .......................................................................................................................................................... 12

8.1. TEST CONDITIONS AND SET-UP....................................................................................................... 12 8.2. RESULTS AND DISCUSSION .............................................................................................................. 14

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

3 of 18

CRESS TEST

1. IDENTIFICATION OF TEST


Project Number

TJO-1/5 Sponsor Scitech Adhesive Systems Ltd. Casle Park Flint CH6 5AX UNITED KINGDOM Monitoring Scientist Mr. Trevor JONES Phone: +44/1352 732007 [email protected] Fax: +44/1352 732045

Testing Facility

ORGANIC WASTE SYSTEMS N.V. Phone: +32/9/233.02.04 Dok Noord 5 Fax: +32/9/233.28.25 B-9000 Gent [email protected] BELGIUM [email protected] Test Item

Standard compost with a test mixture containing 1 test item: a mixture of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis), added in a 2.710% concentration. This concentration is equivalent to a maximum concentration in a compostable material on dry weight basis of 10.0%. Reference Item Standard compost without any addition (= control compost).

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

4 of 18

CRESS TEST


Study Director: Nike MORTIER Replacement Study Director: Bruno DE WILDE Quality Assurance Manager: Mia CLINCKSPOOR

1.3. STUDY SCHEDULE

Before the actual start-up of the cress test the test item was added to mature compost in a defined concentration and composted for 50 days at 58°C ± 2°C and 2 days at ambient temperature in the dark. Study initiation date: Apr-15-11 Study completion date: Jun-30-11 Experimental starting date: Apr-19-11 Starting date of incubation: Jun-10-11 Completion date of incubation: Jun-28-11 Duration of incubation: 18 days Experimental completion date: Jun-30-11

1.4. ARCHIVING

All raw data and records necessary 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:

TJO-1/5

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:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

6 of 18

CRESS TEST


A cress test, which is representative for dicotyledonous plants, was performed on a test mixture consisting of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis) in a specific concentration. A mixture of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis) was tested for a maximum concentration on dry weight in the compostable material of 10.0%. This maximum concentration and the assumption that the compostable material is added to the raw biowaste in a 10% concentration at start of the composting test and is degraded for 90% at the end of the composting test, results in a concentration in the final compost on dry weight of 2.710%. To obtain this concentration, the test item was mixed in an exact amount with mature compost (= test compost). Previous to the plant test a stabilization process was performed during 50 days in the dark at 58°C ± 2°C and 2 days at lab conditions (± 20°C) in order to prevent possible toxic effects because of biological activity and immaturity of the compost due to the addition of the test item. According to EN 13432 (2000), ASTM D 6400-04 and ISO 17088 (2008) such a stabilization could be continued for 12 weeks. The shortening of the stabilization period does make the ecotoxicity test more stringent. The test is executed according to the following standards: the European norm EN 13432 (2000) “Requirements for packaging recoverable through composting and biodegradation - Test scheme and evaluation criteria for the final acceptance of packaging”, the American standard ASTM D 6400-04 “Standard Specification for Compostable Plastics” and the international standard ISO 17088 (2008) “Specifications for compostable plastics”. According to EN 13432 (2000), ASTM D 6400-04 and ISO 17088 (2008) the germination rate and the plant biomass of the test compost should be more than 90% of those from the corresponding blank compost. The test compost easily fulfilled these criteria for cress plants. In conclusion, it can be stated that the addition of a mixture of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis), in a maximum concentration of 10.0% to a compostable material (on dry weight basis), exerts no negative effect on the emergence and growth of cress plants.

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

7 of 18

CRESS TEST

6. INTRODUCTION


The cress test is applied after a preceding composting test. The compost produced at the end of the composting test can eventually contain residuals of the original test item such as metabolites, undegraded components and inorganic components. The purpose of the cress test is to evaluate any toxic effect of the test compost containing the test item residuals in comparison to control compost to which no reference or test item was added at the start of the preceding composting test. The cress plant is chosen as a representative for dicotyledonous plants and for its sensitive germination. The test involves germination and growth of cress in the test compost. At the end of the test the fresh and dry weight of the plants are determined for each test series and compared. Also the germination rate is measured.


The test is executed in line with the European norm EN 13432 (2000) “Requirements for packaging recoverable through composting and biodegradation - Test scheme and evaluation criteria for the final acceptance of packaging”, the American standard ASTM D 6400-04 “Standard Specification for Compostable Plastics” and the international standard ISO 17088 (2008) “Specifications for compostable plastics”.

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

8 of 18

CRESS TEST


7.1. TEST ITEM

Name: ST6093G + ST6097 Sample preparation: Mixing ST6093G + ST6097 in a ratio 500 g + 11 g Total solids: 39.1% Name: ST6093G Batch number: 1840L/1 Description: Liquid (Adhesive) Colour: Whitish Name: ST6097 Batch number: 100809/10/4 Description: Viscous liquid (Cross-linker) Colour: Transparent

7.2. TEST COMPOSTS

Following types of compost were tested for their ecotoxicological effect: Control compost: < 10 mm fraction of mature compost. Test compost: < 10 mm fraction of the control compost + 2.710% of a mixture of

97.8% ST6093G and 2.2% ST6097 (on wet weight basis). Table 1. Composition of control and test compost.

Component Control compost (g)

Test compost (g wet weight)

Test compost (g dry weight)

% test item in test compost

(%) Mature compost ST6093G & ST6097

3500.00 -

3405.20 242.82

1511.57 94.85

- 2.710

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

9 of 18

CRESS TEST


The cress test is done in flower pots of 500 ml, containing a mixture of compost and reference substrate. Each compost is tested in 2 mixing ratios of compost and reference substrate: 1/3 and 1/1 on a volumetric basis. Each mixture is tested in 4 replicates. At the start of the test, each flower pot is filled with 500 ml of compost/reference substrate mixture and 100 ml demi water is added. Subsequently, 100 cress seeds are put on top of the mixture and covered with a thin layer of siliceous sand. Finally, an extra amount of demineralised water can be added to assure optimal moisture content. After the flower pots have been completely prepared, they are covered with a glass plate and incubated at a constant temperature of 20 ± 2 °C in the dark. After germination, the plate is removed and the pots are exposed to a light intensity of at least 3000 lux during at least 12 hours per day. During the test, extra water is added if needed, and visual perceptions are noted. In order to avoid side effects, the position of each pot is changed a few times during the testing period, according to a logical rotation scheme. The test is finished 14 days (± 2 days) after 50% of the control seedlings have emerged. At the end of the test the total fresh and dry weight of the above-soil plant material is determined for each flower pot separately. Also the germination rate is measured. The toxicity of possible residuals of the test item is evaluated by comparing the results on germination and plant yield of test compost to blank compost. More details on the procedure for the particular test reported, are given in the Study Plan.

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

10 of 18

CRESS TEST


Weight Determination During the test two balances were used. A Sartorius AC 210 S with internal calibration (max. 200 g, d = 0.1 mg) for the determination of dry and volatile matter and weighing of the plants. A Mettler PJ12 (max. 12000 g, d = 0,1 g) was used for weighing of the composts and reference substrate and the determination of the density. Dry Matter or Total Solids

The dry matter is determined by drying at 105°C during at least 16 hours and weighing, as described in “METH L.009 Compost - Determination of moisture content”. The dry matter is given in percent on wet weight. Volumetric Density The volumetric density is determined by filling a 1 l cylinder and measuring the weight after compression with a 650 g plunger for 180 s. The exact procedure is described in “METH L.011 Compost - Determination of volumetric density”. Germinative Capacity Five ml of demineralized water are added to a petri dish with a filter paper on top of a layer of cotton. Twenty seeds of cress are put on top of the filter paper. A second wet filter paper is put on top of the seeds. The petri dish is sealed with parafilm and left in the dark and at room temperature. After 4 days the number of germinated seeds is counted. The germination is given in % on the amount of seeds at start. The germinative capacity is tested in 5 replicates. 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 “METH L.010 Compost - Determination of organic matter and carbon content”. The results are given in percent on dry matter. pH

The pH is measured with a pH meter after calibration with standard buffer solutions (pH = 4.00, pH = 7.00 and pH = 10.00), as described in “METH L.006. Compost - Determination of pH and electrical conductivity”. Before inserting the electrode the sample is diluted with distilled water at a ratio of 5 to 1 (5 parts of demineralised water versus 1 part of sample) and thoroughly mixed. Salt Content (Electrical Conductivity, E.C.) The salt content is measured with a conductivity meter after calibration in a 0.01 M KCl solution, as described in “METH L.006. Compost - Determination of pH and electrical conductivity”. Before inserting the electrode the sample is diluted with distilled water at a ratio of 5 to 1 (5 parts of distilled water versus 1 part of sample) and thoroughly mixed, as described in “METH L.012. Compost – Preparation of extracts and analysis solutions”.

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

11 of 18

CRESS TEST

Ammonium - Nitrogen (NH 4

+ -N)

This analysis is done as described in "METH L.016. Compost - Determination of ammonia-nitrogen by FIA and spectrometric detection". The ammonium-N of compost is determined in an aqueous extract (5 parts of demineralised water versus 1 part of sample; see METH L.012). 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 results are given in g per l wet weight. Nitrate and Nitrite - Nitrogen (NO x

− -N) This analysis is done as described in "METH L.017. Compost-Determination of nitrite and nitrate nitrogen and the sum of both by FIA and spectrometric detection". The determination is performed on an aqueous extract (5 parts of demineralised water versus 1 part of sample; see METH L.012). 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-(1-naphtyl)-ethylene diamine dihydrochloride (NED) to form a purple azo dye. This azo dye is measured at 540 nm. The results are given in g per l wet weight. Total Nitrogen (N) This analysis is done as described in “METH L.005. Compost - Determination of total nitrogen”. In the presence of a catalysing agent (K2SO4-mixture) and under boiling conditions (380°C) with a mixture of sulphuric acid- salicylic acid bound nitrogen is converted into the salt (NH4)2SO4. 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:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

12 of 18

CRESS TEST

8. RESULTS


The test compost was prepared by mixing an exact amount of the test item with mature compost to obtain a concentration of 2.710% for a mixture of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis). This concentration is based on a maximum concentration of 10.0% on dry weight basis in the compostable material and the assumption that the compostable material is added to the raw biowaste in a 10% concentration at start of the composting test and is degraded for 90% at the end of the composting test. During composting only part of the raw waste and the compostable material will end up in the final compost (fraction < 10 mm), while the test item, in the worst case scenario, will end up completely in the final compost (for more details see Study Plan TJO-1/5). Because in normal conditions the test item undergoes a composting process before it comes in contact with plants, a stabilization process of 50 days in the dark at 58°C ± 2°C and 2 days at lab conditions (± 20°C) was performed. Attention is paid to aeration and moisture content. During the composting process the content of the reactors is stirred regularly. During this process the test item can be partially or completely biodegraded. Without the composting, the test item may have a toxic effect to the plants because of biological activity and immaturity of the compost, which is not representative for reality. According to EN 13432 (2000), ASTM D 6400-04 and ISO 17088 (2008) such a stabilization could be continued for 12 weeks. It must be noted that the shortening of the stabilization period does make the ecotoxicity test more stringent. The compost is the < 10 mm fraction of fresh, pretreated municipal solid waste (biowaste) that has been aerobically composted for more than 12 weeks. The characteristics of the compost are given in Table 2. The control and test compost were thoroughly mixed, prior to use. Table 2. Characteristics of the mature compost.

Characteristics Compost Total solids (TS, %) Volatile solids (VS, % on TS) Ash content (% on TS) pH E.C. (µS/cm) NOx

--N (mg/l) NH4

+-N (mg/l) Total N (g/kg TS) C/N

44.4 43.5 56.5 8.4

1650 268 b.r. 19.2 11

b.r. = below reporting limit reporting limit: NH4

+-N = 5.5 mg/l

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

13 of 18

CRESS TEST

In total, 16 flower pots were used. The amounts of reference substrate and compost are given on volumetric basis. In practice, the amounts of compost and reference substrate are added on weight basis, based on measurements of the volumetric density of the compost and reference substrate. Table 3 describes the test set-up. Table 3. Test set-up cress test.

Treatment Volume of Weight of Ref. Sub.

(ml/pot) Compost(a)

(ml/pot) Ref. Sub.

(g/pot) Compost(a)

(g/pot)

4 x Blank compost (1:3) 375 125 144 72

4 x Blank compost (1:1) 250 250 96 144

4 x Test compost (1:3) 375 125 144 77

4 x Test compost (1:1) 250 250 96 155 (a) Volumetric density of Reference substrate = 0.385 kg/l Blank compost = 0.575 kg/l Test compost = 0.619 kg/l

The used reference substrate is “Einheitserde O” (EEO), which is produced by Einheitserdewerk Hameln A. Stangenberg GmbH, Kiebitzweg 3, D-31789 Hameln in Germany. The seeds are cress seeds type “large-leaved” and are derived from AVEVE, Pantserschipstraat 6, B-9000 Gent, Belgium. The cress seeds were examined for their germinative capacity. The germinative capacity was 98%, which is well above the recommended value of 90%.

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

14 of 18

CRESS TEST

8.2. RESULTS AND DISCUSSION

The test was stopped 15 days after 50% of the control seedlings have emerged. Table 4 represents the average germination rate of the different test series as a percentage of the total amount of seeds added at start. The relative germination rate is also shown in Figure 1. Table 5 shows the average fresh and dry weight yield (of above-soil plant parts) for each test series, as well as the standard deviation. The results are shown in Figure 2. For statistical analysis of the results, the ‘Anova single factor test’ at P≤0.05 was used. The results are compared with the lowest significant difference procedure of Fisher. Figure 1 and 2 give a visual presentation of the statistical analyses. Bars, which have at least one letter in common, are not significantly different from each other at the P≤0.05 level of significance. The comparison of plant yield is made on dry weight basis and not on fresh weight because differences in fresh plant yield are not only caused by differences in compost mixtures but also by differences in watering (quantity and timing) and evaporation can have an influence on the fresh weight yield. Dry weight plant yield differences are more directly related to compost and reference substrate conditions. Therefore, dry weight yield is used for further comparison. Table 4. Germination rate of cress (%).

Test series Germination rate (%) AVG STD Blank compost 1/3 95.8 2.2 Blank compost 1/1 95.3 2.1 Test compost 1/3 95.0 0.0 Test compost 1/1 94.3 1.5

With AVG = average, STD = standard deviation.

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

15 of 18

CRESS TEST

Figure 1. Average germination rate (as a percentage to the total amount of seeds added at

start). Bars, which have at least one letter in common, are not significantly different from each other at the P ≤ �0.05 level of significance (Anova single factor Test).

Figure 2. Absolute plant dry weight (g/pot). Bars, which have at least one letter in common,

are not significantly different from each other at the P ≤ �0.05 level of significance (Anova single factor Test).

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

16 of 18

CRESS TEST

Table 5. Absolute fresh and dry weight yield of cress.

Test series Fresh Weight Yield (g) AVG STD Blank compost 1/3 6.50 0.23 Blank compost 1/1 4.27 0.26 Test compost 1/3 6.33 0.20 Test compost 1/1 4.09 0.23 Test series Dry Weight Yield (g) AVG STD Blank compost 1/3 0.57 0.04 Blank compost 1/1 0.47 0.00 Test compost 1/3 0.53 0.04 Test compost 1/1 0.43 0.01 With AVG = average, STD = standard deviation. According to EN 13432 (2000) “Requirements for packaging recoverable through composting and biodegradation - Test scheme and evaluation criteria for the final acceptance of packaging”, ASTM D 6400-04 “Standard Specification for Compostable Plastics” and ISO 17088 (2008) “Specifications for compostable plastics” the germination rate and the plant biomass in the test compost should be more than 90% of those in the corresponding blank compost. From Table 6 it is seen that these criteria are easily reached for both the germination rate and the plant biomass of both mixtures of the test compost. Therefore, it can be stated that the requirements of EN 13432 (2000), ASTM D 6400-04 and ISO 17088 (2008) on ecotoxicity are fulfilled for cress plants.

Table 6. Germination and dry weight plant yield of both mixtures of the test compost as a

percentage of the corresponding mixture of blank compost.

Test series Germination Dry weight plant yield (as % of blank compost) (as % of blank compost) Test compost 1/3 99.2 92.5 Test compost 1/1 99.0 91.3 Figures 3 to 6 give a visual presentation of the plant growth of the cress plants in the 1/3 mixtures (Figures 3 & 4) and the 1/1 mixtures (Figures 5 & 6) of the compost/reference substrate mixtures. The visual observations during and at the end of the test showed no significant difference in growth for the 1/3 mixture of the test compost when compared to the corresponding mixture of the blank compost, while the plants in the 1/1 mixture of the test compost were slightly larger when compared to the plants in the corresponding mixture of the blank compost. Furthermore, no signs of chlorosis and necrosis were seen for the different compost mixtures.

Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

17 of 18

CRESS TEST

As a general conclusion it can be stated that the addition of a mixture of 97.8% ST6093G and 2.2% ST6097 (on wet weight basis), in a maximum concentration of 10.0% to a compostable material (on dry weight basis), exerts no negative effect on the emergence and growth of cress plants.

Figure 3. Overview of the cress plant growth after an incubation period of 12 days (from


Figure 4. Detailed cress plant growth after an incubation period of 12 days (from left to


Code:

Date:

R-TJO-1/5

Jun-30-11

Revision:

Page:

00

18 of 18

CRESS TEST

Figure 5. Overview of the cress plant growth after an incubation period of 12 days (from


Figure 6. Detailed cress plant growth after an incubation period of 12 days (from left to


ADHESIVE ST6093G + ST6097 · OWS NV • Dok Noord 5 • B-9000 Gent • Belgium • tel. +32 9...

Documents

Transcript of ADHESIVE ST6093G + ST6097 · OWS NV • Dok Noord 5 • B-9000 Gent • Belgium • tel. +32 9...