Evaluation of aerobic biodegradability

Grant Agreement number: 101058371 Project acronym: ESTELLA Project title: Design of bio-based thermoset polymer with recycling capability by dynamic bonds for bio-composite manufacturing Grant Agreement number: 101058371 Project acronym: ESTELLA Project title: Design of bio-based thermoset polymer with recycling capability by dynamic bonds for bio-composite manufacturing DELIVERABLE 5.1 Evaluation of aerobic biodegradability Contractual Date of Delivery: 31/07/2025 Actual Date of Delivery: 18/7/2025 Lead contractor for this deliverable: xxxxxxxx Author(s): Mª Ángeles Fontecha Cámara Participants(s): Andaltec (AND) WP contributing to the deliverable: WP 5 Nature: xxxx Version V. 2 Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. REVISION TABLE Document version Date Modified sections - Details V1 09.7.2025 Fist version AND contribution before technical and quality review. V2 18.07.2025 Second version AND contribution before technical and quality review. V3 23.07.2025 Third version quality review CID V4 23.07.2025 Forth version AND Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. Table of Contents TABLE OF CONTENTS ..................................................................................................................... 3 EXECUTIVE SUMMARY .................................................................................................................. 4 1. INTRODUCTION ...................................................................................................................... 5 2. EQUIPMENT AND MATERIALS USED....................................................................................... 6 2.1 PRINCIPLES. ............................................................................................................................. 6 2.1. REACTOR VESSELS. .................................................................................................................. 7 2.2. PROGRAM. ............................................................................................................................ 7 3. EXPERIMENTAL PROCEDURE .................................................................................................. 8 3.1. COMPOST PREPARATION ......................................................................................................... 9 3.2. PREPARATION OF REFERENCE AND TEST MATERIAL ...................................................................... 11 3.3. PRELIMINARY STEPS OF THE EXPERIMENT .................................................................................... 11 3.4. PREPARATION OF THE REACTOR VESSELS ................................................................................... 12 3.5. HUMIDITY ADJUSTMENT .......................................................................................................... 15 3.6. INCUBATION PERIOD .............................................................................................................. 16 3.7. END OF THE TEST .................................................................................................................... 18 4. RESULTS ................................................................................................................................. 18 4.1. INITIAL CHARACTERISATION OF COMPOST, REFERENCE MATERIAL AND RESINS ................................ 18 4.2 EVALUATION OF AEROBIC BIODEGRADABILITY ........................................................................... 19 5. CONCLUSIONS ..................................................................................................................... 22 6. REFERENCES .......................................................................................................................... 23 INTERNATIONAL STANDARD ISO 14855-1. (2005). ................................................................... 23 Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. Executive Summary The objective of this task is the study of two selected developed materials (bio-resins and/or bio-composites) that was assessed under controlled temperature, airing and humidity conditions according to ISO 14855-1 normative, in order to analyse the potential degradation grade of the novel synthesized thermoset. This analysis is bases on CO2 released and the final solid recovered. For this purpose, 50 grams of each resin were mixed with 600 grams of wet compost and taken to the reactors, where they were introduced into the Respirometer to initiate the biodegradability study. The experiment was performed in triplicate and three additional reactors containing only compost were used as blanks to subtract the production of CO₂ and other gases. Three more reactors were prepared with reference material to verify the accuracy of the biodegradability test. The biodegradability of the resins was found to be low, between 5 and 15%. This is not necessarily a negative result, as if they were biodegradable; they could produce toxic substances such as bisphenol A, which could be released into the environment. The resins, which had been carefully separated from the final compost, were sent to SINTEF for further micro-organism analysis. Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. 1. INTRODUCTION Preparation of the test materials used in the Respirometer ER series is summarized according to ISO 14855-1:2012, which applies to the aerobic degradation of samples (International Standard ISO 14855-1, 2005). The main method outlined in this section of ISO 14855 involves a solidphase respirometric test system based on mature compost used as a solid bed, a source of nutrients, and an inoculum rich in thermophilic microorganisms. Mature compost can sometimes be affected by a process known as the ‘priming effect’, where a large amount of organic matter present in the compost can cause a polymer-induced degradation, which affects the measurement of its biodegradability. Mature compost is a very heterogeneous and complex material. Therefore, it can be difficult to quantify the residual polymeric material left in the bed at the end of the test. And so, to detect any low-molecularmass molecules released by the polymeric material and assess the biomass. As a result, it can be difficult to perform a complete carbon balance. To overcome these difficulties and to improve the reliability of the method, mature compost can be replaced with a solid mineral medium used as the composting bed, facilitating analyses. This change can be used to measure the biodegradation in terms of CO2 evolution, quantify and analyse the biomass, and the residues of polymeric material left in the solid bed, and to perform a complete carbon balance. Furthermore, the method is not significantly affected by the priming effect and therefore, it can be used to assess materials that are known with incompatibilities with mature compost. The solid mineral medium used as bed can also be subjected to an ecotoxicological analysis to verify the absence of any ecotoxic activity. The basic principle of aerobic biodegradation is stated in the standard: The test material is mixed with the inoculum and placed in a static composting vessel where it is subjected to intensive composting under optimal conditions of oxygen, temperature and humidity for a test period not exceeding 6 months. During aerobic biodegradation of the test material, carbon dioxide, water, mineral salts and new microbial cellular constituents (biomass) are the end products of biodegradation. The carbon dioxide produced is continuously monitored, or measured at regular intervals, in the test vessels and blanks to determine the cumulative carbon dioxide production. The rate of biodegradation is given by the ratio of carbon dioxide produced from the test material to the maximum theoretical amount of carbon dioxide that can be produced from the test material. The maximum theoretical amount of carbon dioxide produced Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. is calculated from the measured total organic carbon (TOC) content. The percentage of biodegradation does not include the amount of carbon converted into new cell biomass that is not further metabolized into carbon dioxide during the course of the test. 2. EQUIPMENT AND MATERIALS USED A respirometer is an apparatus (figure 1) used to measure the respiration of live organisms. It is used to determine the aerobic or anaerobic biodegradability of solid or liquid samples in a variety of applications. Figure 1. ECHO – 12 channel respirometer 21-RESP/12/ 2.1 Principles. The Respirometer measures O2 and CO2 concentration continuously through the sample under controlled conditions (Additional gases can be measured). Basic system configuration described throughout the manual: - Control unit with 12 sampling channels; PC unit with 12 sampling channels - PC unit - Reactor vessels for solid samples 2L - AP03 pump Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. - Multi-tube connection between control unit and thermostatic cabinet - Thermostatic cabinet ST5 Some of the important components are described hereafter: 2.2. Thermostatic cabinet. The thermostatic cabinet is used to regulate the reactor temperature required by a specific standard. The standard model operates in the temperature range from 0 to 70 °C, while the maximum temperature used for measurements is 58 °C. The thermostatic cabinet is connected to the control unit via a multi-tube. 2.1. Reactor vessels. The reactors are connected inside the thermostatic cabinet. The carrier gas enters the reactor together with the return condensate through the pipes marked ‘IN’. The gas then leaves the reactor through the pipes marked ‘OUT’ back to the distributor. The reactors must be placed inside the thermostatic cabinet with sufficient space between them. This will allow free circulation of air and allow an even temperature distribution. 2.2. Program. Recommended settings at the beginning of the experiment if no previous experiments have been performed to determine the general CO2 values for a specific sample. Measuring interval: 1 min Pause interval: 1 min Leakage flow alarm: 10 Flow rate: 500 ml/min It is recommended to adjust the flow rate and sample mass so that the CO2 levels are around 40-80 % of the sensor range at the time of maximum CO2 production. Normally, the measurement starts with a maximum flow rate of 500 ml/min. After a few days, the CO2 production is at its maximum and the CO2 concentration starts to decrease. When it is below 40 % of the sensor's measuring range, the flow rate should be lower. After a few weeks, the production will be low enough to set the lowest flow rate, which is 20 ml/min. Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. Measurements can be started and stopped to change settings during the experiment and still use the same database. Figure 2. Programme settings 3. EXPERIMENTAL PROCEDURE Figure 3 shows a diagram relating to the use of the ER Series ECHO Respirometer. It is intended to ensure that measurements taken on respirometers are within the operating range. The main objective of this approach is to harmonize the measuring system and the biological component through appropriate adjustments of the air flow and the amount/type of fertilizer. These parameters depend on the quality activity and the amount of biomass. The rapid test of the compost in a reactor can be used to set the test parameters depending on the biomass activity in the compost. Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. Figure 3. Procedure diagram 3.1. Compost preparation It is important to use homogeneous, well aerated compost from wellfunctioning aerobic composting with enough diversity of micro-organisms. It is preferable that the compost is 2-4 months old; it should not contain large inert objects e.g. glass, stones although the compost can contain small wood particles to avoid clogging during the test; compost from plants treating garden or farmyard waste or mixtures of garden waste and municipal solid waste can be used and finallycompost can also be purchased from the garden shop (Figure 4). Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. Figure 18 shows the composting vessels placed in the Respirometer. It is necessary to use an enough high flow rate to ensure that aerobic conditions are maintained during the test in all composting vessels and connect the vessels to the aering tubes. Figure 18. Compost bins placed in the Respirometer chamber 3.6. Incubation period The composting vessels are stirred weekly to avoid extensive channeling and to ensure an even attack of the micro-organisms on the test material as well as an even distribution of moisture. During the stirring phase, the air supply system and the CO2 measurement system are switched off. Stirring can be done by gently shaking the test material with a glass rod (Figure 19). Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. Figure 19. Stirring the test material. The moisture content of the test mixture in the composting bin must be visually checked. Very dry conditions are revealed by the absence of condensate in the headspace of the composting vessel. On the other hand, adequate moisture conditions can be observed by the presence of condensate in the composting vessel ( Figure 20). Optionally, the humidity can be measured and should be kept at around 50%. A more drastic change in moisture content can be obtained by adding distilled water ( Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. Figure 21) or by draining through the air inlet ( Figure 16 and Figure 17). Figure 20. Condensation on the vessel vall Figure 21. Adding water through the central tube The incubation period of compost bins should not exceed 6 months. During incubation a constant of temperature of 58 ± 2 °C must be maintained by measuring the pH at regular intervals as at the start of the test. The last point is important because if the pH is below 7.0, biodegradation could be inhibited due to the compost becoming acidic as a result of the rapid degradation of an easily degradable test material. 3.7. End of the test The following steps are taken to complete the test: • Take samples of the test mixture from all vessels Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. The mass of the test material can be determined by weighing the compost bins with their test mixture. • Determine the total dry solids and the volatile solids • Record any visual observation about the appearance of the test material to assess its degree of disintegration Further investigations of any remaining test material are advisable, including measurement of relevant physical properties, chemical analysis and photography. 4. RESULTS To begin to show the results of the aerobic biodegradability of the resins the previous characterisation of the compost or inoculum used together with the same characterisation for the three resins under study supplied by other partners is started. 4.1. Initial characterisation of compost, reference material and resins The following table shows the results obtained for all of the inoculum (compost) used and the three resins. It can be seen that the moisture content of the inoculum used is lower than the required moisture content, so water was added until the required moisture content of 50-55% was reached. Table 1. Initial characterisation of compost and resins Compost Cellulose NIC Resin WUR Resin ICSO Resin Dry solids 52.90 97.72 97.41 99.60 99.13 Humidity 47.10 2.28 2.59 0.40 0.87 Volatile solids 70.41 100 99.89 99.89 98.59 Ashes 29.59 0 0.10 0.11 1.41 Organic carbon 9.81 41.5 58.76 73.54 66.43 Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. As previously mentioned, cellulose was used as a reference material as its response to biodegradability is known to be over 90%, in this case the moisture content is 2.28% and the volatile solids are almost 100%. The ash content of all materials was determined. As expected, low ash content was obtained for both resins and cellulose, meaning that the volatile solids content is very high for all materials. The pH of the compost at the start and during the test must be above 7 at all times, as lowering the pH biodegradation could be inhibited, due to acidification, this pH was superior at all times. 4.2 Evaluation of aerobic Biodegradability Aerobic biodegradability test has been completed according to the UNEEN ISO 14855-1 standard for the 3 resins developed in WP2. It is currently planning the study of metals in leachates for the remaining compost, as well as other chemical tests. The material to be tested is mixed with the inoculum and placed in a static composting vessel, where it is subjected to intensive composting under optimum Oxygen, temperature and humidity conditions for a test period of 6 months. During the aerobic biodegradation of the material, carbon dioxide, water, mineral salts and new microbial cellular components (biomass) are the end products of biodegradation. The carbon dioxide produced is continuously monitored and measured at regular intervals in test vessels and blanks to determine the cumulative carbon dioxide production. The following figure shows the curves obtained for the percentage (%) biodegradability obtained for the three resins and cellulose as a reference. Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. Figure 22. Biodegradability % The test was carried out for 6 months and as can be seen the reference reaches around 90% biodegradability. However the three resins reached between 5-15% biodegradability. The following table shows the values obtained from an average of three measurements for each resin. Table 2. Final results of the degree of biodegradability obtained after an incubation period of 6 months (maximum allowed by the standard) Reference (cellulose) NIC Resin WUR Resin ICSO Resin Initial mass (g) 50 51.1 50.1 50.4 Degree of biodegradation (%) 90 14.2 4.5 9.0 As for the visual assessment, Figure 23 shows the state of the resin at the end of the biodegradability test. Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. Figure 23. Resin at the end of the biodegradability test. NIC, WUR and ICSO resins The final characteristics of the inoculum are as follows: • No fungal growth in any of the sample references or blanks. • As to structure; aerated and moist soil with a sticky texture. Free water is observed under slight pressure. • Colour is dark brown. • Odour typical composting plant. For resin samples: • During the test, no major changes in the appearance of the sample are observed, apart from a slight and gradual partial disintegration into smaller pieces. • The naked eye can see pieces of the sample of various sizes remaining after the exposure period. • The end of the exposure period. There is also a loss of colour. Further analysis is performed on the two remaining fractions (Figure 24 and 25). On the one hand, Sothe resins were carefully separated to avoid possible contamination and sent to SINTEF for analysis of heavy metals by ICP-MS and screening of untargeted scan for volatile and semi-volatile compounds by GC-MS. On the other hand, the remaining compost is analyzed at ANDALTEC in order to detect liberated substances: heavy metals are analyzed by ICP-MS, volatile and semi-volatile compounds, as well as aromatic amines are analyzed by GC-MS, and bisphenol A is analyzed by HPLC. Complete descriptions of these analyses and results will be collected in deliverable 5.2. Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. Figure 24. Separation of resins from compost Figure 25. Original resins and resins following aerobic biodegradation 5. CONCLUSIONS The main conclusion is that the resins do not meet the requirements for being a biodegradable and therefore not compostable material. However, this is not necessarily a negative result, as if they were biodegradable; they could produce toxic substances such as bisphenol A, which could be released into the environment. Therefore, this would be a problem for the product rather than an advantage. Of the ESTELLA project objectives, it is more important that the resins are recyclable than biodegradable or compostable. Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein. 6. REFERENCES International Standard ISO 14855-1. (2005). 7. ANEXX List of the exchanged samples Partners Sample description Quantity What for From to CID AND ICSO + hemp (38%) 110 g Chemical recycling test CID AND ICSO 1-1 (0.5%) 175 g Chemical recycling test CID AND ICSO 1-1 (1%) 90 g Chemical recycling test CID AND ICSO 1-8 (0.5%) 180 g Chemical recycling test AND SINT NIC resin before b.test 50 g Micriobiological tests after biodegradability test. AND SINT NIC resin M8 50 g Micriobiological tests after biodegradability test. AND SINT NIC resin after biodegradability test 50 g Micriobiological tests after biodegradability test. AND SINT ICSO resin before b. test 50 g Micriobiological tests after biodegradability test. AND SINT ICSO resin M11 50 g Micriobiological tests after biodegradability test. AND SINT ICSO resin M12 50 g Micriobiological tests after biodegradability test. AND SINT WUR before b.test 50 g Micriobiological tests after biodegradability test. AND SINT WUR resin M10 50 g Micriobiological tests after biodegradability test. AND SINT WUR resin M9 50 g Micriobiological tests after biodegradability test. AND SINT WUR resin M9.2 50 g Micriobiological tests after biodegradability test. AND SINT WUR resin M10.2 50 g Micriobiological tests after biodegradability test. Grant Agreement 101058371 – Project ESTELLA ESTELLA_D5.1_V1 ©ESTELLA - This is the property of ESTELLA Parties: shall not be distributed/reproduced without formal approval of ESTELLA General Assembly. This reflects only the author’s views. The Community is not liable for any use that may be made of the information contained therein.