Polímeros: Ciência e Tecnologia (Polimeros) 2nd. issue, vol. 31, 2021 by Polímeros: Ciência e Tecnologia (Polimeros)

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A ss o ci at e E d i t o r s Alain Dufresne Bluma G. Soares César Liberato Petzhold José António C. Gomes Covas José Carlos C. S. Pinto Paula Moldenaers Richard G. Weiss Rodrigo Lambert Oréfice

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Polímeros / Associação Brasileira de Polímeros. vol. 1, nº 1 (1991) -.- São Carlos: ABPol, 1991Quarterly v. 31, nº 2 (Apr./June 2021) ISSN 0104-1428 ISSN 1678-5169 (electronic version)

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1. Polímeros. l. Associação Brasileira de Polímeros. Polímeros, 31(2), 2021

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Editorial Section News....................................................................................................................................................................................................E3 Agenda.................................................................................................................................................................................................E4 Funding Institutions.............................................................................................................................................................................E5

O r i g in a l A r t ic l e Mechanical and thermal properties of polystyrene and medium density fiberboard composites Juliana Cristina Kreutz, Paulo Ricardo de Souza, Viviane Prima Benetti, Adonilson dos Reis Freitas, Paulo Rodrigo Stival Bittencourt, Luciana Gaffo .................................................................................................................................................................................................. 1-7

Development of a bio-based adhesive from Protium heptaphyllum resin Marcos Danilo Costa de Almeida, João Antonio Pessoa da Silva, Felipe Fernando da Costa Tavares, Ludmila Leite Araujo, Jefferson de Souza Zeferino and Ruth Marlene Campomanes Santana ............................................................................................................................. 1-8

Determination of antioxidant and antimicrobial activity of sweetgum (Liquidambar orientalis) leaf, a medicinal plant

Hatice Ulusoy, Şule Ceylan and Hüseyin Peker ............................................................................................................................................. 1-7

Extraction and characterization of nanofibrillated cellulose from yacon plant (Smallanthus sonchifolius) stems Romaildo Santos de Sousa, Alan Sulato de Andrade and Maria Lucia Masson ............................................................................................ 1-8

Migration of phthalates and 2,6-diisopropylnaphthalene from cellulose food packaging Leda Coltro, Elisabete Segantini Saron, Thiago Ivan Pessoa, Julia Morandi and Bruna Santos Silva ......................................................... 1-8

Effect of molar weight of gelatin in the coating of alginate microparticles Joelma Correia Beraldo, Gislaine Ferreira Nogueira, Ana Silvia Prata and Carlos Raimundo Ferreira Grosso......................................... 1-9

Chitosan-based hydrogel for treatment of temporomandibular joint arthritis Fabianne Lima, Wanderson Gabriel Melo, Maria de Fátima Braga, Ewerton Vieira, João Victor Câmara, Josué Junior Pierote, Napoleão Argôlo Neto, Edson Silva Filho and Ana Cristina Fialho ............................................................................................................................... 1-6

PVC plasticizer from trimethylolpropane trioleate: synthesis, properties, and application Laura de Andrade Souza, Edson Luiz Francisquetti, Rafael Domingos Dalagnol, Celso Roman Junior, Maria Telma Gomes Schanz, Martin Edmund Maier and Cesar Liberato Petzhold ............................................................................................................................................... 1-10

Development of electrically conductive polymer nanocomposites for the automotive cable industry Miguel Guerreiro, Joana Rompante, André Costa Leite, Luís Paulo Fernandes, Rosa Maria Santos, Maria Conceição Paiva and José António Covas .................................................................................................................................................................................................. 1-8

Increasing the physical and combustion performance of Oriental beech by impregnating borates and coating liquid glass Yilmaz Anil Gunbekler, Hilmi Toker, Caglar Altay, Mustafa Kucuktuvek and Ergun Baysal .......................................................................... 1-8

Silane-coupled kenaf fiber filled thermoplastic elastomer based on recycled high density polyethylene/natural rubber blends Cao Xuan Viet, Hanafi Ismail, Abdulhakim Masa and Nabil Hayeemasae ..................................................................................................... 1-8

Modification of poly(lactic acid) filament with expandable graphite for additive manufacturing using fused filament fabrication (FFF): effect on thermal and mechanical properties João Miguel Ayres Melillo, Iaci Miranda Pereira, Artur Caron Mottin and Fernando Gabriel da Silva Araujo .......................................... 1-9 Cover: Scanning electron microscopy images of Expandable graphite after heat treatment. Arts by Editora Cubo.

Polímeros, 31(2), 2021

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Polymer Biomaterial Global Market Report 2021: COVID-19 Growth and Change to 2030

NSF Creates Polymer Chemistry Optimization Center at Duke for Future Materials

This report was announced by Reportlinker.com in Sept. 24, 2021 and can be seen at https://www.reportlinker.com/ p06151666/?utm_source=GNW The polymer biomaterial market consists of sales of polymer biomaterial and related services by entities (organizations, sole traders and partnerships) that produce polymer biomaterial used for enhancing the functionality of tissues and organs that are damaged in various disease therapies. Polymer biomaterials are inert pharmacological substances made up of natural and synthetic origin. Stringent regulatory systems concerning the biocompatibility of the polymer biomaterials is expected to limit the global polymer biomaterials market. Although biomaterials undergo rigorous premarket evaluations, considerable adverse events and complications are reported regarding the biocompatibility of polymer biomaterials. Biomaterial implantation may result in immunological and inflammatory reactions due to the induction of cellular and molecular events in the host, which may lead to excessive inflammation, impairment of healing, fibrotic encapsulation, tissue destruction, or even isolation and rejection of the implant. To overcome such adverse immune reactions, stringent regulations are imposed by the regulatory authorities, restricting market growth. Increasing applications of polymeric biomaterials in tissue engineering are driving the global polymer biomaterials market. Polymers are extensively used in regenerative medicine and tissue engineering due to their flexibility and versatile properties such as tailoring the damaged tissue’s physical, chemical and mechanical properties by modification of functional groups during synthesis, according to the regeneration capability of tissues of the organs. Besides being biodegradable, they offer different geometry and structures, thus meeting the needs of specific tissue engineering applications. The polymer biomaterial market covered in the report is segmented by type into nylon, silicone rubber, polyester, polymethyl methacrylate (PMMA), polyethylene (PE), polyvinyl chloride, others and by application into cardiovascular, ophthalmology, dental, plastic surgery, wound healing, tissue engineering, orthopedics, neurological disorders/central nervous system, others. The major players in the polymer biomaterial market are BASF, Corbion, Zimmer Biomet, Royal DSM, Koninklijke DSM, Covestro, Evonik Industries, Starch Medical, Victrex, and W. L. Gore & Associate. Countries covered in the market report are Australia, Brazil, China, France, Germany, India, Indonesia, Japan, Russia, South Korea, UK, USA. The global polymer biomaterial market is expected to grow from $47.05 billion in 2020 to $53.87 billion in 2021 at a compound annual growth rate (CAGR) of 14.5%. The growth is mainly due to the companies resuming their operations and adapting to the new normal while recovering from the COVID-19 impact, which had earlier led to restrictive containment measures involving social distancing, remote working, and the closure of commercial activities that resulted in operational challenges. The market is expected to reach $93.98 billion in 2025 at a CAGR of 15%. The full report can be seen at: https://www.reportlinker. com/p06151666/?utm_source=GNW Source: Yahoo!Finance – www.finance.yahoo.com

The National Science Foundation has awarded a five-year, $20 million grant to Duke University researchers to explore and optimize the chemical structure and physical properties of individual molecules in a polymer network. “The long-term potential of this research includes cost- and time-efficient optimization of the polymers used in products like biomedical implants, building materials and even automobile tires,” said principal investigator Stephen Craig, the William T. Miller distinguished professor of chemistry at Duke. “We hope to discover new ways to make tougher, longer-lived materials with improved end-of-life properties that reduce waste while being perfectly tailored to their intended uses.” The NSF Center for the Chemistry of Molecularly Optimized Networks (MONET) will bring together experts in polymer chemistry, synthetic methods, photochemistry, multi-scale modeling and bioconjugation. While led by Duke, the center will include researchers from Columbia University, Johns Hopkins, MIT, Northwestern, University of California – San Diego, University of Illinois Urbana-Champaign, University of Michigan, and University of Washington. The senior investigator team includes Duke’s Michael Rubinstein, the Aleksandar S. Vesic distinguished professor of mechanical engineering and materials science, chemistry, physics, and biomedical engineering, who specializes in polymer modeling. “The scientific path being charted by the MONET team will further national priorities in Advanced Manufacturing, Sustainability and Artificial Intelligence,” said NSF Division Director David Berkowitz. In addition to its scientific activities, MONET will work to broaden participation and improve student training in the sciences and advance the public appreciation and commercial translation of its findings. Source: Mirage News. - www.miragenews.com

Polímeros, 31(2), 2021

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A A A A A A A A A A A A A A A A A A A A A

February World Congress on Carbon and Advanced Energy Materials Date: February 07-08, 2022 Location: Webinar Website: global.materialsconferences.com ICPMPS 2022: 16. International Conference on Polymeric Materials and Polymer Synthesis Date: February 07-08, 2022 Location: Melbourne, Australia Website: waset.org/polymeric-materials-and-polymer-synthesisconference-in-february-2022-in-melbourne Polymer Colloids Date: February 19-22, 2022 Location: San Diego, United States Website: www.polyacs.net/22polycolloids 18th International Plastics and Petrochemicals Trade Exhibitions Date: February 21-24, 2022 Location: Riyadh, Saudi Arabia Website: saudi-pppp.com/saudi-plastics-petrochem International Conference on Material Science and Engineering Date: February 24-25, 2022 Location: Prague, Czech Republic Website: materialsscience.conferenceseries.com

March ICFPPMA 2022: 16. International Conference on FlameRetardant Polymeric Materials and their Applications Date: March 11-12, 2022 Location: Miami, United States Website: waset.org/flame-retardant-polymeric-materials-and-theirapplications-conference-in-march-2022-in-miami

April 37th International Conference of the Polymer Processing Society (PPS-37) Date: April 11-15, 2022 Location: Fukuoka, Japan Website: www.pps-37.org International Conference on Polymer Science and Composite Materials Date: April 14-16, 2022 Location: Porto, Portugal. Website: www.pagesconferences.com/polymer-sciencecomposite-materials/index.php Fire Retardants in Plastics - 2022 Date: April 26-27, 2022 Location: Houston, USA Website: www.ami.international/events/ event?Code=C1189#15909

May Polymers for Fuel Cells, Energy Storage and Conversion Date: May 15-18, 2022 Location: Napa, United States Website: www.polyacs.net/2022fuelcells 23rd World Congress on Materials Science and Engineering Date: May 18-19, 2022 Location: London, United Kingdom Website: materialsscience.insightconferences.com 7th Annual Conference and Expo on Biomaterials Date: May 18-19, 2022 Location: London, United Kingdom Website: biomaterials.insightconferences.com POLY-CHAR 2022 — World Forum on Advanced Materials and “Short Course on Polymer Characterization” Date: May 22-25, 2022 Location: Halle and Siegen, Germany Website: poly-char2022.org

MOMPS-X — 10th International Symposium on Molecular Order and Mobility in Polymer Systems Date: May 23-27, 2022 Location: Saint Petersburg, Russia Website: momps2020.macro.ru Polymers 2022 — New Trends in Polymer Science: Health of the Planet, Health of the People Date: May 25-27, 2022 Location: Turin, Italy Website: polymers2022.sciforum.net

June Polymers and Fire Date: June 5-8, 2022 Location: Napa, United States Website: www.polyacs.net/22fipo Chemical Recycling - 2022 Date: June 15-16, 2022 Location: Cologne, Germany Website: www.ami.international/events/ event?Code=C1185#15657

Polymer Sourcing & Distribution – 2022

Date: June 28-30, 2022 Location: Hamburg, Germany Website: www.ami.international/events/event?Code=C1186 2nd Global Conference on Advances in Polymer Science and Nanotechnology Date: June 27-28, 2022 Location: Berlin, Germany Website: polymerscience.peersalleyconferences.com EPF – European Polymer Congress Date: June 26 - July 1, 2022 Location: Prague, Czech Republic Website: www.epf2022.org/

July 49th World Polymer Congress – MACRO2022 Date: July 17-21, 2022 Location: Winnipeg, Canada Website: www.macro2022.org/ PVC Formulation Asia - 2022 Date: July 19-20, 2022 Location: Bangkok, Thailand Website: www.ami.international/events/event?Code=C1178 84th Prague Meeting on Macromolecules – Frontiers of Polymer Colloids Date: July 24-28, 2022 Location: Prague, Czech Republic Website: www.imc.cas.cz/sympo/84pmm/ 8th International Conference on Chemical and Polymer Engineering (ICCPE’22) Date: July 31 - August 2, 2022 Location: Prague, Czech Republic Website: www.imc.cas.cz/sympo/84pmm

August The Global Meet on Bio-Polymers and Polymer Science (GMBPPS2022) Date: August 25-27, 2022 Location: Paris, France Website: primemeetings.org/2022/polymer-science

September Polymer Physics Meeting — Retirement Conference for Dame Athene Donald Date: September 12-13, 2022 Location: Cambridge - United Kingdom Website: events.iop.org/polymer-physics-meeting-retirementconference-dame-athene-donald

Polímeros, 31(2), 2021

ABPol Associates Sponsoring Partners

Polímeros, 31(2), 2021

ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.07120

Mechanical and thermal properties of polystyrene and medium density fiberboard composites Juliana Cristina Kreutz1* , Paulo Ricardo de Souza2, Viviane Prima Benetti3, Adonilson dos Reis Freitas1, Paulo Rodrigo Stival Bittencourt3, Luciana Gaffo1*  Departamento de Química, Universidade Estadual do Oeste do Paraná – UNIOESTE, Toledo, PR, Brasil 2 Departamento de Química, Universidade Estadual de Maringá – UEM, Maringá, PR, Brasil 3 Departamento de Química, Universidade Tecnológica Federal do Paraná – UTFPR, Medianeira, PR, Brasil

*juli_cristinakreutz@hotmail.com; lugaffo@yahoo.com.br

Abstract Virgin polystyrene (PS) composites were reinforced with medium density fiberboard (MDF) residue, considering the influence of fiber content. The composites were evaluated for their morphology, identification of functional groups and thermal behavior. Mechanical tests and a degradation study under ultraviolet radiation (UV) were also performed. The results showed that the best properties were obtained for composites with 4% by mass of MDF waste. The addition of residue was found to increase thermal stability of polystyrene compared to its pure form. The morphology of the composites showed homogeneity of the material. In the degradation tests under ultraviolet (UV) radiation, it was found that the presence of MDF residue slows down the matrix degradation process when evaluated by means of tensile strength. Polystyrene composites reinforced with MDF residues showed good mechanical properties and can be applied in the development of materials that do not need a good appearance. Keywords: waste valuation, pollution, sustainability. How to cite: Kreutz, J. C., Souza, P. R., Benetti, V. P., Freitas, A. R., Bittencourt, P. R. S., & Gaffo, L. (2021). Mechanical and thermal properties of polystyrene and medium density fiberboard composites. Polímeros: Ciência e Tecnologia, 31(2), e2021013. https://doi.org/10.1590/0104-1428.07120

1. Introduction The amount of solid waste that has been generated by humanity in recent years raises attention to the problem associated with its disposal, challenging researchers and companies to seek effective solutions to the issue, combined with social awareness[1]. In this sense, polystyrene (PS) is one of the general-purpose plastics with a wide variety of applications due to its good mechanical properties, anti‑corrosion capacity and processing performance[2]. However, it generates a lot of waste, as it is used in low-cost articles, disposable parts, such as cups and plates, transparent packaging and housewares, with very short use time. Every year, 13 million tons of PS are produced worldwide[3]. One of the ways to reuse these residues is incineration, but this contributes to the emission of greenhouse gases such as NOx, SOx, COx, which cause climate change and release carcinogenic compounds[4]. Another widely produced waste comes from medium density wood panels (MDF), which are made up of lignocellulosic fibers and synthetic adhesive, joined under high heat and pressure[5,6]. The main application of MDF is in the furniture industry, due to its easy processing and low cost. The global production of MDF together with that of high density panels (HDF) reached 100 million m3 in 2016[7], however, currently, no method is commercially

Polímeros, 31(2), e2021013, 2021

applied to recycle MDF waste and thus it is burned or landfilled[8] after service life. In the literature there are bit studies about recycle or apply MDF waste. It´s an alternative has been using it as a source of energy in furnaces, but it is still a small percentage in relation to production. The use of these MDF residues as fillers in polymeric matrices has been studied by several researchers with satisfactory results in the production of composites. Hillig et al.[9] characterized composites made of virgin high density polyethylene (v-HDPE) and different types of sawdust from furniture industry, including MDF waste, observing that the inclusion MDF sawdust provided composites with greater resistance to flexion and impact than those manufactured with other types of waste. Gomes et al.[10] analysed the feasibility of using waste from the manufacture of MDF panels as reinforcement in orthophthalic polyester resin, for the development of materials for industrial application. The results pointed out a decrease in mechanical properties for composites as a function of addition of residue. Souza et al.[11] carried out studies on MDF powder waste in order to identify the best method to recover it. For this, they carried out an environmental diagnosis of a small furniture factory and proceeded with the characterization of MDF waste in the form of powder. The results were similar to other reinforcement loads applied in polymeric matrices. Some limitations found for the application of

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Kreutz, J. C., Souza, P. R., Benetti, V. P., Freitas, A. R., Bittencourt, P. R. S., & Gaffo, L. this residue were the hygroscopicity and the difference in density between the residue and the polymeric matrix. This can be mitigated with previous heat treatment and the use of a coupling agent, respectively. In general many polymers exhibit some disadvantageous intrinsic properties, such as fragility and flammability[12,13]. In this sense, studies have been carried out to improve its performance of polymers, with materials being added as reinforcement[7]. Possible reinforcement could be wood waste discarded by the furniture industries (included MDF), transforming them into new products[14]. These products could be used as engineering material, or as a new product that can be sold in the furniture industry itself, resulting in environmental and financial benefits. The aim of this study is to evaluate the mechanical and thermal performance of a polystyrene (PS) polymer composite, associating MDF waste as a reinforcement, and subjected to conditions of accelerated environmental aging by ultraviolet radiation (UV).

2. Materials and Methods 2.1 Utilized materials Commercial waste of MDF was donated by N. J. Móveis Sob Medida. Polystirene P.A. grade was purchased from Sigma-Aldrich® and used as received.

2.5 Tensile strength test Tensile strength test were performed in triplicate using the Texturometer TA equipment HD Plus, branded Stable Micro Systems, according to ASTM D638-10 and traction speed of 5 mm/min.

2.6 Scanning electron microscopy (SEM) The SEM images were taken from test body in a microscopy FEI, model Quanta 250. The samples were first fractured and sputter coated with a thin layer of gold and then observed at magnification of 1000x. All the SEM images were taken at 23 °C and accelerating voltage was 10.00 kV.

2.7 UV-accelerated aging effect The UV-accelerated aging effect was studied with help of the equipment Bass model “UVV Simulador Acelerado de Intemperis”. The procedure was performed according to cycle 6 from ASTM G154-06. The analysis were realized in triplicate, the exposure time to UV light was 12 hours, divided in two parts. First, the sample were exposed at 1,55 W/m2 and wavelength of 340 nm for 8 hours at 60 ºC, and after they were exposed for 4 hours of condensation at 50 ºC. The total exposure time was 2016 hours (12 weeks) divided into two, four, eight and twelve weeks of photo-exposure.

2.2 Acquisition of composite material

3. Results and Discussions

MDF residue was dried in an oven at 120 °C until constant mass. It solid dry material was granulometrically classified using the mesh tyler Bertel, model ASTM 35, operating for 30 min, obtaining particles with size up to 0,500 mm. The PS-MDF composites were prepared by mix of PS and MDF on the proportion described in Table 1. This mix was loaded in an extrusor machine AX PLASTICOS, model LAB-16. Heating zone were programmed to 160, 175 and 200 °C respectively, operating at velocity of 45 rpm. The extruded material was transferred to the AX Plasticos injector, model LHS 150-80, with the engine head temperature in operation, 220 ºC and 20 ºC for the mold.

3.1 Fourier-transform infrared spectroscopy (FTIR) Analyses were performed for the MDF powder after drying and uniformity of the particles; for the polymeric matrix and for composites developed using MDF and polystyrene matrix. Figure 1 shows the comparative results of the FTIR analyses. FTIR spectrum for pure MDF powder shows absorption bands characteristic of the constituents of the material. The 3338 cm-1 band, attributed to the O-H stretch, corresponds to the adsorbed moisture for the cellulose and urea-formaldehyde resin (one of the constituents of MDF)[15]. The bands at

2.3 Fourier Transformed Infrared (FTIR) analysis The body test was characterized by Fourier transformed infrared (FTIR). Sample spectra were obtained in spectrophotometer Perkin Elmer, model Frontier, working in the ATR mode. Spectra were recorded in the range of 400-4000 cm-1, resolution 4 cm-1 and 64 accumulations.

2.4 Thermogravimetry analysis (TGA) Thermogravimetric analysis (TGA) was carried out using a Perkin Elmer STA-6000 thermoanalyzer at a heating rate of 10 ºC per min, under N2 atmosphere. The TGA analysis was performed in the temperature range of 30–600 ºC. Table 1. Body test composition. Steps

Sample Cod

PS-M0 PS-M4 PS-M8

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%PS on the matrix 100 96 92

%MDF on the matrix 4 8

Figure 1. FTIR spectra of the MDF residue; virgin PS matrix and PS composites with 4 and 8 wt % MDF. Polímeros, 31(2), e2021013, 2021

Mechanical and thermal properties of polystyrene and medium density fiberboard composites 1246 cm-1 and 1035 cm-1 correspond to the C-O stretch of the acetyl group, present in lignin and hemicellulose[16]. For the virgin polymer matrix, characteristic absorption bands were also observed and assigned according to the literature[17,18-20]. PS presentes bands at 3025 cm-1, associated with the C-H stretch of the aromatic ring, 2920 and 2848 cm-1 are related to CH2 strech, asymmetric and symmetrical, respectively. Bands at 1607 and 1490 cm-1 are attributed to the C-C stretch of the aromatic ring, 1450 cm-1 is attributed to the CH2 stretch of the aromatic ring. The C-H stretch vibrations, also of the aromatic ring, can be observed at 1073, 1020, 750 and 690 cm-1. For the polymeric matrix, the band at 1743 cm-1 corresponds to the aromatic ring monosubstitution[21]. For the polymeric composites obtained, modification of polymeric matrix was verified at 1027 cm-1, related to the C-O and O-H vibrations of the polysaccharides in cellulose[17,18-20]. This indicates a modification of the virgin polymeric matrix when the MDF residue was added, which may cause changes in the mechanical properties of the composites in relation to the pure matrix.

Figure 2. TGA and DTG curves of MDF waste.

3.2 Thermo analyses Thermo analyses of the samples were performed, monitoring their mass as function of temperature. Figure 2 shows the TGA for MDF, where the first mass loss was shown to occur between 50 and 116 °C, which can be attributed to the loss of moisture present in the residue, a typical hygroscopic characteristic of materials consisting of cellulose[22]. The extrapolated temperature of the beginning of mass loss (Tonset) for the MDF residue was 284 °C. Khanjanzadeh et al.[23] verified a similar result in their study, showing that the loss of MDF mass starts at 285.4 °C. In addition, it was found that at 231 °C only 5% of the mass is lost and 10% of loss of mass occurs at 268 °C. The second event of mass lost in composites using MDF occurs between 250 and 380 °C and can be associated with the release of volatile matter, which consists of toxic and carcinogenic chemical compounds added to wood and its derivatives, such as formaldehyde, which is harmful to human health and the environment[24]. Above 380 °C there occurs degradation of carbonaceous constituents such as lignin, for example. The results of the thermogravimetric analyses were compared for virgin polystyrene and for samples with different levels of MDF residue, shown in Figure 3. For the virgin polymeric matrix, the temperature of the beginning of the degradation was 387 °C. Botan at al. showed that this material started to degrade at temperature around 360 °C[25]. Dominguini et al.[26] showed results where the thermal decomposition of the pure polystyrene started at 380 °C. In our study, we found that the residual mass resulting from the PS burning process is practically zero, and all samples containing MDF presented a single mass loss event, as well as the polystyrene matrix. The results show that the addition of MDF waste slightly increases the temperature at which degradation starts compared to Tonset of the pure PS. The percentage of MDF did not influence the degradation temperature of the polymeric composite. As reported by Spinacé et al.[27], phenols present in lignin Polímeros, 31(2), e2021013, 2021

Figure 3. DTG curves to MDF composites and polymeric matrix.

may eventually act as scavengers of free radicals, delaying the thermal degradation of the polymer.

3.3 Tensile strength test Tensile strength results, performed in triplicate analyses, for the body test of virgin polystyrene with different levels of MDF residues were compared. For the studied samples, Young’s modulus was obtained, as shown in Figure 4. The literature shows that the Young modulus obtained for commercial virgin polystyrene is between 2.28-3.34 GPa[28]. It was observed that the tensile strength of virgin polystyrene in this study showed a reduced value compared to commercial virgin polystyrene, due to the processing to which it was subjected in the extruder. As observed, composites with a content of 4% suffered lower elongation of the composite and, therefore, have a greater Young’s modulus when subjected to tensile tests. For the 8% MDF residue content, Young’s modulus remained unchanged compared to virgin polystyrene, around 2.0 GPa. Borsoi et al.[29] have observed that Young’s modulus increased compared to pure PS, a result that gets being more pronounced for the 20% cotton fiber using compatibilizing agent. With the increase in the fiber content, the stresses become more evenly distributed, with this, the incorporation of discontinuous fibers in the thermoplastic polymer matrix improves the rigidity 3/7

Kreutz, J. C., Souza, P. R., Benetti, V. P., Freitas, A. R., Bittencourt, P. R. S., & Gaffo, L. and resistance properties of the obtained composites[30]. In our study, this was not observed, which may be related to inefficient homogenization of samples, with more residue content inside of the extruder. Although incorporating MDF waste does not improve the material’s Young’s modulus compared to pure PS, it should be taken into account that this method promotes the encapsulation of waste, which is commonly burned and generates gases that are toxic to human health and the environment. It is important to note that the developed composites have properties that make their commercial use feasible, as required by the normative document ANSI A 208.1[31]. According to these standards, the value of Young’s modulus should be 2,300 MPa (2.3 GPa),

Figure 4. Elasticity modules to composite as a function of % MDF.

indicating that the polystyrene composite with 4% MDF residue can be used in making plastic wood.

3.4 Evaluation of degradation to accelerated aging in a UV chamber The main changes that a polymeric material degraded by UV radiation can acquire are yellowing, changing the surface appearance of the material and reducing its mechanical properties[32]. Thermal properties of pure polystyrene and samples with 4 and 8% by weight of MDF powder subjected to accelerated degradation by UV were analyzed. The results are showed in Figure 5. The DTG graphs show that all samples presented a single mass loss event. Temperature of degradation onset for pure polystyrene and for the composites did not show significant changes with the aging time. This indicates that photodegradation does not alter the thermal properties of the material, regardless of the MDF content used in the composite. Figure 6 shows the morphology of the PS, PS-M4 and PS-M8 samples, exposed to accelerated aging in a UV chamber after 90 days, compared to the samples before exposure. The micrographs were taken from the fractured samples after performed mechanical test, at a magnification of 1000 x. Analyzing the images, it is founded that the samples have a good homogeneity, not was observed the presence of agglomerates of MDF particles (originally in the range of 500 µm). Also, not was found cavity, which can originate

Figure 5. DTG curves to pure PS, composite PS-MDF 4% and 8% of MDF, no aging and after 90 days accelerated aging. 4/7

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Mechanical and thermal properties of polystyrene and medium density fiberboard composites when the dispersing material has low adhesion to the matrix, suggesting a good dispersion and adhesion[33] of the MDF powder in polystyrene matrix. It´s a few changes were observed in both pure polystyrene matrix and composite material. After photo-irradiation, greater roughness and cracks were observed in all samples. These cracks were less pronounced for PS-M8 sample, suggesting that adhesion between the components is impaired after UV irradiation[34]. According to Matuana et al.[35], the exposure of the composite to moisture (water in the form of mist) causes swelling in the fiber, causing micro-cracks in the matrix, accelerating oxidation reactions and facilitating the penetration of light. According to Joseph et al.[36], the photooxidation process occurs mainly in the amorphous regions of the polymer due to the greater permeability of oxygen in this region of the material. Figure 7 shows the sample module before and after exposure to accelerated aging in a UV chamber for up to 2160 hours (90 days). It was observed a change in Young’s modulus for all samples subjected to accelerated aging, under 90 days exposure.

This property is very sensitive to structural changes, such as the mass of the polymeric matrix, the density of crosslinking and fiber/matrix interfacial adhesion[37]. Considering the standard deviation related to the measurements, polymeric composites reinforced with MDF powder presented a linear increase in module up to 60 days of exposure, whereas after 90 days decreased, indicating that crosslinking with subsequent splitting of the chains may have occurred. Studies show that this increase in Young’s modulus is caused by the photodegradation process, that is, due to the crosslinking reactions that can happen during the composite photodegradation process[37,38]. The reduction of this property is attributed to oxidative reactions that lead to the scission of chains that, together with the formation of superficial cracks and loss of interfac adhesion, cause deterioration in resistance[37,39]. Fernandes et al.[38] studied the photodegradation of high impact polypropylene/polystyrene blends. In the case of high impact polystyrene, the authors observed a small increase in Young’s modulus. They observed that Young’s modulus is obtained in a very small deformation range (elastic region) and, within this range, both split and crosslink reactions,

Figure 6. Scanning Electron Microscope (SEM) images of a fracture surface of no aging and after 90 days accelerated aging to pure PS, PS-M4 and PS-M8. Polímeros, 31(2), e2021013, 2021

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Kreutz, J. C., Souza, P. R., Benetti, V. P., Freitas, A. R., Bittencourt, P. R. S., & Gaffo, L.

6. References

Figure 7. Young’s module as function of aging time to composite PS-M0, PS-M4 and PS-M8.

even if only at a small degree of intensity, are reflected in this property[38]. This increase in Young’s modulus was also observed in the study by Borsoi et al.[40] explaining that this process occurs with some thermoplastics subjected to certain degradation processes. According to the ASTM G154-06, 1000 h of accelerated aging is equivalent to 1 year of natural exposure to UV. Polystyrene composites with 4% by weight of residue MDF showed values of Young’s modulus about 2.3 GPa, after 90 days of exposition, which equates to 2160 h. Considering the normative document, ANSI A 208.1, this material could be used as plastic wood in good mechanical conditions for up to 2 years. Despite the good mechanical conditions, there was a change in the color of the material, becoming whitish over time with accelerated exposure. With such characteristics, the material could be used where good aesthetic conditions would not be necessary, for example, for rural applications.

4. Conclusions According to the results here obtained, we can conclude that the incorporation of MDF waste in the virgin polystyrene to obtain composites, was quite effective, without the use of compatibilizing agents. This may be a technically viable alternative for using these residues in products with higher added value for different destinations, such as for rural applications. It was possible to promote the encapsulation of MDF waste, which is usually disposed of in landfills or burned, releasing toxic gases and contributing to environmental pollution. Future work may be carried out with MDF waste and polystyrene utensils after use, which are discarded. Thus, in addition to reusing MDF, a better destination would be given to post-use PS, which is one of the major generators of environmental pollution today.

5. Acknowledgements The authors are thankful for LADUR (Laboratório de Durabilidade) of LADEMA (Laboratório de Desempenho, Estruturas e Materiais) at UNILA for the experimental support as well as for Fundação Araucária. 6/7

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29. Borsoi, C., Scienza, L. C., Zattera, A. J., & Angrizani, C. C. (2011). Obtainment and characterization of composites using polystyrene as matrix and fiber waste from cotton textile industry as reinforcement. Polímeros: Ciência e Tecnologia, 21(4), 271279. http://dx.doi.org/10.1590/S0104-14282011005000055. 30. Antich, P., Vázquez, A., Mondragon, I., & Bernal, C. (2006). Mechanical behavior of high impact polystyrene reinforced with short sisal fibers. Composites. Part A, Applied Science and Manufacturing, 37(1), 139-150. http://dx.doi.org/10.1016/j. compositesa.2004.12.002. 31. American National Standards Institute – ANSI. (2009). ANSI A2081 - Mat-formed wood particleboard: specification. United States: National Particlepanel Association. 32. Şahin, T., Sinmazcelik, T., & Şahin, Ş. (2007). The effect of natural weathering on the mechanical, morphological and thermal properties of high impact polystyrene (HIPS). Materials & Design, 28(8), 2303-2309. http://dx.doi.org/10.1016/j. matdes.2006.07.013. 33. Gadioli, R., Waldman, W. R., & De Paoli, M. A. (2016). Lignin as a green primary antioxidant for polypropylene. Journal of Applied Polymer Science, 133(45), 1-7. http://dx.doi.org/10.1002/ app.43558. 34. Darie, R. N., Bodirlau, R., Teaca, C. A., Macyszyn, J., Kozlowski, M., & Spiridon, I. (2013). Influence of accelerated weathering on the properties of polypropylene/polylactic acid/eucalyptus wood composites. International Journal of Polymer Analysis and Characterization, 18(4), 315-327. http://dx.doi.org/10.1 080/1023666X.2013.784936. 35. Matuana, L., Jin, S., & Stark, N. (2011). Ultraviolet weathering of HDPE/wood-flour composites coextruded with a clear HDPE cap layer. Polymer Degradation & Stability, 96(1), 97-106. http://dx.doi.org/10.1016/j.polymdegradstab.2010.10.003. 36. Joseph, P. V., Rabello, M. S., Mattoso, L. H. C., Joseph, K., & Thomas, S. (2002). Environmental effects on the degradation behaviour of sisal fibre reinforced polypropylene composites. Composites Science and Technology, 62(10), 1357-1372. http:// dx.doi.org/10.1016/S0266-3538(02)00080-5. 37. Angrizani, C. C., Oliveira, B. F., & Amico, S. C. (2015). Evaluation of the durability performance of glass-fiber reinforcement epoxy composites exposed to accelerated higrothermal ageing. Journal of Materials Science and Engineering with Advanced Technology, 11(2), 31-47. http:// dx.doi.org/10.18642/jmseat_7100121507. 38. Fernandes, L. L., Freitas, C. A., Demarquette, N. R., & Fechine, G. J. M. (2012). Influence of the type of polypropylene on the photodegradation of blends of polypropylene/high impact polystyrene. Polímeros: Ciência e Tecnologia, 22(1), 61-68. http://dx.doi.org/10.1590/S0104-14282012005000013. 39. Fechine, J. M., Santos, A. B., & Rabello, M. S. (2006). The evaluation of polyolefin photodegradation with natural and artificial exposure. Quimica Nova, 29(4), 674-680. http:// dx.doi.org/10.1590/S0100-40422006000400009. 40. Borsoi, C., Berwig, K. H., Scienza, L. C., Zoppas, B. C. D. A., Brandalise, R. N., & Zattera, A. J. (2014). Behavior in simulated soil of recycled expanded polystyrene/waste cotton composites. Materials Research, 17(1), 275-283. http://dx.doi. org/10.1590/S1516-14392013005000167. Received: Sept. 20, 2020 Revised: Mar. 15, 2021 Accepted: May 15, 2021

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ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.10020

Development of a bio-based adhesive from Protium heptaphyllum resin Marcos Danilo Costa de Almeida1* , João Antonio Pessoa da Silva2 , Felipe Fernando da Costa Tavares1 , Ludmila Leite Araujo1 , Jefferson de Souza Zeferino1  and Ruth Marlene Campomanes Santana1  Laboratório de Materiais Poliméricos – LAPOL, Departamento de Materiais, Escola de Engenharia, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brasil 2 Laboratório de Tecnologia de Polímeros – LATEP, Departamento de Engenharia Química, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brasil

*marcos.almeida@ueap.edu.br

Abstract In this work, a bio-based adhesive is prepared from Protium heptaphyllum resin. The resin is first characterized by 1 H and 13C nuclear magnetic resonance spectroscopy and the bioadhesive is then prepared using a simple mixture of the resin with linseed oil, catalyzed by cobalt octanoate, to induce crosslinking. The precursors and bioadhesive obtained are characterized by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). The NMR analysis shows the presence of groups of triterpenes, such as α- and β-amyrins, and diols, such as brein and maniladiol. Thermogravimetric analysis reveals that the resin has less thermal stability than the bioadhesive. Mechanical tests indicate that the bioadhesive has greater adhesion strength compared to the commercial adhesive, reaching an average stress at break of 7.66 and 0.113 MPa for the wood and carbon steel substrates, respectively. In conclusion, the bioadhesive can be used for the production of composites. Keywords: adhesive, Protium heptaphyllum, reticulation reaction, linseed oil. How to cite: Almeida, M. D. C., Silva, J. A. P., Tavares, F. F. C., Araujo, L. L., Zeferino, J. S., & Santana, R. M. C. (2021). Development of a bio-based adhesive from Protium heptaphyllum resin. Polímeros: Ciência e Tecnologia, 31(2), e2021014. https://doi.org/10.1590/0104-1428.10020

1. Introduction Adhesives are polymeric materials capable of interacting, both chemically and physically, with a substrate in such a way that the stresses are transferred between both components[1]. Commercially available adhesives, such as polychloroprene, polyvinyl, cyanoacrylate and vinyl acetate, are generally produced from non-renewable sources. Furthermore, some of the most used petroleum-based adhesives contain dangerous chemicals, e.g., formaldehyde, which have a negative impact on the environment and human health[2]. In order to produce more environmentally friendly materials, significant research has been focused on using renewable resources to obtain adhesives. The adhesives prepared from the modification of linseed oil[3], soy protein[4,5], castor oil plant[6,7] lignin and chitosan[8-10] are good examples of this approach. The employment of these raw materials is possible as a result of their active sites, such as hydroxyl groups and unsaturated bonds. These groups can react, in the presence of a catalyst, with crosslinking agents that have chemical groups compatible with the substrate (wood, metals or plastics). Natural resins are extractive substances secreted by plants as a protective mechanism to injury. Most natural resins are composed of terpenes, which are macromolecules derived

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from isoprene (2-methyl-1,3-butadiene). These compounds present actives sites (double bonds and hydroxyl groups) that are prone to chemical attack and can be exploited for the development of adhesives. In the Amazon region of Brazil, there are many species of trees that secrete natural resins with the potential for the development of adhesives. Among these natural resins, Protium heptaphyllum is highlighted for having an established market due to its application in the cosmetics industry. This resin is mostly composed of α- and β-terpenes[11] and is used in folk medicine as an anti-inflammatory, analgesic, stimulant, cough remedy and for clearing airways[12]. Another important application of this resin is in the caulking of wooden boats, a classical craftwork realized by riverains. The use of natural resins in their crude form for the preparation of adhesives results in poor mechanical resistance, due to their relatively small molecular weight and absence of interchain bonds. In this context, crosslinking with vegetable oils has been a strategy used for improving the mechanical performance of adhesives. Addis et al.[13] performed a chemical modification of linseed oil from various crosslinking agents and tested it as an adhesive in three types of wood. They evaluated the curing time, the type of crosslinking agent

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Almeida, M. D. C., Silva, J. A. P., Tavares, F. F. C., Araujo, L. L., Zeferino, J. S., & Santana, R. M. C. and the rate of adhesive impregnation and reported that the material is a promising bioadhesive for wood. Although Protium heptaphyllum resin has long been used by riverains for craftwork, its scientific investigation is very limited. Only the work of Vieira et al.[11] reports the application of Protium heptaphyllum resin as an adhesive. The authors demonstrated that the pure resin can be used for the adhesion of wood; however, they verified that it has low mechanical resistance and becomes brittle after curing, which decreases its adhesion strength on the substrate surface. In this work, a bioadhesive is prepared from the crosslinking of Protium heptaphyllum resin and linseed oil and catalyzed by cobalt octanoate (12Co). Linseed oil is selected because of its high content of double bonds, which is important for the formation of a consistent chemical network in the cured material. In addition, linseed oil acts as a plasticizer, which resolves the problem of brittleness described by Vieira et al.[11]. The resin and bioadhesive are initially characterized and their chemical composition, thermal stability and main thermal transitions determined. Subsequently, the adhesion force of the bioadhesive is evaluated through shear stress assays in specimens of wood and carbon steel. A commercial adhesive from polychloroprene is used for comparison. On this basis, the aim of this study is to develop a bioadhesive suitable for the production of composites.

2. Materials and Methods 2.1 Materials Protium heptaphyllum resin was acquired from a local market in Macapá, Brazil. The material was received as a dark gray solid, with some vegetable residues of branches and leaves dispersed among the solids. Linseed oil (Corfix, Porto Alegre) was acquired from a local market in Porto Alegre, Brazil, and received as a clear, crude and unpolymerized light yellow oil. The 12Co 12% catalyst was supplied by Miracema Nuodex (Guarulhos, Brazil). Chloroform P.A. 99.98% and deuterated chloroform (CDCl3) were purchased from Sigma Aldrich®.

2.2 Methods 2.2.1 Purification of Protium heptaphyllum resin The resin was first crushed with a hammer and vegetable residues (branches and leaves) were manually removed. The resin was then purified through solid/liquid Soxhlet

extraction with chloroform at 70 °C for 8 h. Finally, the extract was concentrated in an oven at 105 ºC for 24 h, resulting in a red-orange translucent solid (Figure 1) with a mass yield of 92%. 2.2.2 Adhesive formulation and preparation of specimens for mechanical tests The bioadhesive was produced from the crosslinking of the Protium heptaphyllum resin and linseed oil in the presence of the 12Co catalyst. 12Co is considered the most efficient catalyst among transition metals and it can induce a crosslinking reaction of linseed oil at 82 ºC[14]. The bioadhesive preparation involved the simple mixing of the components at 100 ºC in a mass ratio of 80:20 (resin/linseed) with 0.5% of 12Co. In a typical procedure, 16 g of the purified Protium heptaphyllum resin were weighed in a porcelain crucible and heated to 100 ºC until complete liquefaction was achieved. Subsequently, linseed oil (4 g) and 0.5% of 12Co (0.01 g) were added and the mixture was homogenized for 5 min. This mixture was allowed to cure for 72 h in an oven at 60 °C. After curing, samples of the adhesive were collected for FTIR, TGA and DSC characterization. 2.2.3 Characterization of precursor materials and bioadhesive 1 H and 13C nuclear magnetic resonance (NMR) spectroscopies were performed to identify the main compounds present in the purified Protium heptaphyllum resin. Samples were dissolved in deuterated chloroform at 60 mg/mL and analyzed at 50 ºC using a Bruker® spectrometer, Avance III, 300 MHz. The thermal stability of the purified resin, linseed oil and adhesive was evaluated using TGA. The analysis was performed using a TGA Q50 V20.13 Build 39 under a N2 flow rate of 100 mL/min from room temperature to 840°C at a heating rate of 20 °C/min. An alumina pan was used and the approximate sample mass was 15 mg. DSC was performed on the purified resin, linseed oil and adhesive using a TA Instrument calorimeter with a N2 cooling module. Samples of ~5 mg were analyzed from 0 to 200 ºC at a heating rate of 10 ºC/min and in a N2 flow rate of 50 mL/min. FTIR spectroscopy was employed to evaluate the main functional groups of the Protium heptaphyllum resin and linseed oil. The spectra were collected with a Perkin‑Elmer Frontier spectrometer in transmittance mode using the KBr pellet method in the range of 4000–650 cm-1. For the bioadhesive of Protium heptaphyllum, the analysis was

Figure 1. Protium heptaphyllum resin (a) before and (b) after purification. 2/8

Polímeros, 31(2), e2021014, 2021

Development of a bio-based adhesive from Protium heptaphyllum resin performed in total attenuated reflection using a wavenumber range from 4000 to 700 cm-1, 16 scans and a 4 cm-1 resolution. 2.2.4 Mechanical shear testing To determine the adhesion force, the bioadhesive was prepared in a similar manner as described in section 2.2.2. The mixture (crude resin, linseed oil and catalyst) was impregnated in specimens of wood or carbon steel that were placed in an oven with air recirculation (DELEO, model ABAR-FC) at 60 ºC for 72 h for curing. The carbon steel specimens were based on the ASTM D1002‑10[15] standard, with dimensions of 100 mm×25 mm and thickness of 1.5 mm. The bioadhesive was impregnated on the specimen with a grammage of 144 g/m2, in a bonding area of 12.5×25 mm2. After the contact of both specimen sides, they were attached with double metal clips. For wooden specimens, the modified ASTM D906[16] standard was employed. Laminated eucalyptus wood with dimensions of 100 mm×25 mm and a thickness of 4 mm was used. The bioadhesive was applied on the substrate with a grammage of 154 g/m2 in a bonding area of 30×25 mm2. The specimens were again attached with double clips. The tests were carried out using a universal machine EMIC, model 23-5D. The specimens were attached by two flat plate claw screws and tensioned. For the metal substrate, a shear rate of 1.27 mm/min was used, according to ASTM D1002-10[15], while for the wooden specimens, a shear rate of 10 mm/min was used, according to ASTM D906[16]. The test with a commercial polychloroprene adhesive was made using the same procedure, except for the curing time, which was 72 h. Statistical analyzes were performed using the free PAST software, version 2.17c. Variance (ANOVA) and Tukey’s test were used at the 95% significance level.

as β-amirinone, α-amirinone and lupenone[17]. The peaks at β-79.07 ppm and α-77.03 ppm in the 13C NMR spectrum are related to carbons in position C3 linked to hydroxyls (C-OH), which is confirmed by the double doublet at 3.25 ppm in the 1H NMR spectrum, which is characteristic of protons attached to carbons adjacent to hydroxyl groups. In addition, in the 13C NMR spectrum, the peaks at 144.6 ppm (C13) and 121.26 ppm (C12) are characteristic of β-amyrin. The peaks at 139 ppm (C13) and 124 ppm (C12) are from α-amyrin[17,18]. The peaks at 67.4 ppm and 66.4ppm in the 13 C NMR spectrum are attributed to carbinolic carbons, at position C16, and the double doublet at 4.2 ppm in the 1 H NMR spectrum is characteristic of diols, such as brein and maniladiol[17,19]. The TGA curves and their respective derivatives (DTG) of the purified Protium heptaphyllum resin, linseed oil and bioadhesive are shown in Figure 4. Table 1 presents the values of the thermal stability at 5% mass loss (T5%), and the mass loss (ΔmI) and peak temperature (Tmax, which is the temperature of the maximum rate of decomposition obtained from the DTG curves) of each thermal event observed for the curves.

3.1 Characterization of precursor materials and bioadhesive

Figure 4 shows four thermal events for the resin and adhesive, while for the linseed oil, the degradation occurs in a single step. The first thermal event appears between 106 and 201 ºC and is attributed to the loss of volatile components. It is noted that the resin (Δm1 = 9.18%) presents a higher variation in mass loss than the adhesive (Δm1 = 4.10%), which may be due to the thermal processes of heating at 100 °C until liquefaction and curing at 60 °C used for preparing the adhesive. The second thermal event is observed between 201 and 435 °C and is related to the loss of the main components (amyrins and diols) of the Protium heptaphyllum resin, as reported by Silva Junior et al.[20] and Vieira Junior et al.[17]. Silva Junior et al.[20] showed that the isomers α- and β-amyrins, the major components of Protium heptaphyllum resin are degraded in the range of 210 to 380 ºC. Vieira Junior et al.[17] obtained similar results for the Protium heptaphyllum resin. In this step, the remaining mass of the resin (Δm2 = 90.65%) is lost, while for the bioadhesive, a broadening of the peak (Δm2 = 89.48%) is observed. From analyzing the Tmax of the both peaks, it is observed that the resin has the highest value; however, this does not mean that it has a greater thermal stability, since the peak broadening is displaced for a higher temperature

The 1H and 13C NMR spectra of the Protium heptaphyllum resin are shown in Figures 2 and 3, respectively. Since techniques for separating the components of the Protium heptaphyllum resin were not used, the spectra are only discussed qualitatively, with the primary purpose of identifying functional groups and structures. For this, the chemical shifts of the compounds found in the Protium heptaphyllum resin depicted in the literature are used. In the region between 0.8 and 2.0 ppm for the 1H NMR and 0 to 56 ppm for the 13C NMR spectra, a set of chemical shifts characteristic of carbon chains is noted; however, it is not possible to identify these compounds. In addition, no chemical deformations above 140 ppm were observed, which is typical of triterpenoids with alkene groups, such

Figure 2. 1H NMR spectrum of Protium heptaphylum resin.

2.2.5 Optical microscopy The micrographs of the fracture area in the specimens were observed using a digital optical microscope model U1000X with a magnification of 1000×.

3. Results and Discussions

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Almeida, M. D. C., Silva, J. A. P., Tavares, F. F. C., Araujo, L. L., Zeferino, J. S., & Santana, R. M. C.

Figure 3. 13C NMR spectrum of Protium heptaphyllum resin.

The DSC results of the Protium heptaphyllum resin, linseed oil and bioadhesive are shown in Figure 5. Linseed oil showed an exothermic peak between 75 and 200 ºC, with a maximum close to 150 ºC. Lazzari and Chiantore[21] demonstrated that this exothermic peak, in the range of 100 to 200 ºC, is a result of the formation of peroxides that react with conjugated double bonds characteristic of oils that have undergone some type of treatment or prepolymerization. Thus, it is inferred that the commercial linseed oil used to prepare the bioadhesive had already been oxidized to some level, probably due to exposure to the environment. The oxidation of triglycerides, using transition metals, such as cobalt, as catalysts, occurs from the hydrogen abstraction of methylene groups present in the unsaturation. The cobalt catalyst provides both the formation of hydroperoxides and their decomposition due to the formation of stable ions. The combination of these radicals initiates the crosslinking reaction[22]. Other possible termination reactions described by Charamzová et al.[23] suggest that alkoxide groups, formed from hydroxyl groups (major components of Protium heptaphyllum resin), can be combined with radicals from unsaturations.

Figure 4. TGA (a) and DTG (b) curves for resin, adhesive and linseed oil.

and third and fourth additional peaks are identified for the bioadhesive. The third (shoulder) and fourth thermal events were observed only for the bioadhesive. They occurred in the temperature range of 435 to 498 ºC for the third peak (Δm2 = 3.36%) and from 498 to 607 ºC for the fourth peak (Δm2 = 2.84%). These peaks may be the decomposition from macromolecules or oligomers formed from the crosslinking of the Protium heptaphyllum resin with linseed oil, since they have Tmax higher than the crude resin and linseed oil. 4/8

The resin presented two endothermic events, compatible with the results reported by Vieira Junior et al.[17] for Protium heptaphyllum resin. The first one appears close to 52 ºC, with an enthalpy of 3.3 J/g, and is associated with the softening of the material. The second one arises in the range of 100 to 160 ºC and is more intense, with an enthalpy of 24.89 J/g. This is attributed to the loss of volatile materials, such as water or solvent, which are likely to be residues from the purification process. For the adhesive, the DSC curve exhibited two exothermic events, with the first one occurring in the range of 63 to 126 ºC, with an enthalpy of 15.32 J/g, and the second one in the range of 126 to 190 ºC with an enthalpy of 7.50 J/g. Both events may indicate a two-step curing of the adhesive, in which in the first step occurs via the oxidation of the linseed oil or reactions of OH groups from resin with unsaturated groups of linseed oil, while the second step occurs with crosslinking between the linseed Polímeros, 31(2), e2021014, 2021

Development of a bio-based adhesive from Protium heptaphyllum resin Table 1. Main thermal events in the samples. Samples

(ºC) Resin Linseed Bioadhesive

Thermal events

T 5%

155 331 222

1º

∆m wt.% 9.28 100 4.10

2º Tmax (ºC) 152 404 129

∆m wt. % 90.65 89.48

Figure 5. DSC curves for samples of Protium heptaphyllum resin, linseed oil and bioadhesive.

3° Tmax (ºC) 357 341

∆m wt.% 3.36

Ash

4º Tmax

(°C) 457

∆m wt.% 2.84

Tmax

(°C) 556

(%) 0.07 0.00 0.22

Figure 6. DSC curves for bioadhesive samples at 0, 24 and 48 h.

oil chains or between the linseed oil and components of the Protium heptaphyllum resin (amyrins and diols). To assess whether the exothermic event observed for the bioadhesive in Figure 5 is from curing, thermograms from DSC were evaluated at 0, 24 and 48 h of curing. The results are exhibited in Figure 6. In general, the curves also showed two exothermic events and an evolution in the energy released was observed. The first event (70 to 125 °C) released 5.22 J/g at 0 h and 8.60 J/g at 48 h. Similarly, the second exothermic event (125 to 190ºC) increased from 4.80 J/g at 0 h to 8.61 J/g at 48 h. Thus, the fact that this energy increases with the elapsed curing time of the bioadhesive is an indication that chemical bonds are formed (crosslinking), since this behavior is characteristic of a curing process. From comparing the resin and bioadhesive spectra, Figure 7, the main difference is the decrease in the intensity of the bands at 3430 and 3100 cm-1, corresponding to the OH groups of the resin components (amyrins, brein and maniladiol) and the =C-H groups of the linseed oil, respectively. The reduction of the OH band is possibly a consequence of the reaction of OH with double bonds through an oxidation mechanism[23]. The =C-H band is indicative of the crosslinking of the resin with linseed oil, since the bioadhesive does not show this band. Such processes can be correlated with the DSC results, in which a double-step curing was observed.

3.2 Adhesion strength in wood and steel The adhesion strength of the bioadhesive was evaluated through shear stress assays in carbon steel and wood substrates. In addition, a commercial adhesive (polychloroprene) was used Polímeros, 31(2), e2021014, 2021

Figure 7. FTIR spectra for resin, adhesive and linseed oil.

for reference. Figure 8 shows the results of the mechanical shear stress tests and Table 2 the ANOVA of the values obtained. Comparing the bioadhesive and polychloroprene, it is observed that the adhesion strength of the first one is 3 times the second one for the wooden substrate, and 2 times for the metallic substrate. In terms of performance between substrates, the difference was significantly higher, being the shear stress of the wooden substrate 67 times of the metallic substrate for the bioadhesive, and 47 times for the polychloroprene.This result is related to the greater affinity of the resin with the wood material, which has surface groups that facilitate physical interactions and a roughness that favors mechanical anchoring. Besides, the penetration of the 5/8

Almeida, M. D. C., Silva, J. A. P., Tavares, F. F. C., Araujo, L. L., Zeferino, J. S., & Santana, R. M. C. Table 2. ANOVA results for substrates. Sum of Substrate Factor squares Metal

Wood

Mean square 13

Between groups

13018

Within groups

10489

Between 85.6104 groups

Within 8.60465 groups

P-valor

14.89 0.00272

85.6104 109.4

4.702.10-7

0.78224

DF= degrees of freedom; F= value from the F distribution; P-valor= probability of significance.

Figure 8. Comparative results of shear stress of samples evaluated on steel and wood substrates.

adhesive into the pores of the substrate (absorption effect) provides greater resistance, and this effect appears for the wood but is absent for the metallic substrate[24].The values of tensile shear stress are much higher than those obtained by Vieira et al.[11], who employed crude Protium heptaphyllum resin as an adhesive and reached only 0.14 MPa of shear stress using wood as a substrate. This better performance is mainly attributed to the crosslinking with linseed oil. In addition, the linseed oil acts as an internal lubricant, providing gains in deformation and eliminating the problem of brittleness reported by Vieira et al.[11]. Compared with the polychloroprene adhesive, the bioadhesive of Protium heptaphyllum was superior, as evidenced by the higher average values of shear stress for both substrates that were statistically different (p<0.05) according to ANOVA (Table 2). The difference in performance was more visible for the wood substrate, since the bioadhesive was three times higher than the polychloroprene. Figure 9 shows micrographs of the fractured area of samples from adhesion strength tests for both substrates. According to Wei et al.[25], there are three different failures that can occur in adhesives: adhesive failure, cohesive failure and mixing failure. In this work, different failures were observed in the tested substrates. For the metallic substrate, there were areas where the adhesive completely detached from the substrate (delimited area), characterizing adhesive failure and parts where the contact area still had adhesive, showing a cohesive fracture. The mechanical interlocking, adsorption, and chemical bonding are the three primary mechanisms that control the adhesion[26]. Besides, the roughness of the metallic surface is another decisive factor for the interaction[27]. Therefore, one reason that may have contributed to the adhesive having presented

Figure 9. Micrography of the fractured surface area of the adhesion strength specimen tests of the Protium heptaphyllum adhesive, realized in carbon steel (a) and wood as substrates (b). 6/8

Polímeros, 31(2), e2021014, 2021

Development of a bio-based adhesive from Protium heptaphyllum resin adhesive failure is the fact that the substrate has undergone no kind of pre-treatment to increase adhesion. For the wood adherent, cohesive failure is perceived in regions of the bonding area with part of the adherent fibers (Figure 9b), which for ASTM D5573-99[28] standard can be defined as Light-Fiber-Tear failure (LFT failure). In both cases, the occurrence of cohesion failures may be related to a slight reticulation of the bioadhesive[29], which did not form a consistent interlacing among the molecules that are required to hinder an early cohesion fracture during the shearing of the bioadhesive. On the other hand, it indicates that the Protium heptaphyllum adhesive has a fixation to the substrate stronger than the internal resistance (cohesion), which is required for adherence behavior[25].

4. Conclusions A bioadhesive was prepared from the crosslinking of Protium heptaphyllum resin with linseed oil using cobalt octanoate as a catalyst. Through 1H and 13C NMR spectroscopy analyzes it was confirmed the main chemical structures of the Protium heptaphyllum resin. From the thermal analyzes it was identified a curing procress, which was additionally noted in the FTIR spectra. The mechanical assays results demonstrated that the bioadhesive prepared has greater mechanical properties than the commercial material (polychloroprene) used as reference. It is highlighted that the adhesion strength of the bioadhesive was three times that of the commercial adhesive used as reference for the wood substrate. Finally, taking in to account the good results obtained for the wood substrate, we consider that the bioadhesive prepared from the Protium heptaphyllum adhesive has potential to be applied in the production of wood-based composites.

5. Acknowledgements The authors would like to thank the technicians, scholarship and professors responsible for the Polymeric Materials Laboratory-LAPOL of the Federal University of Rio Grande do Sul of PPGE3M.

6. References 1. Conner, A. H., & Bhuyan, M. S. H. (2017). Wood: adhesives. Reference Module in Materials Science and Materials Engineering, 1-17. https://doi.org/10.1016/b978-0-12-803581-8.01932-9. 2. Norström, E., Fogelström, L., Nordqvist, P., Khabbaz, F., & Malmström, E. (2015). Xylan - a green binder for wood adhesives. European Polymer Journal, 67, 483-493. http:// dx.doi.org/10.1016/j.eurpolymj.2015.02.021. 3. Sahoo, S. K., Khandelwal, V., & Manik, G. (2019). Synthesis and characterization of low viscous and highly acrylated epoxidized methyl ester based green adhesives derived from linseed oil. International Journal of Adhesion and Adhesives, 89, 174-177. http://dx.doi.org/10.1016/j.ijadhadh.2019.01.007. 4. Mo, J., Wang, F., Xu, Z., Feng, C., Fang, Y., Tang, X., & Shen, X. (2019). Characterization and performance of soybean protein modified by tyrosinase. International Journal of Adhesion and Adhesives, 92, 111-118. http://dx.doi.org/10.1016/j. ijadhadh.2019.04.013. Polímeros, 31(2), e2021014, 2021

5. Pang, H., Zhao, S., Wang, Z., Zhang, W., Zhang, S., & Li, J. (2020). Development of soy protein-based adhesive with high water resistance and bonding strength by waterborne epoxy crosslinking strategy. International Journal of Adhesion and Adhesives, 100, 102600. http://dx.doi.org/10.1016/j. ijadhadh.2020.102600. 6. Moghadam, P. N., Yarmohamadi, M., Hasanzadeh, M., & Nuri, S. (2016). Preparation of polyurethane wood adhesives by polyols formulated with polyester polyols based on castor oil. International Journal of Adhesion and Adhesives, 68, 273282. http://dx.doi.org/10.1016/j.ijadhadh.2016.04.004. 7. Oliveira, P. R., May, M., Panzera, T. H., Scarpa, F., & Hiermaier, S. (2020). Reinforced biobased adhesive for eco-friendly sandwich panels. International Journal of Adhesion and Adhesives, 98, 102550. http://dx.doi.org/10.1016/j.ijadhadh.2020.102550. 8. Ji, X., Li, B., Yuan, B., & Guo, M. (2017). Preparation and characterizations of a chitosan-based medium-density fiberboard adhesive with high bonding strength and water resistance. Carbohydrate Polymers, 176, 273-280. http:// dx.doi.org/10.1016/j.carbpol.2017.08.100. PMid:28927608. 9. Ji, X., & Guo, M. (2018). Preparation and properties of a chitosan-lignin wood adhesive. International Journal of Adhesion and Adhesives, 82, 8-13. http://dx.doi.org/10.1016/j. ijadhadh.2017.12.005. 10. Vargas Villanueva, J. G., Sarmiento Huertas, P. A., Galan, F. S., Esteban Rueda, R. J., Briceño Triana, J. C., & Casas Rodriguez, J. P. (2019). Bio-adhesion evaluation of a chitosan-based bone bio-adhesive. International Journal of Adhesion and Adhesives, 92, 80-88. http://dx.doi.org/10.1016/j.ijadhadh.2019.04.009. 11. Vieira, R. K., Vieira, A. K., Kim, J. T., & Netravali, A. N. (2014). Characterization of Amazonic White Pitch (Protium heptaphyllum) for potential use as ‘green’ adhesive. Journal of Adhesion Science and Technology, 28(10), 963-974. http:// dx.doi.org/10.1080/01694243.2014.880220. 12. Bandeira, P. N., Deusdênia, O., Pessoa, L., Teresa, M., Trevisan, S., & Gomes, L. (2002). Secondary metabolites of Protium heptaphyllum march. Quimica Nova, 25(6b), 1078-1080. http:// dx.doi.org/10.1590/S0100-40422002000700006. 13. Addis, C. C., Koh, R. S., & Gordon, M. B. (2020). Preparation and characterization of a bio-based polymeric wood adhesive derived from linseed oil. International Journal of Adhesion and Adhesives, 102, 102655. http://dx.doi.org/10.1016/j. ijadhadh.2020.102655. 14. Juita, Dlugogorski, B. Z., Kennedy, E. M., & Mackie, J. C. (2011). Oxidation reactions and spontaneous ignition of linseed oil. Proceedings of the Combustion Institute, 33(2), 2625-2632. http://dx.doi.org/10.1016/j.proci.2010.06.096. 15. American Society for Testing and Materials (2019). ASTM D 1002-10: Standard Test Method for Apparent Shear Strength of Single-Lap-Joint Adhesively Bonded Metal Specimens by Tension Loading (Metal-To-Metal). West Conshohocken: ASTM. 16. American Society for Testing and Materials (2017). ASTM D 906: Standard Test Method for Strength Properties of Adhesives in Plywood Type Construction in Shear by Tension Loading. West Conshohocken: ASTM. 17. Vieira, G. M., Jr., Souza, C. M., & Chaves, M. H. (2005). The Protium heptaphyllum resin: isolation, structural characterization and evaluation of thermal properties. Quimica Nova, 28(2), 183-187. http://dx.doi.org/10.1590/S0100-40422005000200003. 18. Vásquez, L. H., Palazon, J., & Navarro-Ocaña, A. (2012). The pentacyclic triterpenes e α, β-amyrins: a review of sources and biological activities. In: L. H. Vázquez (Ed.), Phytochemicals: a global perspective of their role in nutrition and health (pp. 487-502). United States: IntechOpen. 7/8

Almeida, M. D. C., Silva, J. A. P., Tavares, F. F. C., Araujo, L. L., Zeferino, J. S., & Santana, R. M. C. 19. Maia, J. G., & Zoghbi, M. G. B. (1998). Óleos essenciais da Amazônia: inventário da flora aromática. In: L. G. J. Farias & C. M. L. Costa. Tópicos especiais de produtos naturais (pp. 147-162). Brasil: POEMA. 20. da Silva Júnior, W. F., Pinheiro, J. G. O., Moreira, C. D. L. F. A., Rüdiger, A. L., Barbosa, E. G., Lima, E. S., da Veiga Júnior, V. F., da Silva Júnior, A. A., Aragão, C. F. S., & de Lima, Á. A. N. (2017). Thermal behavior and thermal degradation kinetic parameters of triterpene a, b amyrin. Journal of Thermal Analysis and Calorimetry, 127(2), 1757-1766. http://dx.doi. org/10.1007/s10973-016-6046-x. 21. Lazzari, M., & Chiantore, O. (1999). Drying and oxidative degradation of linseed oil. Polymer Degradation & Stability, 65(2), 303-313. http://dx.doi.org/10.1016/S0141-3910(99)000208. 22. Lima, G. E. S., Nunes, E. V., Dantas, R. C., Simone, C. A., Meneghetti, M. R., & Meneghetti, S. M. P. (2018). Catalytic behaviors of coiI and MnII compounds bearing α-Diimine ligands for oxidative polymerization or drying oils. Journal of the Brazilian Chemical Society, 29(2), 412-418. http://dx.doi. org/10.21577/0103-5053.20170155. 23. Charamzová, I., Vinklárek, J., Kalenda, P., & Honzícek, J. (2018). Application of oxovanadium complex stabilized by N,N,N,N-chelating ligand in air-drying paints. Coatings, 8(6), 204. http://dx.doi.org/10.3390/coatings8060204. 24. Gardner, D. J., Blumentritt, M., Wang, L., & Yildirim, N. (2015). Adhesion theories in wood adhesive bonding. In K.

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L. Mittal (Ed.), Progress in adhesion and adhesives (pp. 125-168). United States: Scrivener Publishing. http://dx.doi. org/10.1002/9781119162346.ch5. 25. Wei, H., Xia, J., Zhou, W., Zhou, L., Hussain, G., Li, Q., & Ostrikov, K. (2020). Adhesion and cohesion of epoxy-based industrial composite coatings. Composites. Part B, Engineering, 193, 108035. http://dx.doi.org/10.1016/j.compositesb.2020.108035. 26. Baldan, A. (2012). Adhesion phenomena in bonded joints. International Journal of Adhesion and Adhesives, 38, 95-116. http://dx.doi.org/10.1016/j.ijadhadh.2012.04.007. 27. Chen, P., Wang, Y., Li, J., Wang, H., & Zhang, L. (2018). Adhesion and erosion properties of epoxy resin composite coatings reinforced with fly ash cenospheres and short glass fibers. Progress in Organic Coatings, 125, 489-499. http:// dx.doi.org/10.1016/j.porgcoat.2018.09.029. 28. American Society for Testing and Materials – ASTM. (2019). ASTM D5573-99: standard practice for classifying failure modes in fiber-reinforced-plastic (FPR). West Conshohocken: ASTM. 29. Silva, L. F. M., Ochsner, A., & Adams, R. D. (2011). Handbook of adhesion technology. Berlin: Springer. http://dx.doi. org/10.1007/978-3-642-01169-6. Received: Dec. 21, 2020 Revised: Apr. 24, 2021 Accepted: May 17, 2021

Polímeros, 31(2), e2021014, 2021

ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.04221

Determination of antioxidant and antimicrobial activity of sweetgum (Liquidambar orientalis) leaf, a medicinal plant Hatice Ulusoy1 , Şule Ceylan2  and Hüseyin Peker2*  Department of Forestry, University of Muğla Sıtkı Koçman, Muğla, Turkey Department of Forest Industry Engineering, University of Artvin Çoruh, Artvin, Turkey 1

*peker100@hotmail.com

Abstract In the study, sweetgum tree (Liquidambar orientalis), which is an endemic species that grows in Mugla, Köyceğiz and is applied for medicinal purposes among the public, its leaves was examined. The antioxidant ability of the extract obtained from dried plant leaves has been evaluated using a variety of methods which are Total Phenolic Substance, Total Flavonoid, FRAP, CUPRAC, DPPH, and ABTS+. Simultaneously, the antimicrobial activity of the plant extract was examined using disk diffusion and microdilution methods to determine the minimum inhibitor concentration (MIC). While the total phenolic content of Liquidambar orientalis extract was 96.34 mg GAE/g, the total amount of flavonoid was 2.15 mg QE/g. When the results of the antioxidant analysis were examined, it was observed that it had a good level of antioxidant activity with the results of 49.25 ± 0.54 mmol TEAC/g according to the CUPRAC method, 39.83 ± 0.25 µmol Fe/g according to the FRAP method, 80.34 μg/mL according to the DPPH method and 51.20 μg/mL according to the ABTS+ method. As a result of the antimicrobial analysis, it was indicated that L. orientalis extract was more effective on Staphylococcus aureus (S. aureus), which is a gram-positive bacterium and causes a wide variety of clinical diseases. Even, L. orientalis extract with an MIC value of 10 mg/mL has been found to have a higher antibacterial effect than Amoxicillin+Clavulanic acid, which is used as a standard drug in that field. This research is significant because it is the first to report the determination of all biological activities for L. orientalis, including total polyphenols, flavonoid contents, antioxidant content, and antimicrobial activity. Keywords: sweetgum tree, diary tree, medicinal plant, antioxidant, antimicrobial. How to cite: Ulusoy, H., Ceylan, Ş., & Peker, H. (2021). Determination of antioxidant and antimicrobial activity of sweetgum (Liquidambar orientalis) leaf, a medicinal plant. Polímeros: Ciência e Tecnologia, 31(2), e2021015. https:// doi.org/10.1590/0104-1428.04221

1. Introduction From past to present, people have benefited from plants for nutrition, shelter, heating, healing wounds and curing diseases. It has been determined that there are 250 plants that people used in treatments in the 5000s B.C. Hittites, Egyptians, Sumerians, Assyrians and Mesopotamians have used herbs for years.The introduction of synthetic drugs into production over time has led to a decrease in the use of medicinal and aromatic plants. However, after the 1900s, when people discovered the side effects of synthetic drugs and became aware of the harmful effects of synthetic substances in food and beverages on human health, the demand for natural products increased[1]. Many medicinal plants have been discovered by humankind by trial and error. In Turkey, medicinal aromatic plants are commonly used in daily life to treat a variety of diseases. Turkey is an attractive source of medicinal plants because of its diverse flora and attention to a variety[2]. It is known that plant extracts and components exhibit important biological activities, especially antimicrobial[3], antifungal[4], antibacterial[5], and antioxidant activities[6]. Most medicines

Polímeros, 31(2), e2021015, 2021

are now manufactured pharmacologically, with herbal origins accounting for 25% of them. Free radicals are generated throughout the body by the toxic air we breathe during the day, poisonous compounds in spoiled foods, additives, unconscious eating, and inactivity. Oxygen atoms broken off by these harmful effects from outside circulate freely in the body, breaking down hydrogen atoms and causing tissue damage. Free radicals especially attack the cell and immune system. Molecules that minimize and block the effect of free radicals in the body and prevent chain reactions that may cause many diseases and premature aging are called “antioxidant” substances[7]. Prevention of free radical-mediated reactions that lead to difficult-to-treat problems such as aging, cancer, and diabetes is only possible with the help of antioxidant compounds[8]. As is known, antioxidants are mostly found in green and red-leaved plants. At the same time, vitamins A, C, and E show natural antioxidant properties[7]. Due to the increase of microorganisms with multiple antibiotic resistance in recent years, the treatment of the infection caused by these microbes has become increasingly

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O O O O O O O O O O O O O O O O

Ulusoy, H., Ceylan, Ş., & Peker, H. intractable. Drug resistance increases and spreads in bacteria that develop resistance to all known antibiotics. As a result, using medicinal plants as an alternative to medications is recommended, and some popular herbs are used as antimicrobials[9]. Many phytochemical compounds are found in vegetable-derived products, and they have been shown to have high antioxidant and antimicrobial properties in the literature[10]. Plant extracts and essential oils have been studied extensively for their antioxidant and antimicrobial properties[11]. Many phytochemical compounds are found in vegetablesourced products, and they have been shown to have high antioxidant properties as well as good antimicrobial activity in the literature[10]. There are many studies on the antioxidant and antimicrobial properties of plant extracts and essential oils[11]. There have been studies that show the sweetgum tree, which is an endemic species to the Muğla area, has medicinal properties. Liquidambar orientalis, known locally as the sweetgum tree, and its products (leaves, bark, sweetgum oil, etc.) have the natural protective potential[12-14]. Liquidambar orientalis[15], one of the four sweetgum tree species present in Turkey today, has been around for about 60 million years. People living in the province of Muğla and its surroundings apply sweet gum trees for shortness of breath, bronchitis, and so on. It is also applied as incense because it helps the healing process of respiratory system diseases. In addition, the essential oil extracted from sweetgum trees is used in the formulation of many natural perfumes, as well as in the soap industry and in the fragrance of gum and tobacco. It is also claimed that it is used in the form of pomade and patch against skin diseases such as scabies, fungus, stomach, and duodenal ulcers, as well as being a healthy antiseptic and parasitic[12-14]. In light of this knowledge, the antioxidant and antimicrobial properties of the leaf of the sweetgum tree (L. orientalis), which plays an important role as a medicinal aromatic plant, were investigated in this research.

2. Materials and Methods 2.1 Material (chemicals/plant) 2,4,6-tripyridyl-s-triazine(TPTZ), Folin-Ciocalteu’s phenol reagent, Methanol, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid), 2,2’-azinobis (3-ethyl-beothiazoline 6 sulfonate) (ABTS) were acquired from Sigma Chemical Co. (St. Louis, MO, USA). Neocuproine (2,9-dimethyl1,10-phenanthroline), acetic acid, ammonium acetate, aluminium nitrate nonahydrate, potasyum persülfat (K2S208), and sodium carbonate, were bought from Merck Chemical Co. (Darmstadt, Germany). The chemicals were analytical degrees. The subject of the study is L. orientalis (sweetgum) leaves and the herbal material was obtained from MuğlaKöyceğiz. L. Orientalis plant was authenticated by Prof. Dr. Temel Göktürk, a forest engineer. The dried sweetgum leaves are powdered. Powdered sweetgum leaves were extracted by brewing method with distilled hot water (80 °C). After the mixture had cooled, filtration was carried out with Whatman grade 1 filter paper. The obtained filtrate was then dried under vacuum with a freeze-dryer (lyophilizer) system, 2/7

model Christ Freeze-Dryer Alpha 1-4 LD, and the crude extract was obtained. Solutions of different concentrations of the extract obtained were prepared and biological activity analyzes such as antioxidant and antimicrobial were tested.

2.2 Total phenolic assay The total phenolic amount of samples was detected by using the Folin-Ciocalteu test[16]. Gallic acid (1; 0.5; 0.25; 0.125; 0.0625 and 0.03125 mg/mL) was used as a standard in this work. Shortly, 400 µL of 0.5 N Folin-Ciocalteu tests, 20 µL methanolic plants (1 mg/mL), 680 µL of distilled water, and 20 µL of different concentrations of gallic acid were mixed and the mixture was vortexed. 400 µL of Na2CO3 (10%) solution was added after 3-minute incubation and again vortexed. Then the mixture was incubated for 2 hours. Following the incubation time at room temperature, absorbances of the mixtures were determined at 760 nm. The concentrations of total phenolic compounds were measured as mg of gallic acid equivalents per g of the dry weight of the sample.

2.3 Total flavonoid assay The total flavonoid amount was determined by using the aluminum chloride test[17]. Quercetin was used as a standard. 4.3 mL methanol, 0.1 mL 1 M NH4CH3COO, 0.5 mL of Quercetin (1; 0.5; 0.25; 0.125; 0.0625 and 0.03125 mg/mL), and 0.1 mL 10% Al(NO3)3 were put in the tubes and then they were mixed. Mixtures were incubated for 40 minutes. Following incubation, absorbance was determined at 415 nm. The total flavonoid contents of plants were defined as mg quercetin equivalents per g of dry weight sample.

2.4 The determination of antioxidant activity The antioxidant activities of the samples were determined using by FRAP and CUPRAC methods. The FRAP method was used for the determination of total antioxidant capacity, based on the reduction of yellow Fe3+-TPTZ complex to the blue Fe2+-TPTZ complex by electron-donating substance under acidic conditions[18]. The 3 mL of FRAP reagent (containing TPTZ, FeCl3, and acetate buffer) and 100 µL of the test sample or the blank (solvents used for extraction) were added to the test tube and mixed. Maximum absorbance values at 593 nm were recorded for 4 min at 25 °C. The final absorbance was compared with the standard curve (100-1000 µmol/L). The data were expressed as µmol FeSO4.7H2O equivalents per gram of dry matter. The CUPRAC method is comprised of mixing the antioxidant solution (directly or after acid hydrolysis) with a copper (II) chloride solution, a neocuproine alcoholic solution, and an ammonium acetate aqueous buffer at pH 7, and subsequently measuring the developed absorbance at 450 nm after 60 minutes[19] 1 mL 10 mM CuCl2, 1 mL 7.5 mM Neocuproine and 1 mL 1M NH4Ac were added to test tubes, then 0.2 mL sample and 0.9 mL H2O were added and mixed. The final volume was 4.1 mL. Then, the final absorbance was measured at 450 nm after incubated 1 h. The test results were evaluated by Trolox® equivalent antioxidant capacity (TEAC). The scavenging activity of DPPH• radical was determined using the method of Molyneux[20]. Different concentrations Polímeros, 31(2), e2021015, 2021

Determination of antioxidant and antimicrobial activity of sweetgum (Liquidambar orientalis) leaf, a medicinal plant of 0.75 mL of sample extracts were mixed with 0.75 mL of a 0.1 mM of DPPH• solution (dissolved in methanol). Then, extracts were incubated at room temperature in the dark for 50 min. Absorbance was measured by a spectrophotometer at 517 nm. Trolox is used as standards and the values are expressed as IC50 (mg sample per mL). The method developed by Re et al.[21] was applied to assess ABTS+ removal activity. This method is based on the principle that the colored ABTS•+ cation radical changes color after treatment with the extract. 5 mL ABTS•+ cation radical was prepared by mixing ABTS (7 mM) solution with 2.45 mM potassium persulfate (K2S208) solution and incubated for 16 hours in the dark and at room temperature. By diluting 1 mL of this radical solution with ethanol, the absorbance was adjusted. At concentrations ranging from 500 g to 4000 g, 4 mL of ABTS solution prepared in ethanol was applied to samples containing 1 mL of sample. As a control, 1 mL of ethanol was used. At 734 nm, radical scavenging activity was measured after a 10-minute incubation period at room temperature.

2.5 The biological materials

controls, as was sterile distilled water in which plant extract was dissolved as a negative control. All three experiments were conducted in parallel. 2.6.2 Broth microdilution test for bacteria The minimum inhibition concentration (MIC) values of the extract on S. aureus, which was used in the study and whose inhibition effect was detected by the disk diffusion method, were determined by the broth microdilution method[23]. Serial dilutions of the extract (80, 40, 20, 10, and 5 mg/mL) were performed using (Mueller Hinton Broth (Merck) with a final volume of 2 mL. Bacterial suspensions were prepared at a concentration of 106 CFU/mL (using Mac Farland No: 0.5) with turbido for each test bacteria and 20 µL of suspension was added to each test tube. Tubes were incubated at 37 ± 0.1 °C for 48 hours and the lowest concentration without bacterial growth was determined as MIC. Measurements were carried out in triplicate and the results of the antimicrobial test were compared with standard Amoxicillin+ Clavulanic acid as antibacterial agents.

All three microorganisms were used in this work. As bacteria; Escherichia coli (E. coli) ATCC 25922, Staphylococcus aureus (S. aureus) ATCC 6538P, as yeast: Candida albicans (C. albicans) ATCC 14053. All test microorganisms were got from the American Type Culture Collection (ATCC), the Faculty of Science of Muğla University, and the commercial culture collections. All microorganisms were stored at -85 °C (Ultrafreezer, New Brunswick) in 15% glycerol and protected on nutrient agar (Merck, 1.05450) and malt extract agar (Merck, 1.05398) slants at 4 °C, respectively. They were subcultured in Petri dishes before use for purity check. The microorganisms chosen for the antibacterial property studies are between significant herb and man pathogens and biofilm giving microorganisms.

3. Results and Discussions

2.6 In vitro antimicrobial activity

At the same time, total phenolic and flavonoid contents were determined for the extract. In these tests, the UV spectrophotometric method was applied. Spectrophotometric strategies are frequently utilized for the standardization of natural raw materials. Total phenolic and total flavonoid contents, FRAP and CUPRAC values are presented in Table 1 and the results shown in the tables refer to the average ± SD of three parallel measurements. IC50 values determined from DPPH and ABTS+ analyzes are given in Figure 1 and Figure 2. IC50 values were calculated from linear regression analysis (Microsoft Excel, Microsoft Corporation®, USA).

2.6.1 Disk diffusion method The disk diffusion approach was used to investigate the effects of beef extract on Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538P, and Candida albicans ATCC 14053[22]. 1000 mL of liquid cultures that had reached 0.5 McFarland normal turbidity were transferred to sterile petri dishes, along with approximately 20 mL of Mueller Hinton Agar (Merck) for bacteria and Sabouraud Dextrose Agar (Merck) for C. albicans, and planting was done using disc diffusion method. Then, disks soaked with 20 µL of extract (at 400 mg/mL concentration) were appropriately placed on the agar. Sowed petri dishes were incubated for S. aureus and E. coli for 24 hours at 37 ± 0.1 °C and for C. albicans at 37 ± 0.1 °C for 48 hours. The diameter of the inhibition zones formed around the discs was measured in mm at the end of the incubation. Bacteria, Amoxicillin+Clavulanic acid disc (Oxoid) for E. coli and S. aureus, and nystatin (Oxoid) for yeast strain C. albicans were used as positive

3.1 Extract (solution) feature There are numerous diverse antioxidants in plants, and it is very difficult to measure each antioxidant component individually. The chemical complexity of the extracts, often a mixture of dozens of compounds with different functional groups, polarity, and chemical behavior, can lead to varying results depending on the test used. Therefore, it is more informative to utilize diverse tests to assess the antioxidant potential of each test[24,25]. In this research, four fundamental strategies, CUPRAC (copper decreasing control), FRAP (ferric decreasing control), DPPH• and ABTS•+ radical scavenging activity strategies were utilized.

The total polyphenol content of L. orientalis leaf extract, which is endemic in the Muğla region, was 96.34 ± 1.75 mg GAE/g, and the total flavonoid content was calculated as 2.15 ± 0.36 mg QE/g. According to the antioxidant activity results, it was found to have 49.25 ± 0.54 mmol TEAC/g according to CUPRAC analysis and 39.83 ± 0.25 µmol Fe/g according to FRAP analysis. According to the antioxidant activity results, it was found to have 49.25 ± 0.54 mmol

Table 1. Results of phenolic contents, flavonoid contents, FRAP and CUPRAC for L. orientalis medicinal plant. Sample L. orientalis

Total phenolics (mg GAE/g) 96.34 ± 1.75

Polímeros, 31(2), e2021015, 2021

Total flavonoid (mg QE/g) 2.15 ± 0.36

CUPRAC (mmol TEAC/g) 49.25 ± 0.54

FRAP (µmol Fe/g) 39.83 ± 0.25

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Ulusoy, H., Ceylan, Ş., & Peker, H. TEAC/g according to CUPRAC analysis and 39.83 ± 0.25 µmol Fe/g according to FRAP analysis (Table 1). When DPPH and ABTS+ radical scavenging activity results are examined, it is given that it has IC50 concentrations of 80.34 µg/mL for DPPH and 51.20 µg/mL for ABTS+ (Figure 1 and Figure 2). According to these results, it is observed that ABTS+radical scavenging activity of L. orientalis leaf is higher than DPPH radical scavenging activity. In the antimicrobial activity studies of the aqueous extract tested against microorganisms in the current study, the inhibition diameters obtained according to the disk diffusion method are given Figure 3 and Table 2, and the minimum inhibition concentrations (MIC) results are presented in Table 3. 3.1.1 Staphylococcus aureus (S. aureus) ATCC 6538P When the antimicrobial activity of L. orientalis leaf extracts was evaluated in vitro against three test microorganisms known to cause some diseases in foods. According to the antimicrobial activity results obtained, It has been found,

Figure 1. The result of DPPH for L. Orientalis extract.

Figure 2. The result of ABTS+ for L. Orientalis extract.

while L. orientalis plant extract does not show any activity against E. coli, a gram-negative bacterium, and C.albicans, a yeast strain, L. orientalis plant extract shows very good activity against S. aureus, which is a gram-positive bacterium, causing various clinical diseases. While the MIC value obtained for L. orientalis has a concentration of 10 mg/mL, the MIC value of Amoxicillin+Clavulanic Acid, which is used as a standard drug in that area, is 40 mg/mL. Therefore, L. orientalis extract antibacterial activity is much higher than the standard drug used in that field. Medicinal plants are traditionally applied worldwide for the treatment of various human diseases[26]. Many of these have proven to be abundant sources of biologically active compound ingredients used as a tool to develop new pharmaceutical compounds[27]. Although the antibacterial and antioxidant activities of many plant species have been extensively investigated, the antimicrobial and antioxidant mechanism of the L. orientalis plant included in this research has not been reported in detail. The research conducted in İzmir, Turkey[28], on L. orientalis leaves obtained from Mugla like in our study, the total polyphenol amount for the leaves of the plant was found as 0.37 mg GAE/g and this value was found to be much higher as 96.34 ± 1.75 mg GAE/g in our study. Although the plant was obtained from the same region, the extraction solvent was a 70% aqueous ethanol solution in the study conducted in Izmir, while only water was used as the extraction solvent in our research. The number of phenolic compounds found in plants; solvents are affected by many factors such as temperature, extraction time, particle size, sample type, and extraction methods[29]. According to the antimicrobial activity results, while the MIC value against S. aureus was 0.4 mg/mL in the literature[28], this value is 10 mg/mL in our study. L. orientalis leaf extract has shown the highest antibacterial activity against S. aureus, which causes various diseases, both in our study and in the literature. Additionally, according to the study conducted in İzmir, ABTS+ radical removal activity was 8.009 TEAC (mmol/g sample), whereas in our study it was investigated to have antioxidant activity with a value of 51.20 µg/mL. In a research conducted by Saraç and Şen[30], DPPH radical scavenging activity was investigated to be 3.11 ± 0.024 mg/mL for the ethanol extract of L. orientalis leaf obtained from Köyceğiz, Muğla. In our study, for the extract of L. orientalis leaf in water, DPPH radical scavenging activity value was measured as 0.080 ± 0.42 mg/mL and it can be accepted as higher DPPH antioxidant activity than the literature.

Figure 3. Photos of Inhibition Zone Diameters (mm) for L. Orientalis extract. 4/7

Polímeros, 31(2), e2021015, 2021

Determination of antioxidant and antimicrobial activity of sweetgum (Liquidambar orientalis) leaf, a medicinal plant Table 2. Antimicrobial activity of L. orientalis medicinal plant. Samples

E.coli 16 -

L. orientalis Amoxicillin+Clavulanic Acid Nystatin

Zone of Inhibition Diameters (mm) S. aureus 13 20 -

C. albicans 19

Notemean, Escherichia coli (E. coli) ATCC 25922, Staphylococcus aureus (S. aureus) ATCC 6538P, as yeast: Candida albicans (C. albicans) ATCC 14053, (-): no activity of test concentrations.

Table 3. MIC values of L. orientalis medicinal plant against the bacterial strains tested. Samples L. orientalis Amoxicillin+Clavulanic Acid

Minimal Inhibition Concentration Values (mg/mL) S. aureus 10 40

In a study conducted by Sağdıç et al.[31], the antibacterial effect of the extract of the juice obtained from the wood and inner bark of the sweetgum tree in ethanol against various microorganisms was examined according to the disc infusion method and according to the result, L. orientalis secretion did not have any activity against E.coli while it has good antibacterial activity against S. aureus with its inhibition zone diameter of 14 mm. Similarly, in our research, while the extract of L. orientalis leaf in water did not show any effect against E. coli, it showed a similar amount of activity against S. aureus with its inhibition zone diameter of 13 mm. In another study[32], the antioxidant activities of L. orientalis plant extract obtained from Mugla Köyceiz using acetone, ethanol, and methanol solvents, as well as antimicrobial activities against eight test microorganisms, were investigated using the DPPH method, and it was discovered that they had strong antimicrobial and DPPH activity. It is assumed that this difference may be due to the change in extraction solvent and method used[29]. Factors such as biological activity studies, composition and amount of active ingredients available in the plant, genetics (i.e. genus, species, cultivar/genotype) and geographical areas, growth conditions of plant material, climatic factors, ripening stage, harvest time, storage condition and postharvest management are also affects[33-35]. Although there are antioxidant and antimicrobial studies on strains Liquidambar styraciflua L[36-38]. ve Liquidambar formosana[39-41], there are few reports on Liquidambar orientalis, an endemic species that we investigated. Especially studies on antioxidant activity are very limited, and only a few antioxidant activity studies have been conducted by using DPPH and ABTS+ methods. This study is significant in that it is the first study reporting the determination of both total flavonoid content and antioxidant content of L. orientalis plant leaf according to CUPRAC and FRAP methods, unlike the previous studies in the literature. In other words, it will be the first research to report the determination of all biological activities such as total polyphenol, flavonoid contents, antioxidant content, and antimicrobial activity for L. orientalis together, and will shed light on the scientists who will work on this species. Phenolic compounds are very essential and significant Polímeros, 31(2), e2021015, 2021

components of plants and the ability of phenolic compounds to scavenge radicals is due to their hydroxyl groups. Phenolic compounds can directly contribute to the antioxidative effect[42]. Flavonoids are well-known antioxidants and natural phenolic compounds. The antioxidant efficacy of plant extracts rich in flavonoids is very high in various studies[43].

4. Conclusions This study demonstrated that aqueous extracts of L. orientalis have good antioxidant and antimicrobial activity. With a MIC value of 10 mg/mL, it was found to have a higher antibacterial effect on S. aureus, a gram-positive bacterium that causes a wide variety of clinical diseases, even than Amoxicillin + Clavulanic acid, which is used as a standard drug in that field. Therefore, L. orientalis can be used as sources of natural antimicrobial agents. The antioxidant and antimicrobial properties of extracts obtained from a variety of plants are of great interest to academics as well as the food, cosmetics, and pharmaceutical industries. Since there is a growing trend to substitute synthetic preservatives with natural ones, they can be applied as natural additives. In this respect, it is very critical to work with endemic plant species and to reveal unknown bioactive properties. The results of this study indicate that L. orientalis plant leaf, which is an endemic species, contains compounds with antioxidant and antibacterial activity. Due to these activities, the leaf of this plant can be applied in the preparation of medicinal and nutritious products.

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Determination of antioxidant and antimicrobial activity of sweetgum (Liquidambar orientalis) leaf, a medicinal plant 35. Lee, J., Dossett, M., & Finn, C. E. (2012). Rubus fruit phenolic research: the good, the bad, and the confusing. Food Chemistry, 130(4), 785-796. http://dx.doi.org/10.1016/j. foodchem.2011.08.022. 36. Franco Mancarz, G. F., Pareja Lobo, A. C., Baril, M. B., Assis Franco, F., & Nakashima, T. (2016). Antimicrobial and antioxidant activity of the leaves, bark and stems of Liquidambar styraciflua L. (Altingiaceae). International Journal of Current Microbiology and Applied Sciences, 5(1), 306-317. http:// dx.doi.org/10.20546/ijcmas.2016.501.029. 37. Mancarz, G. F. F., Laba, L. C., Silva, E. C. P., Prado, M. R. M., Souza, L. M., Souza, D., Nakashima, T., & Mello, R. G. (2019). Liquidambar styraciflua L.: a new potential source for therapeutic uses. Journal of Pharmaceutical and Biomedical Analysis, 174, 422-431. http://dx.doi.org/10.1016/j. jpba.2019.06.003. PMid:31220700. 38. Cordier, W., Steenkamp, V., & Rashed, K. (2016). An evaluation of antioxidant, anticholinesterase and antimicrobial activities of Liquidambar styraciflua L. leaves. Pharmaceutical Research, 14(2), 57-63. Retrieved in 2021, May 15, from https://tphres.innovesen.co.in/an-evaluation-of-antioxidantanticholinesterase-and-antimicrobial-activities-of-liquidambarstyraciflua-l-leaves 39. Liu, Y. M., Liu, Y. M., & Li, P. X. (2009). Study on antimicrobial activities of essential oil from leaves of Liquidambar formosana

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Hance as well as its antioxidant activity. Shipin Kexue, 30(11), 134-137. Retrieved in 2021, May 15, from http://www.spkx. net.cn/EN/Y2009/V30/I11/134 40. Shang, H.-J., Li, D.-Y., Wang, W.-J., Li, Z.-L., & Hua, H.-M. (2014). Three new diterpenoids from the resin of Liquidambar formosana. Natural Product Research, 28(1), 1-6. http://dx.doi. org/10.1080/14786419.2013.825915. PMid:23962240. 41. DeCarlo, A., Zeng, T., Dosoky, N. S., Satyal, P., & Setzer, W. N. (2020). The essential oil composition and antimicrobial activity of Liquidambar formosana oleoresin. Plants, 9(7), 822. http://dx.doi.org/10.3390/plants9070822. PMid:32629822. 42. Duh, P.-D., Tu, Y.-Y., & Yen, G.-C. (1999). Antioxidant activity of water extract of harn jyur (Chyrsanthemum morifolium Ramat). Lebensmittel-Wissenschaft + Technologie, 32(5), 269-277. http://dx.doi.org/10.1006/fstl.1999.0548. 43. Cakir, A., Mavi, A., Yıldırım, A., Duru, E., Harmandar, M., & Kazaz, C. (2003). Isolation and characterization of antioxidant phenolic compounds from the aerial parts of Hypericum hyssopifolium L. by activity-guided fractionation. Journal of Ethnopharmacology, 87(1), 73-83. http://dx.doi.org/10.1016/ S0378-8741(03)00112-0. PMid:12787957. Received: May 15, 2021 Revised: June 01, 2021 Accepted: June 02, 2021

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ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.09620

Extraction and characterization of nanofibrillated cellulose from yacon plant (Smallanthus sonchifolius) stems Romaildo Santos de Sousa1* , Alan Sulato de Andrade2  and Maria Lucia Masson1  Laboratório de Tecnologia de Alimentos, Departamento de Engenharia Química, Universidade Federal do Paraná – UFPR, Curitiba, PR, Brasil 2 Laboratório Química da Madeira, Departamento de Engenharia Florestal, Universidade Federal do Paraná – UFPR, Curitiba, PR, Brasil

*romaildosantos@gmail.com

Abstract This study aimed to evaluate the process of cellulose extraction from yacon stem using combined pulping and bleaching processes to produce nanofibrillated cellulose (NFC). First, a chemical pulping process with NaOH was applied and, subsequently, the pulp obtained was bleached. From the chemical pulp (CP) bleached, NFC was obtained by the mechanical defibrillation in a colloidal grinder. Then, chemical composition, and infrared analysis of the pulps were performed. The pulping process showed a lower amount of extractives and lignin content, as a low yield and an excessively dark pulp. The CP bleached with NaClO2 showed the best results increased whiteness of the pulp. A suspension of NFC with fibers of 5-60 nm in diameter, high crystallinity index, and thermal stability was obtained. The results are promising and demonstrate the technical feasibility of obtaining NFC from yacon stems waste which is ideal to apply to other materials of the industry. Keywords: biopolymers, bleaching, nanotechnology, chemical process, lignocellulosic biomass. How to cite: Sousa, R. S., Andrade, A. S., & Masson, M. L. (2021). Extraction and characterization of nanofibrillated cellulose from yacon plant (Smallanthus sonchifolius) stems. Polímeros: Ciência e Tecnologia, 31(2), e2021016. https:// doi.org/10.1590/0104-1428.09620

1. Introduction Yacon (Smallanthus sonchifolius) is a perennial plant native to the Andes that belongs to the Asteraceae family. The plant is adaptable to different altitudes, types of climatic conditions, and soil because it is grown both at sea level (Brazil, Germany, Japan, New Zealand, Russia, and the United States) and in the Andean mountains, which reach up to 3200 m in altitude[1,2]. Also, presents a very branched root system underground, and stems, leaves, and flowers in the aerial part plant. The stem represents the largest fraction of the aerial part of the yacon plant, about 74%, the rest is made up of leaves and flowers. According to Kamp et al.[3], the density of the yacon plantation can vary from 12500 to 30000 plants.ha−1, as it depends on the propagation method. Also, each plant has 4 to 12 stems that can reach up to 3 m in height[1,4]. It is composition includes 23.82% to 26.85% fiber, 9.73% to 11.37% protein, 9.60% to 10.23% ash and 1.98% to 2.26% lipids[5]. The researches with its stem are more scarce than its leaves and roots, and its stem is actually discarded or used as animal feed[5,6]. Nevertheless, there are reports that the young stems are used as a vegetable fresh food, in the form of celery, and dried stems used to make tea infusion along with the leaf[7]. Therefore, the yacon stem is promising biomass to be used as a raw fiber material, as it represents a considerable fraction of its chemical composition.

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The materials derived from lignocellulosic biomass have received great attention because they have a high potential as substitutes for raw material of fossil origin, due to their abundance, availability and renewability, and biodegradability[8]. As well, it is stimulated by different aspects, as its policy, laws, and international treaties. Although the use of these materials may further advance to reduce environmental impacts, it will also require properties similar or superior to those seen in conventional materials[9]. The nanofibrillated cellulose (NFC) obtained from non-wood biomass has gained the attention of several industry sectors, and have been applied in food packaging, biosensors, and drug delivery, because of its biocompatibility, biodegradability, renewability, availability, lower cost of raw material, lower weight, higher technical and mechanical strength[10-12]. The NFCs are a tangled of nanofibrils with a diameter within nanoscale dimensions – i.e. up to 100 nm – and with several length micrometers[13]. However, the choice of cellulose source and the production process has a significant impact on the quality and characteristics of NFC. To obtain NFC, the lignocellulosic biomass is submitted to pre-treatment processes such as pulping and bleaching[14,15], followed by a refinement treatment[16]. The chemical pulping process is the most employed in the industry, where the alkaline chemical process with

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O O O O O O O O O O O O O O O O

Sousa, R. S., Andrade, A. S., & Masson, M. L. sodium hydroxide (NaOH) is the most widely used and known[17]. As advantages, alkaline treatments can efficiently remove lignin, in addition to reducing the solubilization of hemicelluloses and be applied in mild temperature conditions[18]. After the alkaline pulping, the cellulose pulp has dark-colored, requiring the subsequent application of bleaching processes. Bleaching occurs when chemical agents oxidize the non-cellulosic compounds present in the pulp. Sodium hypochlorite (NaClO), hydrogen peroxide (H2O2) and sodium chlorite (NaClO2) have been used to bleach and, at the same time, promotes the delignification of cellulose pulp[17,19-21]. The fibers obtained in these processes are long, so additional refinement processes can be used to obtain NFC[22]. NFC can be isolated through various processes, one of them being the defibrillation process performed in a colloidal grinder, which has been considered an appropriate method to produce NFC in a more economically viable way[23]. To obtain smaller fibers or to produce a more uniform product, the suspension can be treated by multiple turns through the defibrillator[20,24]. Nevertheless, the characteristics of NFCs depend mainly on the type of raw material and the processes that they were submitted, consequently the investigation of their characteristics is very important. There are recent studies with different types of lignocellulosic biomass to obtain NFC[10,25], but there are no reports in the literature about the use of yacon’s agricultural waste (stem) for this purpose and application. Therefore, the objective of this study was to investigate the feasibility of using yacon stem to produce NFC from the bleached pulp. The processes’ yields were compared, as well as the effects of pulp chemical treatments on the resulting NFC characteristics. The physical, thermal, and chemical properties of NFC obtained from the yacon stem were also evaluated.

were packed in polyethylene bags, sealed, and stored in a dry and ventilated environment until the experiment was performed. The preparation of the biomass for the chemical composition analysis of the YS followed the procedures described in T257-cm02[27].

2. Materials and Methods

2.5 Extraction of nanofibrillated cellulose (NFC)

2.1 Materials

The NFC extraction was performed from the bleached pulp that presented the best results in terms of yield, color, and Klason lignin content, following the defibrillation process proposed by Iwamoto et al.[24], with some modifications. The bleached pulp was dispersed and homogenized in distilled water to a consistency of 1% in dry weight, using a food processor with 450 W of power. The pulp suspension went

The yacon plant stems were supplied by a farmer in São José dos Pinhais, Paraná, Brazil (coordinates: 25° 37’8.37” S 49° 07’15.72” W; at 882 m altitude). The reagents used in the chemical characterization of the plant and the NFC were: Absolute ethyl alcohol 99.8% (Neon©, Brazil), toluene 99.5% (Anidrol©, Brazil), sulfuric acid 98% (Sigma-Aldrich©, Brazil), sodium hydroxide 97% (Neon©, Brazil), sodium hypochlorite in 10-12% solution (Neon©, Brazil), hydrogen peroxide 35% (Neon©, Brazil), sodium chlorite 78% (Neon©, Brazil), glacial acetic acid 99.7% (Dynamics©, Brazil).

2.2 Sample preparation The stems were washed in running water and then manually cut into pieces of approximately 5 cm in length. About 12 kg of samples with 85.96% moisture were dried in a forced-air circulation oven at 40 °C for 48 hours, to reach moisture of 6.79%. The moisture levels were determined according to the T264 method of the Technical Association of the Pulp and Paper Industry, in an oven at 105 °C for 24 hours[26]. Subsequently, the dried yacon stems (YS) 2/8

2.3 Pulping process of cellulosic pulp The process to obtain the chemical pulp (CP) was performed with NaOH, following the operational conditions described by Fortunati et al.[28] with some adaptations. The YS samples were placed in containers on a dry basis proportion of 1:10 (w:v) in NaOH solution 5% and submitted to heat treatment in a laboratory autoclave (Phoenix, Brazil) at 120 °C, under pressure (98 kPa) for 1 hour, with automatic time and temperature control. Subsequently, the CP was washed and disintegrated in the disc refiner with water (1:20, w:v) at room temperature (25 °C), for 5 minutes. Finally, the CP was purified in a Brecht-Holl fiber classifier (Regmed®, model BH-6/12, Brazil) and then centrifuged (3000 rpm for 5 minutes) and stored in polyethylene bags under refrigeration (8 °C).

2.4 Bleaching processes of cellulosic pulp The CP samples were bleached as the experimental conditions described in Table 1. The bleaching treatments were applied in a single stage with a diluted solution of the NaClO (SH), H2O2 (HP), and NaClO2 (SC). A standard consistency was adopted in the proportion of 1:10 (w:v) pulp on a dry basis:solution. Briefly, the pulp was placed in a glass becker, followed by the bleaching solution and the mixture was submerged in a thermostatic bath. After treatment, the bleached pulp (CP-SH; CP-HP; CP-SC) were washed in running water, in order to eliminate possible reagent residues, then centrifuged (3000 rpm for 5 minutes) and stored in polyethylene bags under refrigeration (8 °C).

Table 1. Parameters of bleaching treatments of the chemical pulp (CP). Parameters

NaClO[21]

H2O2[22]

NaClO2[23]

Treatment encoding Solution concentration Solution pH Adjustment of solution pH Temperature Time

SH 5% 11 NaOH 4M 70 °C 60 min

HP 1% 11.5 NaOH 4M 80 °C 45 min

SC 1.7% 4.5 C2H4O2 80 °C 120 min

Source: Adapted from Balea et al. [19], Berglund et al. [20] and Cara et al.[21]. A standard consistency of 1:10 (w:v) dry weight biomass:solution was set. NaClO: Sodium hypochlorite; H2O2: Hydrogen peroxide; NaClO2: Sodium chlorite; NaOH: Sodium hydroxide; and C2H4O2: Acetic acid.

Polímeros, 31(2), e2021016, 2021

Extraction and characterization of nanofibrillated cellulose from yacon plant (Smallanthus sonchifolius) stems through the mechanical defibrillation in a colloidal grinder (Masuko Sangyo®, model MKCA6-2J, Japan) four times, at 1500 rpm, with a 0.1 mm space between the grinding stones. Subsequently, the NFC suspension was placed in polyethylene bottles and refrigerated (8 °C).

2.6 Raw fiber and pulp characterization 2.6.1 Scanning Electron Microscopy (SEM) The morphology was visualized through a scanning electron microscope (TESCAN®, VEGA3 LMU model). The samples were fixed on metal support (stub) covered with copper conductive tape and metalized with a gold thin layer. The images were captured with an acceleration voltage of 15 kV. 2.6.2 Chemical composition The chemical composition was performed in triplicate. The total extractives content was determined by standard method T204-om97[29] and Klason lignin by T222-om02[30]. The holocellulose content (HOLO), which represents the amount of cellulose and hemicellulose, was determined by difference according to the following equation: HOLO ( % ) = 100 − (Total extratives + Klasonlignin ) (1)

and heat flow of 10 °C.min-1, in a temperature range of 30 °C to 800 °C. 2.7.3 X-ray diffraction (XRD) The crystallinity index (CrI) was obtained by XRD using a diffractometer (Bruker©, D8 Venture model). The diffraction curves were obtained by Cu-Kα radiation (λ = 1.54 Å) at 40 kV and 20 mA and with diffraction intensities in a 2θ angular range (Bragg angles) from 10° to 40°. The CrI was calculated by Equation 3, where I200 and Iam represent the peak intensities near 2θ = 22° and the minimum near 18°, respectively[34]. ( I 200 − I am ) / I 200  × 100 CrI ( % ) =  

(3)

2.8 Statistical analysis The results of the experiments were subjected to variance analysis ANOVA and the means compared with the Tukey test at 5% significance level with the support of the StatSoft®, version 13.0 (USA) Statistica software. FTIR, TGA/DTG, and XRD curves were analyzed with OriginPro 8.6 (OriginLab®, Northampton, MA, USA), using the Savitzky-Golay method at 15%-point cut, which reduces possible noises coming from the equipment.

2.6.3 Yield

3. Results and Discussions

The gravimetric yields of the pulps were calculated considering the dry weight of the recovered sample (W2) and the dry weight of the initial sample (W1) according to Equation 2. The yield of the NFC was determined according to by Besbes et al.[31].

3.1 Raw fiber and pulps characterization 3.1.1 Morphological analysis

The functional groups found in the samples were identified by FTIR spectrometer (Bruker, Vertex 70 model, USA), in diffuse reflectance mode (DRIFT), and, for each sample, 512 scans were performed in the 4000 to 400 cm-1 range and with a resolution of 4 cm-1. The spectra were manipulated in Kubelka-Munk units, correcting the baseline using the concave rubber band correction method.

Figure 1 shows the SEM micrographs of yacon stem (YS) and the fibers obtained by chemical process, as well as the bleached pulps. The structure of YS is like that of other plants in the Asteraceae family, which have vascular bundles that form a porous network analogous to honeycombs with a variety of sizes in diameter[28]. The CP presented soft and clustered fibers with smooth connections, but trace residues of the YS remained on the fibers. The morphological characteristics of the CP reflected the chemical composition of the pulp, indicating the efficiency of the chemical process. In general, bleaching processes promoted a change in the pulp surface initially treated with NaOH, making it even smoother.

2.7 Characterization of nanofibrillated cellulose (NFC)

3.1.2 Chemical composition, and yields

Yield = (%)

(W2 / W1 ) × 100 (2)

2.6.4 Fourier Transform Infrared Spectroscopy (FTIR)

2.7.1 Transmission Electron Microscopy (TEM) A transmission electron microscope (JEOL©, JEM 1200EX-II model), with an accelerating voltage of 60 kV, was used to visualize the structure of the NFC. The NFC suspension was dispersed in water solution (1:1000, v:v), and a drop of this mixture was placed on a copper grid, layered with Parlodion film. The ImageJ® program determined the diameter range of cellulose fibers[32]. 2.7.2 Thermogravimetric analysis (TGA/DTG) The TGA/DTG study was performed on a thermogravimetric analyzer (PerkinElmer©, model 4000, USA) with adapting the conditions used by Xie et al.[33]. The assays were carried out under a dynamic nitrogen atmosphere of 20 mL.min-1 Polímeros, 31(2), e2021016, 2021

The YS (24.4% extractives, 14.0% lignin, and 61.5% holocellulose) was submitted to chemical pulping process, and the chemical composition of these materials is presented in Table 2. The lignin content found is lower than in tobacco (23%), sunflower (26%), corn (19%), and bamboo (23-28%) biomass[25,35,36]. This is an advantage because it makes the process of fiber extraction less strict, demanding fewer chemical reagents and time. There are no reports in the literature on the composition of the yacon stem in terms of extractives, lignin, and holocellulose, highlighting the importance and innovation of this research. The chemical process removed a significant (p<0.05) amount of the amorphous extractives of the fibers, which affected other properties, such as yield (low) and resistance to thermal degradation, further discussed. The pulping 3/8

Sousa, R. S., Andrade, A. S., & Masson, M. L.

Figure 1. Image and SEM of the yacon stem (YS), chemical pulp treated with NaOH (CP) and chemical pulp bleached with NaClO (SH), H2O2 (HP) and NaClO2 (SC). Table 2. Chemical composition, yield of pulping and effect of cellulose pulp bleaching processes. Parameters Extractives (%) Lignin (%) HOLO (%) Yield (%)

YS 24.4±0.7a 14.0±0.1a 61.5±0.7d -

CP 1.3±0.1b 2.0±0.2b 96.8±0.4c 33.3±1.4c

CP-SH 0.8±0.1bc 0.9±0.1c 98.3±0.1b 94.9±3.3a

CP-HP 0.1±0.0c 1.0±0.1c 98.9±0.1ab 95.0±2.5a

CP-SC 0.1±0.1c 0.4±0.1d 99.5±0.1a 85.9±3.0b

(-) not determined; (%) on dry basis; YS: yacon stem; CP: chemical pulp treated with NaOH; SH: NaClO-bleached pulp; HP: H2O2-bleached pulp; and SC: NaClO2-bleached pulp; HOLO: Holocellulose. Mean ± standard deviations followed by different letters in the same line denote difference according to the Tukey test (p<0.05).

process applied promoted a significant decrease in the extractives and lignin contents (Table 2). Alkaline pulping of the YS resulted in the removal of considerable amounts of extractives and lignin, 94.6% and 85.7% respectively, showing a pulp with 96.8% holocellulose, but with 33.29% yield. Subjecting the plant matrix to treatment with alkaline solutions at high temperatures causes a disturbance in the cell wall structure due to cleavage of the ester and ether bonds between lignin and hemicellulose, resulting in its solubilization[37]. This explains the significant reduction of these constituents in the matrix, on the other hand, promotes an increase in the purity of cellulose fibers. The bleaching of pulp also allows delignification, as it significantly reduced the lignin content in the pulp. The combined bleaching and hydrolysis helped with cellulose purification and isolation because of the removal of non‑cellulosic components including lignin and hemicelluloses, besides facilitates mechanical defibrillation to obtain NFC[38]. The application of more bleaching stages can be used to obtain pulps with a higher level of whiteness. The bleaching of cellulose pulp in a single stage is an advantage as it reduces costs and process time. 4/8

3.1.3 Fourier Transform Infrared Spectroscopy (FTIR) The effects of pulping and bleaching processes on the chemical composition of YS fibers were assessed by infrared readings (Figure 2). The chemical pulping process with NaOH altered the chemical structure of the fibers as seen in the chemical characterization (Table 2). All infrared spectra of the samples have a high-intensity band around 3600 cm-1 attributed to the vibration of hydroxyl bonds (-OH), a functional group present in cellulose, hemicellulose, and lignins[39]. An elongation of this band is noticeable in the YS until about 3100 cm-1, which may be related to the formation of hydrogen bonds from carboxylic and phenolic groups of the hemicellulose, lignins, and extractives structures[40]. In YS, the band between 2920-2850 cm-1 represents the vibration of the C–H bond present in cellulose, hemicellulose, and lignin. The range of 1750 to 1720 cm-1 reflects the vibration of C=O bonds, with an increase in intensity in this region (1730 cm-1), possibly due to the acetyl groups in hemicellulose[41,42]. The NaOH process, as well as the bleaching treatments, significantly reduced the lignin content of YS, observed in the Polímeros, 31(2), e2021016, 2021

Extraction and characterization of nanofibrillated cellulose from yacon plant (Smallanthus sonchifolius) stems

Figure 3. Image (a) and TEM (b) and (c) of nanofibrillated cellulose (NFC) with magnification of 15000× and 5000×, respectively.

3.2.2 Thermogravimetric analysis (TGA/DTG)

Figure 2. Infrared spectrum of the yacon stem fiber (YS), chemical pulp treated with NaOH (CP); and chemical pulp bleached with NaClO (CP-SH), H2O2 (CP-HP) and NaClO2 (CP-SC).

range of 1600 to 1500 cm-1, where there are less peaks, which are attributed to the vibration of the aromatic structure[42]. The peak at 1250 cm-1 disappears after alkaline treatment on YS fibers due to the vibration of hemicellulose’s C–O[43], corroborating the chemical composition found. Moreover, the peaks at 1170 cm-1 and 1082 cm-1 are attributed to the vibration of the C–O–C group in the pyranose ring in polysaccharides[41]. A considerable inversion of the spectrum signal occurs around 830 cm-1, attributed to the presence of carbohydrates, such as hemicelluloses[44]. Thus, among the bleached pulps and considering yields, color and residual Klason lignin content in the fibers, the NaOH-treated pulp with NaClO2 bleaching (CP-SC) offered a more satisfactory result to proceed with the extraction of NFC.

3.2 Characterization of nanofibrillated cellulose (NFC) 3.2.1 Transmission Electron Microscopy (TEM) Figure 3 gives an overview of the morphological characteristics of the NFC. In Figure 3a, the NFC suspension exhibited a gel-like viscous appearance where a non-phase separation has been verified during storage. Figure 3b and 3c shows how the process of obtaining NFC allowed the individualization of the fibers. Mechanical defibrillation in a colloidal grinder yields a highly branched and interwoven structure with fibers diameters ranging from 5 to 60 nm very smaller than their lengths, which characterizes a nanomaterial, and a yield of 92.7%. No reports were found in the literature about the production of NFC from the stem of the yacon plant. But, fibers of different lignocellulosic materials have a similar appearance and diameter to the fibers obtained[11,12,28,45]. Therefore, it was possible to obtain NFC from the yacon stems, as wished. Polímeros, 31(2), e2021016, 2021

The TGA/DTG curves of YS, CP, CP-SC, and NFC are shown in Figure 4. The constituents present in the analyzed materials exhibit three main stages of thermal degradation (Figure 4a). The first stage starts at 30 ºC and extends to 110 ºC, which is mainly caused by the loss of water mass[13]. The second stage occurs between 150 ºC and 450 ºC, possibly due to the depolymerization of cellulosic components (cellulose and hemicellulose) and due to the traces of lignin in the samples. There are considerable mass losses between 150 °C and 300 °C for YS in the second stage, which are not explicitly seen in chemical pulps and NFC. It can be attributed to the thermal decomposition of extractive materials, such as low molecular weight polysaccharides - e.g. pectic substances[46]. In the third stage, there is a small mass loss at 450 ºC, where the complete degradation of residual lignin mainly occurs[33]. The maximum thermal resistance temperature (Tmax) around 360 °C is attributed to cellulose, as hemicelluloses, as well as the other components, are considered amorphous and have a low degree of polymerization[28]. This characteristic is attractive to NFC, whose purpose is to be applied to materials in which the processing temperature is high, such as to biocomposites that may exceed 200 °C[45]. NFC obtained of source non-wood has been used as the base or auxiliary material to produce paper and board, coatings, packaging, adhesives, sensors, filters, biomedical, among others[47]. In Figure 4b, Tmax increases as YS (335 °C) undergoes alkaline pulping (360 °C) and NaClO2 (380 °C) bleaching treatments, but lowers to 368 °C with ultrafine fibrillation. This lower resistance to thermal degradation of NFC may be related to the defibrillation to which CP-SC was submitted, as this process may cause changes in the crystalline regions of cellulose[48]. This effect can be noted by the XRD analysis. 3.2.3 X-ray diffraction analysis (XRD) The processes’ effect on the crystallinity of samples can be visualized by XRD analysis (Figure 5). Similar intensity peaks were identified in all samples analyzed (YS, CP, CP‑SC, and NFC) via XRD profiles, located at diffraction angles (2θ) near 17º and 22°. Another low-intensity peak is visible in the 34º angle, more evident in the pulps and the NFC. The 5/8

Sousa, R. S., Andrade, A. S., & Masson, M. L.

Figure 4. TGA (a) and DTG (b) of yacon stem fiber (YS), the chemical pulp (CP), NaClO2- bleached chemical pulp (CP-SC) and nanofibrilated cellulose (NFC) as a function of weight loss.

and pulp bleaching increased crystallinity by 24.78% and 36.52%, respectively, in relation to the matrix (YS). Such an increase in crystallinity is related to the removal of pulp amorphous components such as extractives, hemicelluloses and lignin[33], which corroborates the results of Table 2. In addition, a high value of crystallinity means greater rigidity of the fibers and this characteristic can be beneficial for the application as reinforcement for biocomposites[26]. It was also noted that the CrI of the NFC had a slight decrease when compared to CP-SC, which may be related to the effect of the mechanical defibrillation in a colloidal grinder and may have affected the crystal structure[48]. Although the process of obtaining NFC reduces the index, the crystallinity remains high (above 70%).

4. Conclusions

Figure 5. XRD of yacon stem fiber (YS), the chemical pulp treated with NaOH (CP), NaClO2- bleached chemical pulp (CP-SC) and nanofibrillated cellulose (NFC).

samples have a typical diffraction curve of cellulose I, similar to other lignocellulosic materials[32,48,49]. The crystallinity index (CrI), that relate the crystalline phase to the amorphous phase of the material, was calculated according to Equation 3, obtaining 52.21%, 65.15%, 71.28%, and 70.60% for YS, CP, CP-SC, and NFC, respectively, showing a clear increase after the bleaching treatment. The chemical pulping process 6/8

This study was the first to characterize and use yacon plant stem biomass for nanofibrillated cellulose production. The best result obtained in terms of yield, color, and lignin content was the use of the alkaline pulping process with NaOH followed by bleaching with NaClO2. The yacon NFC obtained show high crystallinity index and thermal resistance, which demonstrate the potential application in other materials, for example in biocomposites and packaging, as well as assisting in future research in this area.

5. Acknowledgements The present study was developed with the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES) – Grant Code 001, Projeto FINEP/CT-INFRA/3080/20112011, and Centro de Microscopia Eletrônica at the Universidade Federal do Paraná (UFPR). Polímeros, 31(2), e2021016, 2021

Extraction and characterization of nanofibrillated cellulose from yacon plant (Smallanthus sonchifolius) stems

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40. Pastore, T. C. M., Oliveira, C. C. K., Rubim, J. C., & Santos, K. D. O. (2008). Effect of artificial weathering on tropical woods monitored by infrared spectroscopy (DRIFT). Química Nova, 31(8), 2071-2075. http://dx.doi.org/10.1590/S010040422008000800030. 41. Fiore, V., Scalici, T., & Valenza, A. (2014). Characterization of a new natural fiber from Arundo donax L. as potential reinforcement of polymer composites. Carbohydrate Polymers, 106(1), 77-83. http://dx.doi.org/10.1016/j.carbpol.2014.02.016. PMid:24721053. 42. Morán, J. I., Alvarez, V. A., Cyras, V. P., & Vázquez, A. (2008). Extraction of cellulose and preparation of nanocellulose from sisal fibers. Cellulose, 15(1), 149-159. http://dx.doi.org/10.1007/ s10570-007-9145-9. 43. Orue, A., Eceiza, A., & Arbelaiz, A. (2017). Pretreatments of natural fibers for polymer composite materials. In. S. Kalia (Ed.), Lignocellulosic composite materials (Springer Series on Polymer and Composite Materials, pp. 137-175). Spain: Springer. https://doi.org/10.1007/978-3-319-68696-7_3. 44. Mascarenhas, M., Dighton, J., & Arbuckle, G. A. (2000). Characterization of plant carbohydrates and changes in leaf carbohydrate chemistry due to chemical and enzymatic degradation measured by microscopic ATR FT-IR spectroscopy. Applied Spectroscopy, 54(5), 681-686. http://dx.doi. org/10.1366/0003702001950166. 45. Alemdar, A., & Sain, M. (2008). Isolation and characterization of nanofibers from agricultural residues: wheat straw and soy hulls. Bioresource Technology, 99(6), 1664-1671. http://dx.doi. org/10.1016/j.biortech.2007.04.029. PMid:17566731. 46. Sarasini, F. (2018). Mechanical and thermal properties of less common natural fibres and their composites. In. S. Kalia (Ed.), Lignocellulosic composite materials (Springer Series on Polymer and Composite Materials, pp. 177-213). Spain: Springer. http://dx.doi.org/10.1007/978-3-319-68696-7_4. 47. Dufresne, A. (2019). Nanocellulose processing properties and potential applications. Current Forestry Reports, 5(2), 76-89. http://dx.doi.org/10.1007/s40725-019-00088-1. 48. Lengowski, E. C., Magalhães, W. L. E., Nisgoski, S., Muniz, G. I. B., Satyanarayana, K. G., & Lazzarotto, M. (2016). New and improved method of investigation using thermal tools for characterization of cellulose from eucalypts pulp. Thermochimica Acta, 638, 44-51. http://dx.doi.org/10.1016/j. tca.2016.06.010. 49. Khenblouche, A., Bechki, D., Gouamid, M., Charradi, K., Segni, L., Hadjadj, M., & Boughali, S. (2019). Extraction and characterization of cellulose microfibers from Retama raetam stems. Polímeros: Ciência e Tecnologia, 29(1), e2019011. http://dx.doi.org/10.1590/0104-1428.05218. Received: Oct. 28, 2020 Revised: Feb. 09, 2021 Accepted: June 03, 2021

Polímeros, 31(2), e2021016, 2021

ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.02321

Migration of phthalates and 2,6-diisopropylnaphthalene from cellulose food packaging Leda Coltro1* , Elisabete Segantini Saron1 , Thiago Ivan Pessoa2 , Julia Morandi2  and Bruna Santos Silva1  1

Centro de Tecnologia de Embalagem, Instituto de Tecnologia de Alimentos – ITAL, Campinas, SP, Brasil 2 Instituto de Química, Universidade Estadual de Campinas – UNICAMP, Campinas, SP, Brasil *ledacolt@ital.sp.gov.br

Abstract Recycling systems are unable to remove efficiently all potential contaminants acquired along the recycling chain. Therefore, contaminants may potentially exist in recycled food packaging. The safety of recycled cellulose food-contact materials depends on the toxicity and the ability of post-consumer contaminants to be absorbed by recycled fibers released by the packaging and ultimately absorbed by the food. Furthermore, the migration of different contaminants is related to their levels of contamination, structures and chemical affinity with cellulose fibers. In this study, twenty samples of cellulose packages available in the Brazilian market were evaluated regarding migration of phthalates (dibutyl phthalate – DBP, diisobutyl phthalate – DIBP and bis(2-ethylhexyl) phthalate – DEHP) and 2,6-diisopropylnaphthalene – DIPN into fatty food simulant using GC-FID. Fifty percent of the cellulose packaging samples showed no migration of DIPN or of any phthalates evaluated, whereas 20% showed migration of DIBP, 15% migration of DBP and 40% migration of DEHP. Keywords: cellulose packaging, contaminants, phthalates, migration, recycled fibers. How to cite: Coltro, L., Saron, E. S., Pessoa, T. I., Morandi, J., & Silva, B. S. (2021). Migration of phthalates and 2, 6-diisopropylnaphthalene from cellulose food packaging. Polímeros: Ciência e Tecnologia, 31(2), e2021017. https:// doi.org/10.1590/0104-1428.02321

1. Introduction Packaging recycling is critical to circular economy. Recycling cellulose packaging (paper, cardboard and corrugated paperboard) is a traditional practice in Brazil. Since the 1990s, recycling rates have ranged from 70% to 80%. In 2019, approx. 85% of the total volume of cellulose packaging consumed in the country was recycled[1]. Due to the use of recycled packaging to manufacture food contact packaging, strict regulation of materials is required to prevent risks to food safety, since recycling systems are unable to eliminate all potential contaminants efficiently. The fibers of recycled paper and cardboard may have several contaminants due to: i) recycling of non-food grade paper and cardboard; ii) recycling of printed materials, adhesives or coatings of paper and cardboard still remaining after recycling; iii) additives used in recycling; iv) residues in the paper and cardboard remaining after these materials have been used; and v) degradation products and chemical impurities introduced in the different stages of the chain[2]. Therefore, the potential presence of contaminants in the recycled packaging exists. Such contaminants may migrate into food, thus resulting in risks to consumers[3-7]. The safety of recycled cellulose materials for use in food contact packaging depends on the toxicity and the ability of post-consumer contaminants to be absorbed by recycled fibers, then released by the packaging and ultimately absorbed by the food. Besides, migration of different contaminants

Polímeros, 31(2), e2021017, 2021

is related to their levels of contamination, structures and chemical affinity with cellulose fibers. According to EU Regulation (EC) no. 1935/2004[8], any materials intended to come into direct or indirect contact with food should not transfer substances into food in quantities which could endanger human health, cause unacceptable change in food composition or bring about deterioration in its organoleptic characteristics. Therefore, with the use of recycled paper and cardboard in food contact materials (FCM) a broader range of testing is recommended to ensure consumer health. The European Food Safety Authority – EFSA established a safe level – a group Tolerable Daily Intake (TDI) for dibutyl phthalate – DBP and bis(2-ethylhexyl) phthalate – DEHP of 0.01 and 0.05 mg per kilogram of body weight (mg kg-1 bw) per day, taking into account their effects on the reproductive system. Since the structure of diisobutyl phthalate – DIBP is comparable to DBP, its TDI was also set as 0.01 mg kg-1 bw per day. TDI is an estimate of the amount of a substance that a person can ingest daily during his/her lifetime without any significant risks to health[9]. In 2020, the European Committee for Food Contact Materials and Articles submitted a technical guide on materials and articles made of food-contact paper and cardboard to public consultation, as there are no specific requirements for these materials in Europe; however, they must comply with the national regulations of the country

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O O O O O O O O O O O O O O O O

Coltro, L., Saron, E. S., Pessoa, T. I., Morandi, J., & Silva, B. S. where the products are marketed. This technical guide is based on the regulations for paper and cardboard packaging from Germany, France, Italy and the Netherlands; it sets forth the following specific migration limits – SML: sum of 0.3 mg kg-1 for DBP and DIBP; 1.5 mg kg-1 for DEHP and absence of DIPN[2]. To ensure food safety, packaging in Brazil must comply with the regulations agreed among the MERCOSUR countries (Southern Common Market), according to Resolution RDC no. 88, of June 29th, 2016, issued by the Brazilian National Health Surveillance Agency (Anvisa) of the Brazilian Ministry of Health[10]. This Resolution includes a positive list of components for materials, packages and cellulose equipment intended to come into contact with food, specifying maximum SML for various substances, including inorganic contaminants, when the material includes recycled fibers in its production. The SML for substances evaluated in this study are the same as those adopted by the EU, i.e.: 0.3 mg kg-1 for DBP; 0.3 mg kg-1 for DIBP – taking into account that the sum of DBP and DIBP must not exceed 0.3 mg kg-1; 1.5 mg kg-1 for DEHP and it must be non-detectable for 2,6-diisopropylnaphthalene – DIPN. This Resolution does not apply to secondary packaging made of paper or cardboard, as long as it is ensured that they do not come into contact with food, do not interfere with food integrity and do not transfer unhealthy substances. Phthalates are potential contaminants that can migrate from cellulose packaging into food. Some studies have shown that phthalates cause genetic changes in mice, and DEHP is an endocrine disruptor that can trigger toxic and adverse effects, particularly in animal or human tissues and organs, such as the pituitary gland, liver or testicles[11-13]. However, these additives are often unintentionally incorporated due to the possibility and permission to use recycled pulps[14]. They are a group of organic lipophilic chemicals used primarily as plasticizers to increase flexibility of polymer products and may be found in printing inks, lacquers and adhesives of such packages. DBP, DIBP and DEHP are some of these phthalates that can migrate into food[5]. Contamination may take place when the unprinted and the printed surfaces of the packaging touch when the cellulose material is wound or stacked and stored. As a consequence, printing components can migrate into food. This is known as set-off effect[15,16]. DIPN is mostly found in packaging that has been produced from recycled office papers, since it is used as solvent for dyes in carbonless copy paper in substitution to polychlorinated biphenyls. It can migrate into food through direct contact or even through gaseous transport[5,17]. After several analyses at different concentrations, Zhang, Noonan and Begley observed that at concentrations higher than 20 mg kg-1 DIPN migration into food products is detected[3]. According to estimation made by Coltro and Machado based on analyses of samples contaminated with different levels of DIPN, 12 mg kg-1 is the maximum concentration of DIPN in cellulose packaging to reach the specific migration limit of 0.01 mg kg-1 or to be detected[18]. Geueke and Muncke conducted a systematic literature review of studies that detected phthalate migration into food or food simulants[4]. Among the studies listed by Geueke and Muncke there is a study, from 2012, on migration 2/8

of 33 food packaging items (11 materials) into various food simulants (water, 3% acetic acid or 15% ethanol) in which the authors identified migration of DEHP between limit of detection – LOD and 17.7 µg L-1 (55% detection frequency), DBP between LOD and 1.95 µg L-1 (33% detection frequency) and benzyl butyl phthalate – BBP between LOD and 0.355 µg L-1 (36% detection frequency). Another study, from 2013, on 17 samples of recycled paper and cardboard, the authors quantified DEHP migration in 12 samples, with values between 0.97 and 66.3 mg kg-1. In a study from 2016, conducted with 19 paper cups, the authors analyzed extraction and migration of phthalates into different food simulants. The results showed that all samples contained DEHP (from 0.45 to 58.6 mg kg-1) and DBP (from 0.07 to 3.14 mg kg-1), while BBP was measured in two samples. DBP migrated into 20% and 50% ethanol, 4% acetic acid and n-heptane, but not into water and DEHP migrated only into n-heptane. Graiño et al. analyzed potential migrants in paper-based candy wrappers[19]. The authors analyzed seven samples of paper-based candy wrappers and two samples of popcorn paper packaging. Among the 28 compounds identified in the packaging samples, four phthalates were found: diethyl phthalate – DEP (eight samples), DIBP (eight samples), BBP (three samples) and DEHP (all samples). Therefore, this study aims to assess migration of DIPN and DBP, DIBP and DEHP phthalates from cellulose food packaging commonly available in the Brazilian market into fatty food simulant (n-heptane) to assess the exposure of Brazilians to phthalates and DIPN.

2. Materials and Methods 2.1 Reagents The following reagents were used in this study: 2,6 - diisopropylnaphthalene – DIPN, CAS number 24157‑81-1 (Sigma – Aldrich, 99%); dibutyl phthalate – DBP, CAS number 84-74-2 (Sigma-Aldrich, 99%); diisobutyl phthalate – DIBP, CAS number 84-69-5 (Sigma-Aldrich, 99%); bis(2-ethylhexyl) phthalate – DEHP, CAS number 117-81-7 (Sigma-Aldrich, 99%); benzyl butyl phthalate – BBP, CAS number 85-68-n (Sigma-Aldrich, 98%), used as internal standard; and n-heptane p.a. (Synth), used as fatty food simulant.

2.2 Packaging samples Several types of cellulose packaging (paper and cardboard used as primary and/or secondary dry food packaging were purchased in the retail market in Campinas, São Paulo, Brazil between 2016 and 2019. The packages are listed in Table 1. The tests of specific migration from the packages into fatty food simulant were carried out both with primary (direct contact with food) and secondary packages. The cellulose food packages were characterized as to grammage and thickness. The tests were conducted at 23oC ± 1ºC and 50% ± 2% relative humidity after conditioning the samples under these same conditions for at least 48 hours. The thickness of paper and cardboard packages was measured using mechanical scanning according Polímeros, 31(2), e2021017, 2021

Migration of phthalates and 2, 6-diisopropylnaphthalene from cellulose food packaging Table 1. Cellulose food packages evaluated. Sample code

Description

Type of packaging/paper

1-p 2-p 3-p 4-p 5-p 6-p 7-c 8-c 9-c 10-c 11-c 12-c 13-c 14-c 15-c 16-c 17-po 18-po 19-po 20-po

Paper package of wheat flour A – white, printed Paper package of wheat flour B – white, printed Paper package of wheat flour C – white, printed Paper package of bread Paper package of powdered chocolate B Paper packaging of cornstarch– white Cardboard of powdered chocolate A – printed Cardboard of powdered chocolate B – printed Cornstarch cardboard – printed Cookie cardboard – printed Cardboard package of oatmeal – printed Cardboard package of jelly – produced in 2017 Cardboard, white on both sides – produced in Nov. 2016 Cardboard – produced in Apr. 2018 Cardboard – produced in Jan. 2019 Takeaway pizza box (cardboard) Corrugated paperboard of toast – white Takeaway pizza box A (corrugated paperboard) Takeaway pizza box B (corrugated paperboard) Takeaway pizza box C (corrugated paperboard)

Primary Primary Primary Primary Primary Primary Secondary Secondary Secondary Secondary Secondary Secondary/20% scrap/ 5%PCR Secondary Secondary/30%PCR Secondary/30%PCR Primary Primary Primary Primary Primary

Grammage (g m-2)* 82 ± 2 82 ± 1 80 ± 2 36 ± 1 66 ± 1 56 ± 2 263 ± 2 263 ± 10 236 ± 2 255 ± 3 234 ± 3 256 ± 2 511 ± 19 277 ± 3 268 ± 1 272 ± 2 148 ± 4 533 ± 5 505 ± 6 412 ± 13

Thickness (µm)** 84 ± 4 92 ± 6 96 ± 3 60 ± 0 84 ± 5 82 ± 2 425 ± 5 363 ± 15 330 ± 2 346 ± 5 393 ± 3 n.d. 1379 ± 109 381 ± 7 352 ± 5 372 ± 1 1599 ± 60 1860 ± 12 2032 ± 20 2720 ± 170

*Average ± Standard Deviation relating to 10 specimens. **Average ± Standard Deviation relating to 20 measures. PCR = post-consumer recycled fibers; n.d. = non determined.

to international standard ISO 4593[20]. The thickness of paperboard was determined according to Brazilian standard ABNT NBR ISO 3034[21]. An external digital indicator with maximum 5.1 mm (Starret), 0.001 mm resolution was used. Measurements of the surface of the material were taken at 20 different positions. The mean value is reported in Table 1. The grammage of the cellulose packages was measured by determining the mass of 5 x 5 cm2 test specimens according to Brazilian standard NBR NM-ISO 536[22]. A model AE163 (Mettler Toledo) analytical balance with 0.1 mg resolution was used. Measurements were taken from 10 specimens of the material. The mean value is reported in Table 1.

2.3 Migration tests The analysis was carried out in compliance with the requirements of Resolution RDC no. 88/16[10]. Resolution RDC no. 88/16 incorporates into the Brazilian National Legal Framework all specifications, thresholds and requirements laid down in GMC MERCOSUR Resolution no. 40/15 (GMC means MERCOSUR Common Market Group). This Resolution provides different contact conditions for migration tests, i.e. prolonged contact at room temperature, brief contact at room temperature, momentary contact at room temperature, processing temperature and hot filling. In addition, this Resolution establishes four food simulants: ultra-purified water (aqueous foods), 3% acetic acid (aqueous acidic foods), 10% ethanol or higher content (alcoholic foods) and n-heptane (fatty foods). The fatty food simulant (n-heptane) was selected for all samples under review since phthalates have a greater affinity for fatty foods, and this is the worst scenario to study. Polímeros, 31(2), e2021017, 2021

Dry foods packed in cellulose packaging, such as those evaluated in this study, are usually kept at room temperature and have a long shelf life. For this reason, cellulose packages/materials were evaluated in this study according to Resolution RDC no. 88/16[10] contact condition of 20ºC ± 1ºC/30 min + 1 min. Nevertheless, in some cases food is placed in the packaging at an elevated temperature and can remain in contact with the packaging for a long time, as the case of takeaway pizza boxes evaluated in this study. For these samples, a different contact condition was adopted according to Resolution RDC no. 88/16[10], as follows: , 50ºC ± 2ºC/15 min + 1 min followed by 20ºC ± 1ºC/30 min + 1 min. The specimens were dipped in n-heptane food simulant at a ratio of 0.3 mL cm-2 of the analyzed surface, and both faces of the material were considered in the calculations. Twelve square (5 cm) test pieces of each sample were evaluated, totaling an area of 600 cm2 (considering both sides of the material). 2.3.1 Overall migration into fatty food simulant The quantification of overall migration was based on contact of the samples with extraction solutions for times and temperatures simulating their actual condition of use as described previously (section 2.3). The extract solutions were evaporated, and the residues of overall migration were determined by the difference in weight after the contact employing an analytical balance with 0.01 mg accuracy. 2.3.2 Specific migration into fatty food simulant The cellulose packages/materials were evaluated as to specific migration of DIPN and phthalates employing the method developed in this study and using fatty food simulant 3/8

Coltro, L., Saron, E. S., Pessoa, T. I., Morandi, J., & Silva, B. S. n-heptane, which is the worst scenario due to the chemical affinity among these substances and the simulant. For printed cardboard samples, the outer (printed) layer was removed and only the inner layer (without printing) was analyzed. Benzyl butyl phthalate (BBP) was employed as internal standard to quantify the plasticizers in the food simulant since this plasticizer is not used in food packaging. After the contact, the extracts were concentrated 25 times employing a rotary evaporator from Fisatom 803, Fisatom 826T vacuum pump, filtered in a 0.45 μm polytetrafluoroethylene – PTFE filter and injected into the GC-FID, in triplicate.

2.4 Chromatographic conditions For analysis of migration of target substances from packaging into food simulant, a gas chromatograph with flame ionization detector – GC-FID, from Agilent Technologies 7890A, equipped with an automatic injector Agilent Technologies 7693B was used, operating with a DB-1 capillary column (30 m length x 0.25 mm I.D x 0.25 µm film thickness). The chromatographic conditions followed the method developed by Coltro et al.[23]. The column temperature was set to 60ºC (hold 1 min), a 7ºC min-1 heating rate from 60ºC to 100ºC followed by a 15ºC min-1 heating rate up to 280ºC (hold 5 min). The total run time was 23.7 min. The injector and detector temperatures were 270ºC and 300ºC, respectively. Injection volume was 1 µL. Split mode 35:1.

2.5 Method validation The method was validated according to parameters of selectivity, linearity range, detection limit, quantification limit, recovery, precision and accuracy[24,25]. 2.5.1 Linearity range The calibration curves were built in the 1 – 40 mg kg-1 range (considering a concentration factor of 25 times for solutions of 0.04 – 1.6 mg kg-1) and 2.5 mg kg-1 of BBP (internal standard) taking into account the ratio of the areas (phthalate area / internal standard area) and the respective concentration of phthalate solutions. The coefficients of linear and angular correlation were calculated per the linear regression model. 2.5.2 Detection Limit (LOD) and Quantification Limit (LOQ) Three analytical curves were constructed on different days. With the average peak areas of the compounds under study, it was possible to determine the detection and quantification limits by means of analytical curves using the following equations: LOQ = 10 * linear coefficient

error / angular coefficient and LOD = 3.3 * linear coefficient error / angular coefficient[24]. 2.5.3 Precision and accuracy Two analytical curves were built, being each curve obtained by a different analyst. The intraday repeatability was estimated from the Relative Standard Deviation (RSD) among the replicates of the points of one same curve. The intermediate precision was obtained by calculating the relative standard deviation but considering both analytical curves. The accuracy was assessed via calculation of the relative error (RE), expressed in percentage[26]. 2.5.4 Recovery The recovery rate of the tests of migration from packaging into food simulant was obtained via the ratio between the concentrations obtained in the tests and the expected concentration, as represented by the standard solution[26].

3. Results and Discussion 3.1 Validation of the method 3.1.1 Specific migration from packaging Table 2 shows the validation parameters of the method of specific migration into fatty food simulant for DIPN and the phthalates evaluated. At least five calibration standards with concentrations between 1 mg kg-1 and 40 mg kg-1 were prepared in order to evaluate linearity. This range was selected due to the requirement to ensure linearity at a working range where the specific migration limit – SML – of the phthalates was inserted in the curve (considering a concentration factor of 25 times). All the calibration curves have shown acceptable determination coefficient (r2) values (>0.9900). LOD and LOQ were lower than SML of the phthalates and DIPN studied and sensitive enough to evaluate possible migration of these substances from commercial samples. The results of precision, calculated in terms of intraday repeatability (one analyst) and intermediate precision (two analysts), were below 10% for all substances, indicating an acceptable repeatability of the methods. The recovery ranged from 89% to 104% and the accuracy ranged from -11% to 4%. The Association of Official Analytical Chemists recommends recovery in the range of 75% to 120% for concentrations of the order of 1 mg kg-1[27]. The results obtained meet the acceptable ranges and are similar to those obtained by other authors[28,29].

Table 2. Analytical method validation parameters.* Parameter1 Working range (mg kg-1)** LOD (mg kg-1) LOQ (mg kg-1) Accuracy (%) Intraday repeatability (%) Intermediate precision (%) Recovery (%)

DIPN 1 – 40 0.007 0.022 -8.5 to 2.9 0.1 to 1.3 0.8 to 9.3 91.5 to 102.9

DIBP 1 – 40 0.005 0.014 -7.3 to 3.5 0.1 to 1.6 0.5 to 8.7 92.7 to 103.5

DBP 1 – 40 0.005 0.016 -9.0 to 3.7 0.0 to 1.2 0.8 à 9.1 91.0 to 103.7

DEHP 1 – 40 0.006 0.018 -11.4 to 4.0 0.0 to 4.0 0.7 to 8.3 88.6 to 104.0

*Results for triplicates, except for LOD / LOQ (results from seven replicates). **Taking into account a concentration factor of 25 times, which corresponds to a range of 0.04 – 1.6 mg kg-1 in the sample. LOD = detection limit; LOQ = quantification limit.

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Migration of phthalates and 2, 6-diisopropylnaphthalene from cellulose food packaging As can be seen, the method is suitable to determine specific migration of the phthalates and DIPN under study, since their SML is included in the linearity range and all the other parameters are adequate. Under the chromatographic conditions adopted, the retention times obtained were 14.00, 14.67, 15.23 and 18.54 min for DIPN, DIBP, DBP and DEHP, respectively. The internal standard evaluated was BBP, with retention time of 17.42 min. Therefore, there was no interference in the analysis of phthalates and DIPN in n-heptane, since there were no overlapping chromatographic peaks.

3.2 Migration from packages into fatty food simulant 3.2.1 Overall migration The results for overall migration of all samples that were assessed were below the quantification limit of the method, which corresponds to 2.33 mg dm-2. Resolution RDC no. 88/16[10] establishes an overall migration limit of 8.0 mg dm-2, with analytical tolerance of 10%. Therefore, according to this criterion, all packages evaluated are approved for fatty food contact since all packages showed lower overall migration to fatty food simulant than the limit established by legislation. However, these samples must also be evaluated regarding specific migration limits of the substances specified by this Resolution. 3.2.2 Specific migration Results of specific migration – SM of phthalates and DIPN from five samples of cellulose packages purchased in the retail area of Campinas, SP, Brazil showed absence of DIPN migration (non-detected), DIBP migration levels below limit of quantification, DIBP migration up to 0.020 mg kg-1 and DEHP migration up to 0.033 mg kg-1 [30]. Therefore, with the aim of examining the exposure of Brazilians to phthalates and DIPN, another fifteen cellulose packages were evaluated in this study. Table 3 shows the results of SM of phthalates and DIPN from this broader sampling of cellulose packages purchased in the retail area of Campinas, SP, Brazil. The samples showed DIPN migration levels below the detection limit, but sample 20-po showed migration below the quantification limit due to the higher temperature contact conditions. DIPN is the most harmful substance evaluated in this study since the SML is defined according to the toxicity of the substances and SML for DIPN is close to zero (it should not be detected). Migration levels of phthalates for the contact condition of 20oC for 30 min ranged from below detection limit up to 0.200 mg kg-1 for DIBP, from the limit of detection up to 0.021 mg kg-1 for DBP, and from the limit of quantification up to 0.058 mg kg-1 for DEHP. The specific migration of DIBP was ten times higher than DBP and approximately three times higher than DEHP. Since the structure of DIBP is comparable to that of DBP and they have similar migration rates[18], probably these diverse migration amounts can be due to different degrees of interaction of the contaminants with the cellulosic fibers as well as different concentrations of these substances in the samples. As can be seen in Figure 1, ten samples (50%) showed no migration of phthalates or migration below the limit of Polímeros, 31(2), e2021017, 2021

quantification, three samples (15%) showed migration of DIBP, three samples (15%) showed migration of DBP and eight samples (40%) showed migration of DEHP. Taking into account the packaging material, 33% of the paper packages, 40% of the cardboard packages and 75% of the corrugated cardboard packages showed migration of phthalates. Table 3. Migration of phthalates and DIPN from cellulose packages into n-heptane, at 20oC/30 min, in mg kg-1*. Sample code 1-p 2-p 3-p 4-p 5-p 6-p 7-c 8-c 9-c 10-c 11-c 12-c 13-c 14-c 15-c 16-c 17-po 18-po 19-po3 20-po3 8-c4 10-c4 11-c4 SML5

DIPN

DIBP

DBP

DEHP

n.d. n.d. n.d. 0.036 ± 0.001 n.d. < 0.0142 n.d. < 0.0182 n.d. n.d. n.d. < 0.0182 n.d. n.d. n.d. n.d. n.d. < 0.0142 < 0.0162 < 0.0182 n.d. n.d. 0.021 ± 0.010 0.033 ± 0.007 n.d. < 0.0142 n.d. 0.040 ± 0.002 n.d. n.d. n.d. 0.038 ± 0.001 n.d. < 0.0142 < 0.0162 < 0.0182 n.d. n.d. n.d. < 0.0182 n.d. n.d. n.d. < 0.0182 n.d. 0.200 ± 0.050 < 0.0162 < 0.0182 n.d. n.d. n.d. n.d. n.d. < 0.0142 < 0.0162 0.058 ± 0.005 n.d. 0.018 ± 0.001 < 0.0162 < 0.0182 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.035 ± 0.006 n.d. n.d. n.d. n.d. n.d. 0.043 ± 0.015 0.023 ± 0.003 0.082 ± 0.029 < 0.0222 0.171 ± 0.004 0.048 ± 0.001 0.289 ± 0.041 n.d. n.d. n.d. 0.035 ± 0.001 n.d. 0.019 ± 0.003 n.d. 0.024 ± 0.001 n.d. n.d. n.d. 0.019 ± 0.002 Non 0.36 0.36 1.5 detectable *Results for four replicates, with triplicate injections; 1n.d. = non detected (< LOD = Limit of detection: 0.007 mg kg-1 (DIPN), 0.005 mg kg -1 (DIBP and DBP) and 0.006 mg kg -1 (DEHP); 2 LOQ: Limit of quantification; 3Contact conditions: 50ºC/15 min + 20ºC/30 min; BBP migration was also detected; 4Including printed surface; 5Specific migration limits established in Resolution RDC No. 88/16; 6The sum of the specific migration of these phthalates shall not exceed 0.3 mg kg-1. 1

Figure 1. Migration of phthalates and DIPN from cellulose packages evaluated. (Numbers 21-23 correspond to samples 8, 10 and 11, including printed surface). 5/8

Coltro, L., Saron, E. S., Pessoa, T. I., Morandi, J., & Silva, B. S. Regarding the contaminants, the most harmful phthalate migrated was DIBP since its TDI (0.01 mg kg-1 bw per day) is lower than TDI of DEHP (0.05 mg kg-1 bw per day) and it showed the highest migration among the samples 1 to 18 evaluated at room temperature. On the other hand, at high temperature (samples 19 and 20), DEHP was the phthalate of major concern regarding migration. Fortunately, the migrated amounts are below the SML of these phthalates. Samples 19 and 20 are primary packages of takeaway boxes of pizza, a type of food widely consumed in Brazil. Therefore, an estimation of intake of phthalates was made. Estimating the average consumption of 2 slices of pizza (120 g slice-1) per person with a body weight of 60 kg and the migration values of phthalates from sample 20 (worst case), the following values of intake of phthalates were obtained: 0.68 µg DIBP kg-1 bw, 0.19 µg DBP kg-1 bw and 1.16 µg DEHP kg-1 bw. These values are much lower than the TDI of the phthalates studied (7% of the TDI of DIBP and 2% of the TDI of DBP and DEHP). Therefore, even if these phthalates are ingested from other foods along the day, the TDI will probably not be exceeded. Paper packages (samples 1 to 6 and 16 to 20) are the most critical among the packages evaluated since they are used as primary packaging and therefore, they come into direct contact with foodstuff. Nevertheless, all the migrated amounts are below the SML established (Table 3). Four samples stood out among the 20 samples evaluated, which are samples 12, 14, 19 and 20 (Figure 1). Samples 12 and 14 are cardboard packages with post-consumer recycled PCR fibers in their formulations, i.e. 5% PCR (sample 12) and 30% PCR (sample 14). Although sample 14 had a higher amount of PCR fibers, it showed lower migration of phthalates than sample 12, indicating a lower degree of contamination of sample 14, suggesting that the control of paper recycling of sample 14 is better than sample 12. It is worth highlighting that different manufacturers produced these samples. Corroborating this statement, sample 15 also with 30% PCR fibers in its formulation and produced by the same manufacturer of sample 14 in different years (sample 14 – April 2018 and sample 15 – January 2019) showed migration levels at LOQ or lower. These results indicate the importance of good control of the supply chain of post-consumer cellulose fibers used in these packages in order to have low migration levels. Anyway, all these samples met the established migration limits. The levels of migration of phthalates under contact condition of high temperature (samples 19 and 20) were higher than the levels of long-term storage at room temperature due to the higher temperature, which accelerated the migration process as expected. Migration of DIPN was below the quantification limit and migration of phthalates was up to 0.171 mg kg-1 for DIBP, up to 0.048 mg kg-1 for DBP and up to 0.289 mg kg-1 for DEHP (the highest DEHP migration result among the samples). The larger migration of DIBP in comparison to DBP indicates greater contamination of these samples by DIBP since the migration rate of these substances is similar since their chemical structures are also similar[18]. On the other hand, the larger migration of DEHP is probably due to its apolar long linear chain (C6H6(C=OOR)2, with R = CH2CH(CH2CH3)(CH2)3CH3), 6/8

which reduces its interaction with the polar structure of the cellulosic fibers and promotes its migration. A comparison between the results of specific migration of the samples with the SML values shown in Table 3 leads to the conclusion that all packages analyzed are in conformance with the specific migration limit of phthalates and DIPN, but one sample (20-op). Therefore, these packages should not expose consumers to significant doses of DEHP, which is a phthalate associated with problems in the reproductive and endocrine systems, even taking into account the most critical situation as the hot filling temperature. Regarding DIBP and DBP, the packages showed SM in accordance with the legislation. In relation to DIPN, for which Resolution RDC No. 88/16 establishes that SM should not be detectable, only one packaging (20-po) did not comply with the legislation, since there was a detectable signal for this compound (migration below the quantification limit). In addition, migration of BBP from the sample 20-op was also observed and this phthalate is not listed as an approved substance in RDC Resolution No. 88/16. Samples 8-c, 10-c and 11-c were also tested for specific migration of phthalates and DIPN without removing the printed face of the cardboard to assess the possibility of migration of these substances from printing inks, which might occur due to the set-off effect. As shown in Figure 1 (samples 21, 22 and 23, respectively), the results of these tests showed increased migration of DIBP into sample 10-c and of DEHP into samples 10-c and 11-c, indicating that the formulations of the printing ink of these cardboards probably contained DIBP and DEHP. For sample 8-c, there was no increase in migration; therefore probably the printing ink on this cardboard did not contain these phthalates. As the concentration of phthalates that migrated to the food simulant was low (just above LOQ), even if a set-off effect occurred, the amount that possibly migrated would be lower than the SML of these substances. Therefore, these results indicate that the packages under study are not contaminated by phthalates and DIPN, since all of them met the SML established by Anvisa, except for sample 20-po which showed signs of DIPN when submitted to high temperature, besides migration of BBP (a non-listed substance).

4. Conclusions The GC-FID method adopted in this study enabled the determination of migration of DIPN and phthalates from cellulose packages into fatty food simulant. Twenty cellulose packages used for packing dry foods usually sold in the Brazilian market were evaluated. All packages met the requirements of the legislation regarding overall migration. Taking specific migration into account, 50% of the cellulose packaging samples showed no migration of DIPN or any phthalate evaluated in this study, while 20% showed migration of DIBP, 15% showed migration of DBP and 40% showed migration of DEHP. Regarding the specific migration limits established by Resolution RDC No. 88/16, only one package was rejected due to detection of migration of DIPN and BBP (a non-listed substance) among the samples of paper, cardboard and corrugated Polímeros, 31(2), e2021017, 2021

Migration of phthalates and 2, 6-diisopropylnaphthalene from cellulose food packaging paperboard evaluated. The other nineteen packages met the requirements of the legislation regarding specific migration of DIPN, DIBP, DBP and DEHP and therefore should not be a relevant source of contamination of dry foods. An analysis of migration in printed samples showed that even if the set-off effect occurred, the amount of phthalates migrated would be lower than SML, with no risk of food contamination. Migration of phthalate in samples with 5% to 30% PCR content in their formulations was below the SML values established, enabling their use for food contact (in respect to this requirement). The results showed that the season and the recycling process control affect migration outcomes, so companies need to maintain good control of reverse logistics of cellulose fibers to ensure low migration levels.

5. Acknowledgments This work was supported by the São Paulo Research Foundation (FAPESP) under Grant #2016/24751-3, and by the National Council of Technological and Scientific Development, Brazil (CNPq/PIBIC) under Grant of fellowships. The authors also wish to thank those who provided packaging samples and Cetea’s technical staff for collaborating with the tests.

6. References 1. Compromisso Empresarial para Reciclagem – CEMPRE. (2021). Taxas de Reciclagem. Retrieved in 2021, June 22, from http://cempre.org.br/taxas-de-reciclagem. 2. European Directorate for the Quality of Medicines & HealthCare – EDQM & Council of Europe (2020). Technical guide on paper and board materials and articles for food contact [Draft]. Strasburg: Department of Biological Standardisation, OMCL Network & HealthCare (DBO). Retrieved in 2021, February 28, from https://www.edqm.eu/sites/default/files/medias/fichiers/ Food_contact_materials/food_contact_materials_technical_ guide_on_paper_board_draft_text_for_consultation.pdf. 3. Zhang, K., Noonan, G. O., & Begley, T. H. (2008). Determination of 2,6 – diisoproprylnaphthalene (DINP) and n-dibutylphthalate (DBP) in food and paper packaging materials from US marketplaces. Food Additives and Contaminants, 25(11), 1416-1423. http://dx.doi.org/10.1080/02652030802163380. PMid:19680850. 4. Geueke, B., & Muncke, J. (2018). Substances of very high concern in food contact materials: migration and regulatory background. Packaging Technology & Science, 31(12), 757769. http://dx.doi.org/10.1002/pts.2288. 5. Geueke, B., Groh, K., & Muncke, J. (2018). Food packaging in the circular economy: overview of chemical safety aspects for commonly used materials. Journal of Cleaner Production, 193, 491-505. http://dx.doi.org/10.1016/j.jclepro.2018.05.005. 6. Munoz, C., Eicher, A., Biedermann, M., & Grob, K. (2018). Recycled paperboard with a barrier layer for food contact: set-off during stacking or reeling. Analytical method and preliminary results. Food Additives and Contaminants: Part A, 35(3), 577-582. 7. Vandermarken, T., Boonen, I., Gryspeirt, C., Croes, K., Van Den Houwe, K., Denison, M. S., Gao, Y., Van Hoeck, E., & Elskens, M. (2019). Assessment of estrogenic compounds in paperboard for dry food packaging with the ERE-CALUX bioassay. Chemosphere, 221, 99-106. http://dx.doi.org/10.1016/j. chemosphere.2018.12.192. PMid:30634153. Polímeros, 31(2), e2021017, 2021

8. European Union. Regulation (EC) nº 1935/2004 of the European Parliament and of the Council of 27 October 2004. (2004, November 13). Regulation (EC) nº 1935/2004 of the European Parliament and of the Council of 27 October 2004 on materials and articles intended to come into contact with food and repealing Directives 80/590/EEC and 89/109/EEC, Section L 338. Official Journal of the European Union, Brussels. Retrieved in 2021, February 28, from https://eur-lex.europa. eu/LexUriServ/LexUriServ.do?uri=OJ:L:2004:338:0004:00 17:en:PDF. 9. Silano, V., Barat Baviera, J. M., Bolognesi, C., Chesson, A., Cocconcelli, P. S., Crebelli, R., Gott, D. M., Grob, K., Lampi, E., Mortensen, A., Rivière, G., Steffensen, I. L., Tlustos, C., Van Loveren, H., Vernis, L., Zorn, H., Cravedi, J. P., Fortes, C., Tavares Poças, M. F., Waalkens-Berendsen, I., Wölfle, D., Arcella, D., Cascio, C., Castoldi, A. F., Volk, K., & Castle, L., and the European Food Safety Authority – EFSA (2019). Panel (EFSA Panel on Food Contact Materials, Enzymes and Processing Aids): scientific opinion on the update of the risk assessment of di-butylphthalate (DBP), butyl-benzyl-phthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP), di-isononylphthalate (DINP) and di-isodecylphthalate (DIDP) for use in food contact materials. EFSA Journal, 17(12), e05838. http://dx.doi. org/10.2903/j.efsa.2019.5838. PMid:32626195. 10. Brasil. Agência Nacional de Vigilância Sanitária – Anvisa. Resolution RDC n° 88 from 29 June 2016. (2016, June 30). Approval of technical regulations on cellulosic materials, packages and equipments intended to come into contact with foodstuffs and and makes other provisions, Section 1. Diário Oficial da República Federativa do Brasil, Brasília, DF. Retrieved in 2021, February 28, from https://www. in.gov.br/materia/-/asset_publisher/Kujrw0TZC2Mb/content/ id/23163458/do1-2016-06-30-resolucao-a-rdc-n-88-de-29-dejunho-de-2016-23163247. 11. Magdouli, S., Daghrir, R., Brar, S. K., Drogui, P., & Tyagi, R. D. (2013). Di-2-ethylhexylphthalate in the aquatic and terrestrial environment: a critical review. Journal of Environmental Management, 127, 36-49. http://dx.doi.org/10.1016/j. jenvman.2013.04.013. PMid:23681404. 12. Sant, K. E., Dolinoy, D. C., Jilek, J. L., Sartor, M. A., & Harris, C. (2016). Mono-2-ethylhexyl phthalate disrupts neurulation and modifies the embryonic redox environment and gene expression. Reproductive Toxicology, 63, 32-48. http://dx.doi. org/10.1016/j.reprotox.2016.03.042. PMid:27167697. 13. Muncke, J., Andersson, A.-M., Backhaus, T., Boucher, J. M., Carney Almroth, B., Castillo Castillo, A., Chevrier, J., Demeneix, B. A., Emmanuel, J. A., Fini, J.-B., Gee, D., Geueke, B., Groh, K., Heindel, J. J., Houlihan, J., Kassotis, C. D., Kwiatkowski, C. F., Lefferts, L. Y., Maffini, M. V., Martin, O. V., Myers, J. P., Nadal, A., Nerin, C., Pelch, K. E., Fernández, S. R., Sargis, R. M., Soto, A. M., Trasande, L., Vandenberg, L. N., Wagner, M., Wu, C., Zoeller, R. T., & Scheringer, M (2020). Impacts of food contact chemicals on human health: a consensus statement. Environmental Health, 19(1), 25. http://dx.doi. org/10.1186/s12940-020-0572-5. PMid:32122363. 14. Poças, M. F. F., & Hoog, T. (2007). Exposure assessment of chemicals from packaging materials in foods: a review. Trends in Food Science & Technology, 18(4), 219-230. http://dx.doi. org/10.1016/j.tifs.2006.12.008. 15. Nerín, C., Cacho, J., & Gancedo, P. (1993). Plasticizers from printing inks in a selection of food packaging and their migration to food. Food Additives and Contaminants, 10(4), 453-460. http://dx.doi.org/10.1080/02652039309374168. PMid:8405584. 16. Asensio, E., Peiro, T., & Nerín, C. (2019). Determination the set-off migration of ink in cardboard-cups used in coffee 7/8

Coltro, L., Saron, E. S., Pessoa, T. I., Morandi, J., & Silva, B. S. vending machines. Food and Chemical Toxicology, 130, 61-67. http://dx.doi.org/10.1016/j.fct.2019.05.022. PMid:31102676. 17. Poças, M. F., Oliveira, J. C., Pereira, J. R., Brandsch, R., & Hogg, T. (2011). Modelling migration from paper into a food simulant. Food Control, 22(2), 303-312. http://dx.doi. org/10.1016/j.foodcont.2010.07.028. 18. Coltro, L., & Machado, L. G. S. (2020). Migration of phthalates from cellulose packaging into food simulant: assessment of different levels of contaminants. Macromolecular Symposia, 394(1), 2000070. http://dx.doi.org/10.1002/masy.202000070. 19. Graiño, S. G., Sendón, R., Hernández, J. L., & Quirós, A. R. B. (2018). GC-MS screening analysis for the identification of potential migrants in plastic and paper-based candy wrappers. Polymers, 10(7), 802. http://dx.doi.org/10.3390/polym10070802. PMid:30960727. 20. International Organization for Standardization – ISO. (1993). ISO 4593: plastics – film and sheeting – determination of thickness by mechanical scanning (2 p.). Switzerland: ISO publications. 21. Associação Brasileira de Normas Técnicas – ABNT. (2012). NBR ISO 3034:2011. Corrugated fiberboard — Determination of single sheet thickness. (9 p.). Rio de Janeiro: ABNT. 22. Associação Brasileira de Normas Técnicas – ABNT. (2000). NBR NM-ISO 536:2000. Paper and board – Determination of grammage. (6 p.). Rio de Janeiro: ABNT. 23. Coltro, L., Pitta, J. B., Costa, P. A., Perez, M. A. F., Araújo, V. A., & Rodrigues, R. (2014). Migration of conventional and new plasticizers from PVC films into food simulants: a comparative study. Food Control, 44, 118-129. http://dx.doi. org/10.1016/j.foodcont.2014.03.058. 24. Ribani, M., Bottoli, C. B. G., Collins, C. H., Jardim, I. C. S. F., & Melo, L. F. C. (2004). Validation of chromatographic and electroforetic methods. Quimica Nova, 27(5), 771-780. http://dx.doi.org/10.1590/S0100-40422004000500017.

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25. Ribeiro, F. A. L., Ferreira, M. M. C., Morano, S. C., Silva, L. R., & Schneider, R. P. (2008). Validation spreadsheet: a new tool for estimating the analytical figures of merit for the validation of univariate methods. Quimica Nova, 31(1), 164171. http://dx.doi.org/10.1590/S0100-40422008000100029. 26. Instituto Nacional de Metrologia, Qualidade e Tecnologia – INMETRO. (2018). General Coordination of Accreditation. DOQ-CGCRE-008: guidance on validation of analytical methods (Revision Nº 07). Rio de Janeiro: INMETRO. 27. Association of Official Analytical Chemists – AOAC. (2013). Official methods Analysis. Appendix K: Guidelines Dietary Supplementsand Botanicals. (32 p.). Gaithersburg: AOAC. 28. Bonini, M., Errani, E., Zerbinati, G., Ferri, E., & Girotti, S. (2008). Extraction and gas chromatographic evaluation of plasticizers content in food packaging films. Microchemical Journal, 90(1), 31-36. http://dx.doi.org/10.1016/j.microc.2008.03.002. 29. Bueno-Ferrer, C., Jiménez, A., & Garrigós, M. C. (2010). Migration analysis of epoxidized soybean oil and other plasticizers in commercial lids for food packaging by gas chromatography - mass spectrometry. Food Additives and Contaminants, 27(10), 1469-1477. http://dx.doi.org/10.1080 /19440049.2010.502129. PMid:20635266. 30. Coltro, L., Saron, E. S., Pessoa, T. I., Ferreira, I. A. G., Silva, B. S., Hamdan, M., & Santos, B. B. (2019). Conformity assessment of cellulosic food packaging in relation to phthalates, 2,6-diisopropylnaphthalene and metals. In Proceedings of the 4th International Conference on Food and Biosystems Engineering – FABE (pp. 50-57). Crete Island, Greece: University of Thessaly. Received: Feb. 28, 2021 Revised: May 18, 2021 Accepted: June 07, 2021

Polímeros, 31(2), e2021017, 2021

ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.20210027

Effect of molar weight of gelatin in the coating of alginate microparticles Joelma Correia Beraldo1 , Gislaine Ferreira Nogueira2 , Ana Silvia Prata3*  and Carlos Raimundo Ferreira Grosso1  1

Departamento de Alimentos e Nutrição, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas – UNICAMP, Campinas, SP, Brasil 2 Universidade do Estado de Minas Gerais – UEMG, Passos, MG, Brasil 3 Departamento de Engenharia de Alimentos, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas – UNICAMP, Campinas, SP, Brasil *asprata@unicamp.br

Abstract The protein adsorption on the porous alginate microparticles was evaluated in regards to the coating ability and this protective effect during gastrointestinal assay. The coating was performed at suitable pH for optimized electrostatic interaction between protein and alginate. Concentrations of gelatin (HGE) and their hydrolysates (Collagel® (MGE) (> 10 kDa) and Fortigel® (LGE) (3 kDa)) from 1 to 10% (w/w) were tested. Higher protein adsorption was observed in the highest concentration of protein in solution and the amount adsorbed was inversely proportional to the degree of hydrolysis with 47.3, 41.4 and 29.3% of protein adsorbed when HGE, MGE and LGE were used, respectively. The particles that showed higher protein adsorption were submitted to gastrointestinal in vitro assay. In gastric simulation, 39.1, 41.8 and 49.0% of protein from HGE, MGE and LGE were solubilized while 81.3, 61.5 and 95.2% were solubilized after 5 h under enteric conditions. Keywords: microencapsulation, ionic gelation, electrostatic interaction, layer-by-layer, protein adsorption. How to cite: Beraldo, J. C., Nogueira, G. F., Prata, A. S., & Grosso, C. R. F. (2021). Effect of molar weight of gelatin in the coating of alginate microparticles. Polímeros: Ciência e Tecnologia, 31(2), e2021018. https://doi.org/10.1590/01041428.20210027

1. Introduction Ionic gelation (IGEL) is one of the most used techniques for encapsulation of sensitive, bioactive, and functional compounds[1-4], cells and probiotic bacteria[5,6], due to the mild conditions employed, ie, absence of heating or organic solvents and moderated stirring rate conditions[1]. The interactions of the anionic charge of the polysaccharide (COO-) with cationic ions lead to a tridimensional gel network[7] which is highly porous[6] and undesirable when it is expected a controlled release. There are many examples in the literature reporting on the rapid release of bioactive compounds from gelled microparticle after simulated gastroenteric assays[2,3,8]. Layer-by-layer protein deposition onto gelled particles has been successfully employed aiming to increase the resistance in gastric conditions[8-10] or to reduce the losses of hydrophilic compounds to the product[1]. Gelatin (HGE) has a positive charge below its isoelectric point (IEP) and can interact with alginate, an anionic polysaccharide, above its pKa values[11]. It is obtained from collagen through acid (type A, IEP, 7.0-9.0) or alkaline hydrolysis (type B, IEP, 4.6-5.2)[12] and its molecular weight (MW) varies from 300-200.000 Da depending on the raw material and the process conditions[13]. Globular proteins explored for layer-by-layer deposition[10,14] present topological

Polímeros, 31(2), e2021018, 2021

limitations which prevent their charged groups to optimally contact the rigid anionic polysaccharide chains[15]. The hypothesis is that unfolded protein structures, such as HGE, can form a maximum number of contacts with the charged polysaccharide chain, covering more efficiently particles produced by IGEL. Moreover, the average molecular weight of protein hydrolysates is one of the most important factors which determines their biological properties. The reduced molecular weight in the peptide fractions also better exposure of the amino acid residues, being suggested as a factor that facilitate the interaction with other polymers[16]. However, in the context of layer coating formation, the changes in the structures may reduce the contribution between proteinprotein adsorption, changing the organization of the layer formed[17] and resulting in differences of protein adsorption. Information published on electrostatic interaction (EI) as a consequence of molecular weights are not prevalent. In this work, the effect of the molar weight of gelatin (HGE) was evaluated in regards to the protein adsorption on the porous alginate microparticles and to their protective effect during gastrointestinal assay. The conditions for EI between type A HGE and two commercial hydrolysates of collagen, Collagel® (MGE, > 10 kDa) and Fortigel® (LGE, 3kDa)

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O O O O O O O O O O O O O O O O

Beraldo, J. C., Nogueira, G. F., Prata, A. S., & Grosso, C. R. F. were initially established. Under optimized charge conditions, protein concentrations varying from 1 to 10% (w/w) were tested to perform the coating. The coated microparticles were characterized with respect to morphology, average size, adsorbed protein and moisture contents. The microparticles that showed higher protein adsorption were evaluated for resistance to gastrointestinal conditions in vitro (GIA) by quantifying the solubilized protein content and following their morphology.

2. Materials and Methods 2.1 Materials Sodium alginate (SA) (FMC Biopolymer, lot G470020,SP, Brazil, medium viscosity (200 - 400 mPa.s), and mannuronic to guluronic acid ratio ≥ 1.53); type A – gelatin (HGE) containing 90.15 ± 1.28% of proteins[18] (Gelita, lot 21502 P-04, SP, Brazil); Collagel® (MGE) containing 96.72 ± 0.11%[18] of proteins (MW > 10 kDa, Gelita, lot LF22703 11, SP, Brazil); Fortigel® (LGE) containing 97.38 ± 0.74%[18] of proteins (MW of 3 kDa, Gelita, lot LF897757 09, SP, Brazil) were employed as biopolymers. Commercial sunflower oil (Cargill Agrícola, SP, Brazil); calcium chloride (CaCl2) (Dinâmica, batch 44034, SP, Brazil); sodium hydroxide (NaOH) (Dinâmica, lot 53187, SP, Brazil); hydrochloric acid (HCl) (Merck, SP, Brazil); concentrated sulfuric acid (H2SO4) (Synth, Diadema, SP, Brazil); Pepsin (3180 U/mg of protein), swine pancreatin (3 X USP unit of enzyme activity) and mucin (Sigma-Aldrich, MO, USA). All reagents used were of analytical grade. Deionized water were used to prepare the solutions.

2.2 Characterization of biopolymers 2.2.1 Molar weight (MW) distribution of HGE and hydrolysates HGE and the hydrolysates were mixed (1%, w/v) with a buffer (Tris-HCl 62.5mM; SDS 2%; glycerol 20%; β-mercaptoethanol 5% and bromophenol blue, pH 6.8) and boiled for 5 min. Polyacrylamide gel (SDS-PAGE-Glycine, 0.75 mm) was prepared according to Laemmli[19], with 4% packaging and 7% - separation gels. 4 μL of HGE and 10 μL of hydrolysates solution were poured into the gel wells. The voltage was adjusted to 70 V and the electrophoresis (MiniProtean II Bio Rad equipment, CA, USA) was performed for 2 hours at 23 ± 2 °C. Coomassie brilliant blue G-250 solution (0.1%) was used to stain the protein. To eliminate the background color, it was placed in a bleached solution (methanol (40%, v/v) and acetic acid (10%, v/v)). The MW distribution of the LGE hydrolysate was also determined by using the polyacrylamide-SDS-Tricine gel (1.5 mm, 4% -packaging, 16.5% -separation gels), according to Schägger and von Jagow[20]. The use of tricine allows better resolution for small proteins (less than 14 kDa). 20 μL of sample was applied to the gel channels and the run was carried out at 85 V, at room temperature (23 ± 2 °C). Proteins with MW ranging from 37 to 250 kDa (Code: 161-0375) and 1.42 to 26.62 kDa (Code: 161-0326) from Bio-Rad Laboratories (CA, USA) were used as standard. 2.2.2 Identification of working pH for protein adsorption 2/9

The zeta potential (ZP) of biopolymeric solutions (SA, HGE, MGE, LGE) and SA:HGE mixtures and at 0.1% w/w was measured as a function of pH (3.0-7.0) using a Zetasizer (Nano ZS, Malvern Instrument Ltd., UK) at 25 °C. The solution pH was adjusted by dropwising HCl or NaOH (0.1N). Volumetric ratios of SA:HGE mixtures were prepared (1:1 to 1:10), maintaining the final volume constant in 30 mL. The mixtures were kept under stirring in a tube shaker (AP 22, Phoenix, SP, Brazil) for 1 h. After determining the ZP, the remaining mixtures were kept at rest for 12 h and subsequently photographed. Since greater amount of precipitated coacervate was observed for pH 3.0, only pH 3.0 was used for the continuity of the work. All systems and measurements were realized in triplicate.

2.3 Production of microparticles by ionic gelation Microparticles were obtained following procedures described by Nogueira et al.[10], by using an emulsion produced with 1.65% w/w of sunflower oil and SA solution (2%, w/w) through homogenization at 14.000 rpm for 3 min (Ultra turrax®, IKA Works, RJ, Brazil). The emulsion (pH - 3.0) was atomized in a solution of CaCl2, (2%, w/v) with the aid of a peristaltic pump, flow rate 556 mL/h, a double fluid atomizer nozzle, Ø 1 mm, air pressure of 0.125 kgf/cm2.

2.4 Protein adsorption by electrostatic interaction 100 g of moist microparticles were added to 200 mL of protein solution (pH 3.0) at 45 °C for 15 min under stirring. The final volume was kept constant, and the amounts of HGE or hydrolysates was adjusted to obtain 1, 2, 4, 6, 8 and 10% w/v of protein in the solution. Then, the microparticles were sieved (mesh 53 µm) and three times washed with acidified water at pH 3.0 with HCl 0.1N. Three repetitions were performed.

2.5 Microparticles characterization 2.5.1 Protein, moisture content and average size of microparticles The microparticles were characterized in terms of protein and moisture content, following the methodologies described by AOAC[18] in triplicate. Total nitrogen content (N) was obtained by the Kjeldahl method using a conversion factor of the N x 5.55. The moisture content was determined by oven drying at 105 °C up to constant weight. The average size of the microparticles (D0.5) was determined in a Mastersizer 2000 equipment (Malvern, Worcestershire, UK), using acidified water at pH 3.0 as a dispersant. Size determinations were performed in triplicate. 2.5.2 Optical microscopies of sectioned microparticles Newly processed wet microparticles were soaked in a polymerizable historesin at 40 °C for 2 hours (LEICA HISTORESIN Embedding kit 7022 18500, Solms, Germany). The microparticles embedded in historesin were sectioned in a LEICA RM2245 microtome (LKB, Ultrotome III 8,800, Solms, Germany) using glass knives. The sections of approximately 2-3 μm were placed on glass slides and subjected to the following histochemical methods[21]: Polímeros, 31(2), e2021018, 2021

Effect of molar weight of gelatin in the coating of alginate microparticles a) To check the presence of polysaccharides, the slides were immersed in 1% Schiff’s periodic acid (PAS) for 20 min, washed for 15 min in running water, immersed again in PAS for another 20 min and, finally, washed for 5 min in running water[21] and, after: b) To identify the specific presence of proteins, the slides were immersed in a 0.5% Coomassie brilliant blue aqueous solution G-250 for 60 min and then washed in Clark’s solution (acetic acid and absolute alcohol (1:3)) for 5 min repeatedly. The slides were dried at room temperature and the historesin sections were covered using histological mounting medium or immersion oil for observation and photomicrographic documentation under the NIKON light microscope, Eclipse E 800 (Tokyo, Japan).

2.5.3 In vitro gastrointestinal evaluation of microparticles with protein coating Freshly processed moist microparticles coated with protein solution (HGE, MGE and LGE at 10%, w/w) were employed to in vitro gastrointestinal (GIA) test. Artificial gastric juice (SGA) with pH 2.0 was prepared with the following composition: 1.12 g/L KCl, 2 g/L NaCl, 0.11 g/L CaCl2, 0.4 g/L KH2PO4, 3.5 g/L mucin and 0.26 g/L of pepsin. For pH adjustment, HCl (0.1N) was used[22]. The GIA was carried out in 50 mL glass tubes, using 3 grams of moist microparticles and 30 mL of SGA. The samples were incubated at 37 °C, in a water bath with agitation at 150 rpm, for 2 h. After this time, the samples were centrifuged for 10 min at 17.000 rpm (RC-5C Sorvall Instruments, Wilmington, USA), and a small portion analysed for protein solubility and morphology. Afterwards, the pH of the media was adjusted to 7.0 with a 20% NaHCO3 solution, and pancreatin solution (1.95 g/L) was added for simulation of intestinal conditions. The samples were then re-incubated for

an additional 5 and 17 hours, with morphological observation and determination of the solubilized protein in each period. The morphology of the samples was observed in an optical microscope (JENAVAL, Tokyo, Japan) with objectives of 12.5, 25, 40 and 60 x, and optovar of 0.8, 1.0 and 1.25 x. The images were captured using the EDN-2-Microscopy Image Processing System software. The sample removed for protein quantification and morphology observation were placed in a water bath with ice for 15 min, centrifuged for 20 min at 15.000 rpm (RC-5C Sorvall Instruments, Wilmington, USA). The protein content was quantified in the supernatant by Kjeldahl[18] using N x 5.55, minus the nitrogen amount determined in SGA (blank). The solubilized protein content in relation to the initial protein present in the microparticles is expressed in percentage and on a dry basis. The protein solubility measurement was performed in three independent tests, each test being performed in triplicate. 2.5.4 Statistical analysis The results were analyzed using the SAS 9.2 statistical program to determine the analysis of variance and the comparison between the means was made by the Tukey test with a 95% confidence level. The number of repetitions was specified in each assessment.

3. Results and Discussion 3.1 Characterization of biopolymers 3.1.1 Molar weight (MW) distribution The SDS-PAGE-Glycine for HGE, MGE and LGE is shown in Figure 1a. HGE presented many protein bands distinguishable in molar weights close to ~ 37, ~ 50, between

Figure 1. Polyacrylamide gel-SDS-PAGE electrophoresis patterns. The first lane shows the marker sample (M) with different range of molecular weights. (a) gel with Glycine and standard size markers from 37 to 250 kDa; (b) gel with Tricine and standards size markers from 6.51 to 26.6 kDa. HGE: Gelatin; MGE: Collagel®; LGE: Fortigel®. Polímeros, 31(2), e2021018, 2021

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Beraldo, J. C., Nogueira, G. F., Prata, A. S., & Grosso, C. R. F. 50 and 75, between 100 and 150, ~ 250 and also protein fractions with MW greater than 250 kDa. The diffuse pattern of MGE bands, typical of a hydrolysed product, presented MW between 37 and 150 kDa. LGE could not be detected in the SDS-Glycine polyacrylamide gel. Then, polyacrylamide-SDS-Tricine gel (Figure 1b), with higher density and less porosity, was used to identify the protein fractions of the hydrolysates. The protein patterns adopted for this gel had MW ranging from 1.42 to 26.62 kDa. The intermediate hydrolysate MGE still presented a diffuse pattern, but it was allowed to identify some fractions with MW of ~ 16.9 kDa and higher. Compared to the standard mixture, MGE showed peptide fractions higher than 16.9 kDa. For LGE, which is an intensely hydrolysed material and with a diffuse pattern in electrophoresis, peptide fractions with MW between 6.5 and 26.6 kDa can be identified, as specified by the manufacturer. HGE presented MW greater than 16.9 kDa and protein material present in the stacking gel that was unable to migrate to the separation gel due to its large size. 3.1.2 Determination of the zeta potential of polymers The SA solution showed negative ZP over the entire pH range studied, ranging from -33.1 mV at pH 3.0 to -66.7 mV at pH 7.0 (Figure 2). The HGE solution showed positive ZP from +21.6 mV at pH 3.0 to +3.0 mV at pH 7.0, which confirms the type A HGE, which possesses IEP between pH 7.0 and 9.0[12]. The ZP values of the MGE and LGE varied between +13.3 mV (pH 3.0) to -12.3 mV (pH 7.0) and between +6.6 mV (pH 3.0) to -12.8 mV (pH 7.0), respectively. The respective IEP were identified at pH 4.5 for LGE and at pH 4.0 for MGE. The ZP along pH allowed the determination of the amount of net charge in solution of the polysaccharide and proteins, thus indicating the pH range that satisfies the condition pKa < pH <IEP. EI could occur throughout the studied range (pH 3.0 to pH 7.0) for HGE, but below to the IEP of LGE

(pH 4.0) and MGE (pH 4.5). Then, pH 3.0; 3.5 and 4.0 and different volumetric mixtures between SA: HGE solutions were considered for adsorption study. EI between SA and HGE were reported at pH 3.5[23] and at pH 4.0[24] and with whey proteins (IEP ~ 5) were also previously performed at pH values 3.50 and 3.75[10].

3.2 Identification of working pH for protein adsorption The adsorption of proteins onto alginate microparticles is expected to be driven by electrostatic interation (EI), but the exact determination of the surface charges of the microparticles can not be properly performed due to their large sizes, which present a very rapid sedimentation in the measurement cells. The formation of alginate particles is accompanished by the replacement of monovalent sodium (Na+1) íons from SA by the divalent calcium (Ca2+) ones, so that the particle surface will have less negative charge available for interaction with the protein solutions, in relation to the SA solution. Measurements were realized for very small particles. Opanasopit et al.[25] by using pressure nozzles to produce particles (< 10 µm) found that the pectin microparticles showed about one third (-10.4 mV) of the surface charge in relation to the value of the ZP corresponding to the pectin solution. In another work, ZP values for SA particles, sized around 150 µm, were determined using the diffusion of electrolytic solutions of known charge. The authors observed that the charge of the SA microparticles was -0.68 ± 0.08 mV at pH 4.0 which allows the adsorption of a positive charges protein[26]. Then, aiming to preliminarly find suitable conditions to allow the adsorption takes place, mixture of protein solutions and SA solutions were realized in a more limited range of pH (3.0 to 4.0). The mixture of a diluted solution of positively charged HGE and an anionic polyelectrolyte can lead to phase separation, with one of the phases rich in complexed biopolymers and a second very diluted

Figure 2. Zeta potential of polymers solutions against pH (from 3.0 to 7.0). SA: Sodium alginate; HGE: Gelatin; MGE: Collagel®; LGE: Fortigel®. 4/9

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Effect of molar weight of gelatin in the coating of alginate microparticles phase, practically free of such hydrocolloids[16]. The charge stoichiometry between the biopolymers depends on the ratio between polyelectrolytes and pH. Besides, the concentration of biopolymers is crucial since it strongly has influence on the unfolding and mobility of the molecules[27]. Figure 3 shows different volumetric proportions (1:1 to 1:10) between SA and HGE diluted solutions (0.1%, w/w), at pH 3.0, pH 3.5, and pH 4.0, keeping constant the temperature. The ZP (mV) of Figure 3 indicates the surplus of negative charge increases with the increasing of pH. The behaviour is expected since the -COOH groups of SA became deprotonated by increasing the pH. The requirement of positive charge

to counterbalance the excess of negative charges of the SA raised from 1 part of HGE at pH 3.0, to 6 part of HGE at pH 4.0. The same proportion was found by Bastos et al.[24] at pH 4.0, evaluated by turbidimetry. Moreover, as shown by the schematic line traced to represent the zeta potential, it is observed an increase of ZP with the proportion of HGE, and a saturation of charges is reached right after the turning point (ZP is constant and identical to the HGE solution), with exception of pH 4.0. This condition of unchanged ZP and followed by the turbidity development of the sobrenadant indicates that no more interaction occurs. The turning pH was accompanied by the formation of a precipitated mass of coacervates with transparent supernatant phase. The visual comparison of the coacervate formed at pHs 3.0, 3.5 and 4.0 shows that greater volumes were formed for systems at pH 3.0. This could be consequence of the weaker attraction between HGE and SA at the lowest pH, as shown by the strength of the electrostatic interaction (SEI) values. The SEI was calculated between oppositely charged polyelectrolytes[28,29] in pH 3.0, 3.5 and 4.0 and it is shown inside of Figure 2. The highest SEI values indicate strongest attractions between opposite biopolymers. The SEI increased with the pH for HGE, keeping greater values than for MGE and LGE. Conversely, SEI values for both hydrolisates reduced with increased pH. Therefore, the pH 3.0 was chosen for the protein adsorption in the microparticles and the subsequent evaluations.

3.3 Microparticles characterization 3.3.1 Protein, moisture content and average size of microparticles

Figure 3. Visual aspect and zeta potential (mV) of mixtures at different ratios (1:1; 1:2; 1:4; 1:6; 1:8 e 1:10) between SA:HGE solutions at pH 3.0, pH 3.5 and pH 4.0 . The line illustrate ZP variation and the vertical line, the turning point of pH from negative to positive surface charge. SA: Sodium alginate; HGE: Gelatin.

The amount of protein detected in the microparticles after their immersion in protein solution (Table 1) indicates that interactions occurred between carboxyl groups of SA and positively charged amino groups of the proteins. Contrarily to that observed for solutions, where a “saturation point” was detected by stabilization of ZP values, the amount of protein adsorbed on the particles increased with protein content in solution, regardless of the type of protein used, indicating that, in addition to the protein-polysaccharide EI interaction, protein-protein EI may have occurred, contributing to the high protein adsorption. A similar effect was observed previously[30] and also when whey protein and ovalbumin or a mixture of proteins were adsorbed on IGEL[9,26]. Different surface forces can be associated with interactions between polyelectrolytes including van der Waals forces, hydrogen bonds, and, in particular, electrostatic and hydrophobic

Table 1. Protein and moisture content (%) of IGEL microparticles after protein coating as a function of different concentrations of protein in solution (%, w/w). Protein in solution (%) 1 2 4 6 8 10

HGE 26. 5±0.6Ea* 35.2±2.0Da 38.9±0.7Ca 44.0±2.1Ba 44.4±1.4Ba 47.3±1.1Aa

Protein adsorbed (%) MGE 25.1±0.5Eb 29.8±0.7Db 32.6±0.8Cb 40.1±0.6Bb 40.8±1.4ABb 41.4±0.5Ab

LGE 16.3±0.4Ec 19.6±0.4Dc 21.2±0.9Cc 24.7±0.6Bc 24.2±1.0Bc 29.3±0.5Ac

HGE 86.9±1.0Ac 86.0±0.8Ac 82.6±0.6Bc 78.4±1.0Cc 78.0±1.1CDc 76.6±2.2Dc

Moisture content (%) MGE 89.8±0.6Ab 87.2±0.9Bb 86.1±1.4BCb 83.8±0.8Cb 85.4±0.9Db 81.1±0.9Eb

LGE 91.6±0.8Aa 88.8±0.8BCa 88.8±0.2BCa 89.1±0.5BCa 89.4±0.7Ba 88.2±0.5Ca

*Averages followed by the same letters (upper cases on the same columns and lower cases on the same lines) did not differ according to Tukey´s test (p > 0.05). IGEL: Ionic gelation; HGE: Gelatin; MGE: Collagel®; LGE: Fortigel®.

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Beraldo, J. C., Nogueira, G. F., Prata, A. S., & Grosso, C. R. F. interactions[31]. Molina-Ortiz et al.[32] studied interactions between carrageenan and soy protein and showed that the complexes were formed at both, high and low pH values. According to the authors, EI dominate at low pH whereas hydrophobic interactions are the dominant interactions in complexes at high pH. Significant differences in adsorption between the three protein materials were observed. The adsorbed amount increases with MW. As can be seen in Table 1, in the highest amount of protein in solution (10%), values of 47.3, 41.4 and 29.3% (w/w, dry basis) of protein adsorbed on the microparticles were found when HGE, MGE and LGE were used. Similar behavior was obtained for spray-dried microparticles of SA crosslinked with epichlorohydrin. The adsorption of lysozyme (14.3 kDa) and chymotrypsinogen (25.6 kDa) reached very high protein amounts corresponding to 1880 and 3034 mg of protein/g of SA respectively[33]. The values of ZP (Figure 2) corroborate the electrostatic contribution for adsorption, with the growing order: HGE > MGE > LGE. In addition, the ZP presented by LGE is significantly lower (+6.6 mV) than the ZP observed for HGE and MGE, +21.6 and +13.3 mV, respectively. An adsorption study with human blood proteins indicated that proteins larger than albumin (66.3 kDa) could occupy multiple layers in the adsorption process, while smaller proteins adsorbed completely or partially as a monolayer[17]. In another study[34], protein adsorption at the equilibrium was: albumin (66.3 kDa) < fibrinogen (340 kDa) < fibronectin (450 kDa). Besides size and charge density, many other factors would be included in the adsorption of polyelectrolytes on charged surfaces as non-planar surfaces, porosity of microparticles, chemical structure, protein conformation,

chain length, type of charge, charge density and charge distribution[35]. Another recent review mentioned additional properties of the polymers, such as architecture, density and wettability, chemical and structure’s properties, functional groups, interfacial free energy and conformational flexibility among others[36]. The protein adsorption changed the moisture content of the particles (Table 1). The higher the amount of adsorbed protein, the lower moisture content of the microparticles. Also, the moisture content of particles increased with decreasing the MW of the coating material. The IGEL moist microparticles without coating showed average sizes (D0.5) varying between 83.4±16.6 and 105.2±34.0 µm. Protein adsorption, irrespective of whether HGE, MGE or LGE, produced an increase in the average size of the IGEL microparticles (Table 2). However, the HGE was the only that presented variation in sizes with the protein bulk concentration. Table 2. Average size (D 0.5) of microparticles after protein adsorption (µm) as a function of the amount of protein in solution (%, w/w). Protein in solution (%) 1 2 4 6 8 10

Average size (D0.5, µm) IGEL + HGE 122.9±13.1 Ba* 137.1±11.3 Ba 147.7±31.3Aba 164.9±20.2 Aba 158.3±15.3Aba 200.2±53.5 Aa

IGEL + MGE 132.6±7.8Aa 143.1±6.4Aa 135.0±33.9Aa 110.1±4.0Ab 111.2±6.1Ab 128.9±22.9Aa

IGEL + LGE 139.8±9.7Aa 117.8±5.9Aa 134.9±22.7Aa 132.5±13.4Ab 116.3±6.3Ab 120.5±16.6Aa

Figure 4. Optical microscopy of sectioned IGEL microparticles. Top line: uncoated microparticles stained with Coomassie brilliant blue (CB) (a), and Schiff’s periodic acid (PAS) (b); Bottom line: microparticles coated with the protein stained with CB (c) and with PAS and then with CB (d). 6/9

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Effect of molar weight of gelatin in the coating of alginate microparticles 3.3.2 Optical microscopies of sectioned microparticles Figure 4 shows micrographs of microparticles embedded in the polymerized material and later sliced into the microtome, colored with specific dyes for carbohydrates and proteins. Uncoated IGEL microparticles as expected did not show Coomassie brilliant blue staining by the absence of protein coating material (Figure 4.a). In Figure 4.b, the same particles stained with the PAS acquired a pink color, typical for the PAS-carbohydrate interaction. In Figure 4.c, IGEL microparticles coated with protein showed a blue halo on the perimeter of the microparticles corresponding to the layer of protein adsorbed on the microparticles and oil vesicles without staining. Figure 4.d shows particles containing protein first stained with PAS and then with Coomassie where the interior of the particles is pink and the perimeter shows a blue halo, which means a strong indication of the adsorbed protein. 3.3.3 In vitro gastrointestinal evaluation of microparticles with protein coating The solubility of proteins adsorbed onto microparticles is shown in Table 3. It was observed that 39.1, 41.8 and 49.0% (w/w) of total protein present in the microparticles Table 3. Protein release (%) during gastrointestinal in vitro evaluation (IGEL microparticles +10% of protein in solution). Digestion time Protein

HGE MGE LGE

Simulated gastric conditions pepsin, pH 2 2h 39.1±5.3Ac* 41.8±4.5Ac 49.0±1.9Ab

Simulated intestinal conditions pancreatin, pH 7 7h 81.3±2.5Bb 61.5±6.5Cb 95.2±3.9Aa

24h 96.5±3.5Aa 82.3±6.6Ba 96.1±3.9Aa

coated with HGE, MGE and LGE, respectively, solubilized after 2 h in artificial gastric fluid (Table 3), showing that all coatings were susceptible to gastric conditions. Despite the high protein solubility, the microparticles were still intact, spherical and dense (Figure 5). The coating of microparticles with HGE and its hydrolysates was inefficient compared to results observed when whey protein was adsorbed onto microparticles IGEL[9]. These authors observed a low solubility of the adsorbed layer of whey protein (WPC) under simulated gastric conditions (pH 3.0, 2h, 37 °C). The low susceptibility of WPC to pepsin in gastric conditions was previously observed[37]. After gastric treatment (2h) the microparticle suspensions were sequentially subjected to intestinal conditions and after 7 hours an increase in the percentage of solubility was observed, from 61.5 to 95.2% according to the coating material used (Table 3). The particles became more transparent reflecting the loss of the protein layer, visually more swollen, but still spherical (Figure 5). After 24 hours the protein solubility increased even more, from 82.3 to 96.5% of protein released. For microparticles containing HGE and LGE, the solubilization observed was almost total in relation to the protein initially adsorbed (Table 3). Similar behaviour was previously observed for multilayer particles produced with alginate and whey protein, 30.5% w/w of total nitrogen protein solubilisation occurring after 2 h in artificial gastric fluid; while 86.0% w/w of total nitrogen protein solubilisation after 5 h in the artificial intestinal fluid[10]. Contrasting with the results obtained in this work, in a previous study[16], the gastroenteric resistance assessment of HGE microcapsules containing lycopene resulted in a rapid release of lycopene at pH 5.5 and 7.0, while no lycopene was released at pH 2.0 and 3.5. In agreement with the results obtained here, Wang et al.[38] stated that the HGE could be digested nearly completely into oligopeptides or amino acids, which can be easily adsorbed into the small intestine. The high digestibility and bioactivity of HGE after oral administration reported by the authors

Figure 5. Optical microscopy of IGEL microparticles coated with protein submitted to in vitro gastrointestinal assay. Top line: simulated gastric conditions: 1h pH 2. Bottom line: simulated intestinal conditions: 24h pH 7. Bars represent 50 µm. HGE: Gelatin; MGE: Collagel®; LGE: Fortigel®. Polímeros, 31(2), e2021018, 2021

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Beraldo, J. C., Nogueira, G. F., Prata, A. S., & Grosso, C. R. F. suggest that these particles can serve to delivery bioactive compounds after consumption.

4. Conclusions Appropriate range of interaction between gelatin and their hydrolisates was found to promote their adsorption on the alginate microparticles. The amount of protein adsorbed on IGEL microparticles increased with the concentration of protein in solution (10%) and reduced with MW, with adsorptions of ~ 47.3, 41.4 and 29.3% when HGE, MGE and LGE were obtained, respectively. The coating of fibrous protein and their hydrolisates on microparticles were poorly resistant to solubilization at gastric conditions, with ~ 39 to 49% protein solubilized at pH 2.0 after 2 h. After switching to intestinal conditions, pH 7.0 during 5 h, the solubility increased to ~ 81, ~ 61 and ~ 95% for HGE, MGE and LGE, respectively. These results suggest that these particles can serve to delivery bioactive compounds after oral administration.

5. Acknowledgements The authors are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 135122/2011-2) for the financial support.

6. References 1. Moura, S. C. S. R., Berling, C. L., Garcia, A. O., Queiroz, M. B., Alvim, I. D., & Hubinger, M. D. (2019). Release of anthocyanins from the hibiscus extract encapsulated by ionic gelation and application of microparticles in jelly candy. Food Research International, 121, 542-552. http://dx.doi. org/10.1016/j.foodres.2018.12.010. PMid:31108779. 2. Liu, Q., Cai, W., Zhen, T., Ji, N., Dai, L., Xiong, L., & Sun, Q. (2020). Preparation of debranched starch nanoparticles by ionic gelation for encapsulation of epigallocatechin gallate. International Journal of Biological Macromolecules, 161, 481-491. http://dx.doi.org/10.1016/j.ijbiomac.2020.06.070. PMid:32534085. 3. Dalponte Dallabona, I., de Lima, G. G., Cestaro, B. I., Tasso, I. S., Paiva, T. S., Laureanti, E. J. G., Jorge, L. M. M., Silva, B. J. G., Helm, C. V., Mathias, A. L., & Jorge, R. M. M. (2020). Development of alginate beads with encapsulated jabuticaba peel and propolis extracts to achieve a new natural colorant antioxidant additive. International Journal of Biological Macromolecules, 163, 1421-1432. http://dx.doi.org/10.1016/j. ijbiomac.2020.07.256. PMid:32738324. 4. Paula, H. C. B., Oliveira, E. F., Abreu, F. O. M. S., Paula, R. C. M., Morais, S. M., & Forte, M. M. C. (2010). ALG/Ca beads as an encapsulation agent of croton zehntneri Pax et Hoffm essential oil. Polímeros: Ciência e Tecnologia, 20(2), 112-120. http://dx.doi.org/10.1590/S0104-14282010005000019. 5. de Vos, P., Faas, M. M., Strand, B., & Calafiore, R. (2006). Alginate-based microcapsules for immunoisolation of pancreatic islets. Biomaterials, 27(32), 5603-5617. http://dx.doi. org/10.1016/j.biomaterials.2006.07.010. PMid:16879864. 6. Hansen, L. T., Allan-Wojtas, P. M., Jin, Y.-L., & Paulson, A. T. (2002). Survival of Ca-alginate microencapsulated Bifidobacterium spp. in milk and simulated gastrointestinal conditions. Food Microbiology, 19(1), 35-45. http://dx.doi. org/10.1006/fmic.2001.0452. 8/9

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30. Ramsden, J. J. (1995). Puzzles and paradoxes in protein adsorption. Chemical Society Reviews, 24(1), 73. http://dx.doi. org/10.1039/cs9952400073. 31. Roach, P., Farrar, D., & Perry, C. C. (2005). Interpretation of protein adsorption: surface-induced conformational changes. Journal of the American Chemical Society, 127(22), 8168-8173. http://dx.doi.org/10.1021/ja042898o. PMid:15926845. 32. Molina-Ortiz, S. E., Puppo, M. C., & Wagner, J. R. (2004). Relationship between structural changes and functional properties of soy protein isolates-carrageenan systems. Food Hydrocolloids, 18(6), 1045-1053. http://dx.doi.org/10.1016/j. foodhyd.2004.04.011. 33. Brassesco, M. E., Fuciños, P., Pastrana, L., & Picó, G. (2019). Development of alginate microparticles as efficient adsorption matrix for protein recovery. Process Biochemistry, 80, 157-163. http://dx.doi.org/10.1016/j.procbio.2019.02.016. 34. Yang, J. M., Tsai, R.-Z., & Hsu, C.-C. (2016). Protein adsorption on polyanion/polycation layer-by-layer assembled polyelectrolyte films. Colloids and Surfaces. B, Biointerfaces, 142, 98-104. http://dx.doi.org/10.1016/j.colsurfb.2016.02.039. PMid:26938325. 35. Malinova, V., Freitag, R., & Wandrey, C. (2004). Adsorption of charged macromolecules on oppositely charged porous column materials. Journal of Chromatography. A, 1036(1), 25-32. http:// dx.doi.org/10.1016/j.chroma.2003.10.087. PMid:15139410. 36. Rahmati, M., & Mozafari, M. (2018). Protein adsorption on polymers. Materials Today Communications, 17, 527-540. http://dx.doi.org/10.1016/j.mtcomm.2018.10.024. 37. Kitabatake, N., & Kinekawa, Y.-I. (1998). Digestibility of bovine milk whey protein and β-lactoglobulin in vitro and in vivo. Journal of Agricultural and Food Chemistry, 46(12), 4917-4923. http://dx.doi.org/10.1021/jf9710903. 38. Wang, L., Liang, Q., Chen, Q., Xu, J., Shi, Z., Wang, Z., Liu, Y., & Ma, H. (2014). Hydrolysis kinetics and radicalscavenging activity of gelatin under simulated gastrointestinal digestion. Food Chemistry, 163, 1-5. http://dx.doi.org/10.1016/j. foodchem.2014.04.083. PMid:24912688. Received: Mar. 05, 2021 Revised: June 21, 2021 Accepted: July 15, 2021

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ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.20210026

Chitosan-based hydrogel for treatment of temporomandibular joint arthritis Fabianne Lima1 , Wanderson Gabriel Melo2 , Maria de Fátima Braga2 , Ewerton Vieira3 , João Victor Câmara4* , Josué Junior Pierote5 , Napoleão Argôlo Neto6 , Edson Silva Filho3  and Ana Cristina Fialho1  Departamento de Patologia e Clínica Odontológica, Universidade Federal do Piauí – UFPI, Terezina, PI, Brasil 2 Centro de Ciências Agrárias, Universidade Federal do Piauí – UFPI, Terezina, PI, Brasil 3 Departamento de Química, Centro de Ciências da Natureza, Universidade Federal do Piauí – UFPI, Terezina, PI, Brasil 4 Departmento de Ciencias Biológicas, Faculdade de Odontologia de Bauru, Universidade de São Paulo – USP, Bauru, SP, Brasil 5 Departamento de Odontologia, Universidade de Santo Amaro – UNISA, São Paulo, SP, Brasil 6 Núcleo Integrado de Morfologia e Pesquisas com Células-tronco, Universidade Federal do Piauí – UFPI, Terezina, PI, Brasil 1

*jvfrazao92@hotmail.com

Abstract To produce polysaccharide-based hydrogels and cerium (Ce3+) doped hydroxyapatite plus chitosan and collagen to enable future applications in the treatment of joint degeneration. Hydrogel production and characterization were performed with Fourier transform infrared spectroscopy (FTIR), thermogravimetry analysis (TGA) and cytotoxicity testing with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. A final biomaterial composition was Kelcogel® Gelana (58%), chitosan (22.3%), Ce3+ doped hydroxyapatite (10.7%) and bovine collagen (9%), or selected aspect material gelatinous physical color with whitish color and can be injected. The biomaterial composition was proven in the FTIR and TGA, which also provided the maximum supported temperature. In the MTT assay, despite the reduction in viability of the experimental group compared to the control group, cell viability remained approximately 90%. In the FTIR and TGA tests, the material composition was proven. The material does not present cytotoxic behavior for the MTT test, being an alternative for the treatment of joint diseases. Keywords: hydrogel scaffold, natural polysaccharides, joint arthritis. How to cite: Lima, F., Melo, W. G., Braga, M. F., Vieira, E., Câmara, J. V., Pierote, J. J., Argôlo Neto, N., Silva Filho, E., & Fialho, A. C. (2021). Chitosan-based hydrogel for treatment of temporomandibular joint arthritis. Polímeros: Ciência e Tecnologia, 31(2), e2021019. https://doi.org/10.1590/0104-1428.20210026

1. Introduction Osteoarthritis (OA) is a slow-progressing chronic degenerative joint disease that causes pain and inability to function. It is characterized by degeneration of the articular cartilage and changes in the structure of the cartilage and the underlying subchondral bone. More recent studies have shown that OA affects not only the articular cartilage, but the entire joint, synovial fluid, calcified cartilage and subchondral bone[1]. The treatment of OA is related, basically, to the use of anti-inflammatory drugs, opioids, analgesics, hormonal drugs and Chinese medicine methods[2]. The use of these treatment routes brings with it a wide variety of side effects and, to date, no drug treatment has been able to provide progressive reversibility of the disease[3]. Therefore, patients resort to non-surgical treatments such as arthrocentesis and, in patients refractory to non-surgical treatment, arthroplasty is an alternative that has traditionally been shown to be

Polímeros, 31(2), e2021019, 2021

more efficient. However, surgical techniques often lead to additional complications and new repair surgeries are often necessary[4,5]. Polysaccharides and biopolymers have been important tools for prevention and in situ treatment of bone and cartilage areas affected by OA. Its clinical application is associated with characteristics of biodegradability, biocompatibility, biofunctionality and non-toxicity[2,4]. This biomaterial has the capacity to establish chemical bonds with living bone and cartilage tissue due to its structure and chemical composition, which are similar to the apatite found in the human skeleton[6]. Gellan gum is an anionic bacterial polysaccharide derived from the bacterium Pseudomonas elodea. Its molecular composition has a tetrasaccharide repeat unit, which consists

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O O O O O O O O O O O O O O O O

Lima, F., Melo, W. G., Braga, M. F., Vieira, E., Câmara, J. V., Pierote, J. J., Argôlo Neto, N., Silva Filho, E., & Fialho, A. C. of two molecules of D-glucose, one of L-rhamnose and one of D-glucuronic acid. The gelano is, structurally, a double helix, formed by two triple helical chains, left-handed and interlaced. This helical geometry is promoted by the connection in the gelano repeating unit. It is well known that gellan gum can form hydrogels, which consist of a threedimensional polymeric network that retains large amounts of water and are promising biomaterials in the treatment of joint degenerations[7,8]. The use of biopolymers such as chitosan and collagen for the preparation of hydrogels has proved to be a good alternative. Collagen is the most abundant protein in mammalian tissues and can modify cell morphology and differentiation, enabling significant biocompatibility when applied in tissue engineering. However, it has insufficiency as an injectable property[9-11]. Chitosan, a natural polysaccharide, is a biocompatible polymer that exhibits a wide variety of useful biological properties, such as anti-cholesterol actions and ion sequestration. Due to their molecular structure and a large active surface area, cellulose fibers can be an ideal matrix for the design of bioactive, biocompatible and intelligent materials[12]. Hydroxyapatite (HAp) ensures greater graft stability, as it promotes improved integration of the cartilage projected into the bone matrix by creating an intermediate transition zone rich in calcium phosphate[13]. The addition of Ce3+ salts has been used as an adjunct in the formulation of hydroxyapatite composites due to its good osteoconductive capacity and efficient antimicrobial activity, which allows a significant improvement in the regeneration of bone tissue and a slight improvement in its mechanical properties[14]. This biomaterial is still relatively unknown in the biomedical community and few studies have explored it for tissue engineering. Like alginate, gellan gum can be used for encapsulation and in vitro culture of cells[15,16]. Gellan gum hydrogels were able to develop nasal chondrocytes and, when injected, were efficient in encapsulating and supporting human articulation chondrocytes, in addition to allowing active synthesis of extracellular matrix components[17]. Also, Kelcogel® Gel Gum is a polysaccharide produced by fermentation and used as a gelling agent that forms gels in contact with mono-, di- and multivalent ions. It was used in this work because it has excellent suspension, low impact on viscosity and stabilization. The present study aimed to produce and characterize a hydrogel based on Kelcogel® gellan gum, hydroxyapatite doped with Ce3+, chitosan and bovine collagen, for application in the treatment of joint degenerations in the temporomandibular joint.

2. Materials and Methods 2.1 Hydrogel production The hydrogel was produced at the Interdisciplinary Laboratory of Advanced Materials at the Federal University of Piauí (LIMAV/UFPI). The synthesis of the hydrogel occurred in four stages: 1. Weighing all the components of the hydrogel; 2. Dissolution of chitosan in 0.25% v/v lactic acid solution; 3. Addition of Ce3+ doped hydroxyapatite to form a suspension; 4. Addition of collagen; 5. Addition of gelanine. In all stages, the system was kept under magnetic 2/6

stirring, for about 30 minutes until the complete dissolution of each component (Agitator Fisatom Mod.752A/3). After shaking, Gelana hydrogel, chitosan, hydroxyapatite and collagen were stored in a cooled environment. After the process, the pH of the hydrogel was analyzed with the aid of a pH-meter.

2.2 Hydrogel characterization 2.2.1 Fourier transform infrared spectroscopy To confirm the production of the hydrogel, Fourier transform infrared spectra (FTIR) of the biomaterial and its components were obtained in a spectrophotometer with attenuated total reflectance (ATR). Transmittance mode and wavelength between 400 and 4000 cm-1 were used. 2.2.2 Thermal analysis The technique of thermogravimetric analysis (TGA) was used to evaluate the stability and thermal decomposition of the polymer obtained as a function of the loss of mass. The thermal analyzer was standardized with a heating rate of 10°C/min, in a nitrogen atmosphere, up to a temperature of 600°C and a sample mass of approximately 7 mg. 2.2.3 Cytotoxicity analysis - MTT assay The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] assay was performed to assess the cytotoxicity of the hydrogel using mesenchymal stem cells from adipose tissue of wistar rats (CTMTA) . A sample of the hydrogel was diluted in α-MEM medium in order to obtain a homogeneous mixture. Then, 100 µL of α-MEM medium and 104 CTMTA were added per well in a 96-well plate and incubated for 24 hours for cell adhesion. After two washes with culture medium to remove cells that did not adhere, the hydrogel solution was tested in triplicate, added with α-MEM in each well, with a final volume of 100 μL and incubated for 24, 48 and 72h. Cells in wells without any addition of hydrogel served as a negative control considered 100% viability. Following the incubation period, 10 µL of MTT diluted in α-MEM at 5 mg/mL were added to each well and the plate was incubated again for 5h. The supernatant was discarded and 100 μL of DMSO was added to dissolve the formed formazan crystals, and these were measured at an optical density of 550 nm in a plate reader. The results were compared and analyzed statistically using the Student’s t test.

3. Results and Discussions 3.1 Obtaining hydrogels The final composition of the biomaterial was Gelana Kelcogel® (58%), chitosan (22.3%), Ce3+ doped hydroxyapatite (10.7%) and bovine collagen (9%) and revealed a gelatinous physical appearance with a whitish color and possible to be injected. The pH obtained at the end of the process was 4.7 at a temperature of 29.5°C.

3.2 Fourier transform infrared spectroscopy In Figure 1, it is possible to observe the similarity of the chitosan spectrum with the spectrum presented in Polímeros, 31(2), e2021019, 2021

Chitosan-based hydrogel for treatment of temporomandibular joint arthritis the hydrogel. In both, it is possible to visualize an axial stretching band of OH, attributed to the hydroxyl group present in chitosan between 3440 to 3480 cm– 1; the bands in the 2854 cm–1 region are assigned to the CH2 groups of the pyroses. All characteristic bands are very similar to those reported in the literature[18-20]. For hydroxyapatite, at 3571 cm– 1, we can see in Figure 1 the symmetrical stretching mode, due to the hydroxyapatite OH- groups. The region, which ranges from 3700 to 2500 cm– 1, has wide bands due to the stretching of hydrogen bound to water molecules (H2O). The band around 1638 cm–1 is derived from the deformation mode of water molecules (H2O). The 700 to 500 cm–1 region presents bands at 632 cm–1, referring to the oscillation mode of ions - OH and the bands at 602, 563 and 575 cm–1 are due to the antisymmetric deformation modes of phosphates[21]. From the peaks obtained through the characterization of the gellan gum by FTIR (Figure 2), the assignment of a transmittance band at 3411 cm–1 for the gellan gum, which indicates the stretching vibration of the OH- group in the gelan hydrogel, was demonstrated. The peak at 1051.11 cm–1 in gellan gum is attributed to the stretching vibration of C-O[22].

3.3 Thermal analysis

the hydrogel did not show cytotoxic behavior for the tested concentration, being compatible with previous studies[24,25]. Due to the reaction between the components and the greater composition of water, the infrared spectrum of the hydrogel assumes characteristics of the spectra of its composition. When observing the spectra of the chitosan sample in Figure 1, overlapping bands of the amides and OH groups of the pyranoses are observed in the regions between 1661 to 1671 cm– 1; angular deformation of N-H (between 1583 to 1594 cm–1), the mode referring to the amides is observed in the region of 1500 cm–1 and between 1200-800 cm–1 the vibrations are associated with the chemical bonds of pyraneses. The bands in the 1640 cm–1 region are attributed to the axial deformation C = O of the carbonyl called νC =O, of the acetamide group, which corresponds to the acetylated part of chitosan. The bands in the 1500 cm–1 range correspond to the N-H vibration in the plane called νN-H. The bands around 1300 and 1400 cm–1 correspond to the symmetrical angular deformation of the CH3 group[18-20]. For hydroxyapatite, at 3571 cm– 1, we can see in Figure 1, the symmetrical stretching mode, due to the hydroxyapatite OH- groups. The weak intensity bands in the region between 2200 to 1950 cm–1 are due to the combinations and overlapping of the phosphate stretching modes (PO4).

The thermogravimetry (TG) and derived thermogravimetry (DTG) curves in Figure 3 reveal a peak between 86°C and 96°C characterizing the loss of mass. This loss is related to the vaporization of H2O contained in the polymer and the volatile compounds produced. This loss percentage allows to estimate the amount of water present in the samples, about 90%. This result is in agreement with the results of the OH ligament presented in the FTIR[23].

3.4 Toxicity test - MTT test The MTT assay assesses cellular metabolic activity and, despite the reduction in absorbance, statistically significant only in the first 24 hours, of the experimental group in relation to the control group, the percentage of cell viability increased when it remained in contact with the hydrogel in 48 and 72 hours (Figure 4). Within 72 hours, cell viability approached the control group with 94.7% viable cells. Thus,

Figure 1. FTIR spectra for the hydrogel components. Polímeros, 31(2), e2021019, 2021

Figure 2. FTIR spectra for gellan gum.

Figure 3. Hydrogel TG, DTG and Differential Exploratory Calorimetry (DSC) curves. 3/6

Lima, F., Melo, W. G., Braga, M. F., Vieira, E., Câmara, J. V., Pierote, J. J., Argôlo Neto, N., Silva Filho, E., & Fialho, A. C.

Figure 4. Comparative graph of cell viability.

The intense bands that appear at 1093 cm–1 and the doublet around 1040 cm–1 originate from the anti-symmetric stretching of the phosphates and the band at 962 cm–1 is due to the symmetrical stretching of the phosphates. The 700 to 500 cm–1 region presents bands at 632 cm–1, referring to the oscillation mode of the OH- ions, and the bands at 602, 563 and 575 cm–1 are due to the antisymmetric deformation modes of the phosphates[21]. Hydrophilia, which has been reported to be the main factor for the hydrogel water molecules’ trapping ability, is contributed by the presence of hydroxyl, carboxyl, sulfonic, amidic and primary amidic functional groups. From the peaks obtained through the characterization of the gellan gum by FTIR, the assignment of a transmittance band at 3411 cm–1 for the gellan gum, which indicates the stretching vibration of the OH- group in the gelan hydrogel, was demonstrated. The peak at 1051.11 cm–1 in gellan gum is attributed to the stretching vibration of C-O[22]. DTG results from the TG curve as a function of time or temperature. The recorded peaks represent each mass loss event [9]. The TG / DTG curves of the hydrogels showed a first event between 25°C to 96°C related to the dehydration process. Hydrogel formulated with ASF interpenetrating polymer A B C (Sercin-NIPAAm-AgNPs) showed peaks of decomposition at 303.7°C and 351.2°C associated with the degradation process[25]. In another study, the TG curve in chitosan-based hydrogels showed initial weight loss at 100°C, associated with the dehydration process also present in this study[22]. The samples presented a second mass loss event at 250°C, indicating the beginning of polymer degradation[26]. The DSC curve reveals endothermic and exothermic stages of the samples. The endothermic curves were observed between the ranges of 21°C and 45°C in the hydrogel, the peaks varied between 25 and 35°C. These events help to understand the formation of the hydrogel since they can be associated with the process of the sol-gel transition of the sample. Due to their hydrophilic characteristic, GG hydrogels provide an ideal environment with the necessary hydration for recovery from injuries, not allowing cytotoxic reactions to the body[27]. The result of this work reveals that the mass of the hydrogels is formed, in large part, by H2O, linked to the gellan gum through the junction zones previously mentioned, revealing an extremely important characteristic for this application[8]. The loss of mass at a temperature of 90°C 4/6

makes the sterilization process with humid heat unfeasible, carried out in an autoclave at 121°C. The impossibility of sterilization with moist heat due to the damage to the material’s physical characteristics, suggests the need for further studies to evaluate the use of other methods in order to make its clinical application in the treatment of OA viable[23]. To evaluate the cytotoxicity of cells, the MTT assay was performed, which allowed the quantification of cell viability and proliferation in hydrogels[9]. Test samples that reduce cell viability to values below 70% should be considered cytotoxic[28]. It was necessary to use cells with easy access, which allow in vitro expansion and the potential for differentiation into chondrocytes. The cell types used in this study, stem cells from adipose tissue, meet these criteria[29]. In the initial 24 hours, the decrease in cell viability may occur due to the adaptation of cells in the presence of the material, this initial reduction is also observed in another study with hydrogels[30]. Within 72 hours, the cell viability of the gel group approached the control group, with approximately 95% viable cells. Thus, the hydrogel did not show cytotoxic behavior at the concentrations tested, being compatible with previous studies[31-33].

4. Conclusions During the characterization of the hydrogel, through Fourier transform infrared spectroscopy (FTIR) it was possible to confirm its production. In the thermogravimetric analysis (TGA), the maximum temperature supported by the biomaterial was found to be between 86°C and 96°C. The cytotoxicity analysis by the MTT assay showed low toxicity to cells, allowing cell viability above 90% in 72 hours, positive result for the application of the biomaterial. The production of hydrogels based on polysaccharides and hydroxyapatite doped with Ce3+ plus chitosan and collagen has shown satisfactory results so far and will enable a new low-cost treatment alternative for joint diseases.

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Lima, F., Melo, W. G., Braga, M. F., Vieira, E., Câmara, J. V., Pierote, J. J., Argôlo Neto, N., Silva Filho, E., & Fialho, A. C. (2020). Enriched Gellan Gum hydrogel as visco-supplement. Carbohydrate Polymers, 227, 115347. http://dx.doi.org/10.1016/j. carbpol.2019.115347. PMid:31590845. 30. Meschini, S., Pellegrini, E., Maestri, C. A., Condello, M., Bettotti, P., Condello, G., & Scarpa, M. (2020). In vitro toxicity assessment of hydrogel patches obtained by cation-induced cross-linking of rod-like cellulose nanocrystals. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 108(3), 687-697. http:// dx.doi.org/10.1002/jbm.b.34423. PMid:31134760. 31. Wang, B., Zhang, S., Wang, Y., Si, B., Cheng, D., Liu, L., & Lu, Y. (2019). Regenerated Antheraea pernyi Silk Fibroin/ Poly(N-isopropylacrylamide) Thermosensitive Composite Hydrogel with Improved Mechanical Strength. Polymers, 11(2), 302. http://dx.doi.org/10.3390/polym11020302. PMid:30960286.

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32. Balasubramanian, R., Kim, S. S., & Lee, J. (2018). Novel synergistic transparent k-Carrageenan/Xanthan gum/Gellan gum hydrogel film: mechanical, thermal and water barrier properties. International Journal of Biological Macromolecules, 118(Pt A), 561-568. http://dx.doi.org/10.1016/j.ijbiomac.2018.06.110. PMid:29949745. 33. Dhivya, S., Saravanan, S., Sastry, T. P., & Selvamurugan, N. (2015). Nanohydroxyapatite-reinforced chitosan composite hydrogel for bone tissue repair in vitro and in vivo. Journal of Nanobiotechnology, 13(1), 40. http://dx.doi.org/10.1186/ s12951-015-0099-z. PMid:26065678. Received: Mar. 05, 2021 Revised: July 04, 2021 Accepted: July 18, 2021

Polímeros, 31(2), e2021019, 2021

ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.20200102

PVC plasticizer from trimethylolpropane trioleate: synthesis, properties, and application Laura de Andrade Souza1,2* , Edson Luiz Francisquetti2 , Rafael Domingos Dalagnol2 , Celso Roman Junior3 , Maria Telma Gomes Schanz4 , Martin Edmund Maier4  and Cesar Liberato Petzhold1  Instituto de Química, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brasil 2 Instituto Federal do Rio Grande do Sul, Campus Farroupilha, Farroupilha, RS, Brasil 3 Instituto Federal do Rio Grande do Sul, Campus Caxias do Sul, Caxias do Sul, RS, Brasil 4 Institut fuer Organische Chemie, Universitaet Tuebingen, Tuebingen, Deutschland

*lauradeandradesouza@gmail.com

Abstract A new oleic acid derivative plasticizer, epoxidized trimethylolpropane trioleate (EPO), has been synthesized and its application in PVC formulations compared with di(2-ethylhexyl) 1,2-cyclohexanoate (DOCH/DEHCH), a commercial phthalate-free plasticizer of petrochemical origin. EPO and their blends with DOCH were added to PVC resin (50 PHR) and the plasticized PVC has been characterized by thermal and mechanical measurements. EPO demonstrated good compatibility with the PVC resin improving the thermal stability and elongation at break. Due to EPO high molar mass, a slight increase in the glass transition temperature and hardness was observed as the content of EPO in the plasticizer blend increased. The results indicate that EPO is a potential plasticizer for PVC when pure and, by replacing 50% of DOCH, the PVC compound shows similar properties to pure DOCH, but better elongation at break and thermal stability. Keywords: plasticizers, PVC, renewable sources, epoxidized trimethylpropane trioleate, dioctyl cyclohexanoate. How to cite: Souza, L. A., Francisquetti, E. L., Dalagnol, R. D., Roman Junior, C., Schanz, M. T. G., Maier, M. E., & Petzhold, C. L. (2021). PVC plasticizer from trimethylolpropane trioleate: synthesis, properties, and application. Polímeros: Ciência e Tecnologia, 31(2), e2021020. https://doi.org/10.1590/0104-1428.20200102.

1. Introduction Poly(vinyl chloride), PVC, is the most widely used thermoplastic in commercial and household applications, such as toys, medical supplies, packaging materials, and others[1]. This range of uses is due to the large number of additives that can be incorporated into PVC, originating from rigid to flexible or translucent to opaque products[2]. The most used additives in PVC are plasticizers. These compounds can efficiently interact with the polymer due to the existence of the polarized C-Cl bond in PVC, which allows the association between the plasticizer and polymer chain during the product’s lifetime[3,4]. Many plasticizers used in PVC are derived from petroleum and have phthalates in their structure. Some studies show that these compounds, containing a phthalate fraction, are very toxic and can cause disturbances in the human reproductive system[5-8]. The restriction of the use of phthalates, in particular dioctyl phthalate (DOP), as a plasticizer is increasing. In the United States, for example, the Environmental Protection Agency (EPA) has already banned its use in toys that go in children’s mouths. Currently, there are many alternative plasticizers to DOP, one of which are secondary alcohol esters of cyclohexanecarboxylic acids, that have been used to prepare flexible PVC compounds

Polímeros, 31(2), e2021020, 2021

with enhanced low temperature flexibility, low toxicity, and improved resistance to outdoor aging[9,10]. Among the commercial non-toxic plasticizers used, di(2-ethylhexyl) 1,2-cyclohexanoate (DOCH) stands out as a highly efficient and phthalate-free primary plasticizer[11-13]. Schilling and Kelly[14] found that DOCH is more compatible with PVC in formulations with high contents of plasticizer (100 PHR or more) and, the blend of DOCH with other plasticizers showed a better performance in the range of 60-120 PHR. However, with the increasing restrictions of environmental policies and the imminent depletion of oil, more and more people are opting to replace fossil plasticizers with ones from renewable sources[15]. Vegetable oil-based plasticizers have a high potential to replace petrochemical plasticizers. Among the vegetable plasticizers, epoxidized soybean oil (ESO) stands out, being among the green plasticizers recognized by the Food and Drugs Administration of the United States[16]. Epoxidized soybean oil (ESO) is known as an additive in plasticized PVC since it functions both as a plasticizer and stabilizer. Therefore, Karmalm et al.[17] studied ESO as a primary PVC plasticizer in the formation of plastisols and noticed that after aging the material became stiff and

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O O O O O O O O O O O O O O O O

Souza L. A., Francisquetti E. L., Dalagnol R. D., Roman Junior C., Schanz M. T. G., Maier M. E., & Petzhold, C. L. opaque, due to the formation of a cross-linking network between ESO and PVC. However, using a suspension PVC resin,it was not observed, concluding that the crosslinking reaction was catalyzed by some component of the PVC plastisol preparation, which was avoided by the addition of stabilizers. Mao et al.[18] studied the ability of ESO, tri(2-ethylhexyl) trimellitate, and their blends as plasticizers for PVC. Both were efficient in plasticizing the polymer and, as expected, plasticizer migration decreased with increasing ESO content. In addition, PVC compounds with higher ESO content showed lower Shore D hardness. Given this, ESO can fully or partially replace the fossil plasticizer. Epoxidized soybean oil and its methyl esters were tested as plasticizers for PVC by Chaudhary et al.[19]. The authors showed that ESO, even having a high molar mass, proved compatible with PVC, plasticizing it efficiently. This study shows that soybean oil and its derivatives can be used as sustainable plasticizers for PVC and meets the requirements for coating wires and cables, among others. When PVC is used in food packaging, the migration of plasticizers is of increasing concern in terms of toxicity and environmental pollution. Therefore, Coltro et al.[20] studied the migration of several plasticizers from the PVC matrix, including ESO. The authors used olive oil as a simulant for fatty foods and it was proven that ESO showed the highest rate of exudation. Choi et al.[21] also evaluated the migration of ESO simulating fatty food environment using n-heptane. The samples with ESO showed a high exudation rate, around 35%. These results demonstrate that the migration of plasticizers should be constant. Besides, the search for new plasticizers from ESO or other green sources has been gaining space in recent years[22]. Jia et al.[23] synthesized new polyol esters using soybean oil, with glycerol or pentaerythritol, of which two were acetylated and two were additionally epoxidized. The pentaerythritol-derived esters showed the best performance as a PVC plasticizer. Zheng et al. performed the transesterification of methyl esters from waste cooking oil with 2-ethylhexanol[24]. These epoxidized esters were tested as plasticizers and the obtained PVC compounds showed improved mechanical and thermal performance, and the migration resistance was reduced. In addition, 2-ethylhexyl epoxidized esters from cooking oil proved to be an effective alternative to dioctyl phthalate, replacing about 40% of the total plasticizer. In 2015, Kandula et al.[25] added several functional groups such as epoxy, acetoxy, methoxy, thiirane, and aziridine to soybean oil and evaluated it as a PVC plasticizer. The high viscosity and darker color of the aziridine and thiirane derivatives limited their usefulness, while the physical properties of the other derivatives were acceptable. Methoxy and acetoxy plasticizers of soybean fatty acid esters (methyl and n-butyl) showed good PVC compatibility, high efficiency, and gelling properties comparable to the properties of the commercial plasticizer, di-isononyl phthalate. Owing to environmental concerns and the impending depletion of petroleum, this study developed a new green, phthalate-free plasticizer. This bio plasticizer was synthesized from the epoxidation of a vegetable triester, trimethylolpropane 2/10

trioleate. The difference to a triglyceride is the absence of hydrogen-β, which increases the thermal stability in relation to soybean oil derivatives[26]. In addition, epoxy groups were added for better compatibility of the bioplasticizer with PVC. This product was tested as a plasticizer, either pure or mixed with DOCH at different concentrations (10 - 50 PHR), and the plasticizer efficiency was investigated through the mechanical and thermal properties.

2. Materials and Methods 2.1 Materials PVC was purchased from Braskem (Norvic® SP1000) having a volumetric density of 0.52 ± 0.03 g.cm-3. Di(2-ethylhexyl)-1,2-cyclohexane dicarboxylate (DOCH) was purchased from Elekeiroz® (EKFLEX® 8815), with a density of 0.952 g.cm-3 and a molar mass of 396 g.mol-1. Trimethylolpropane trioleate (Emulchem OTMP) was purchased from Chemax® with a molar mass of 924 g.mol-1. Formic acid and 30% hydrogen peroxide were purchased from Synth and Nuclear, respectively. External lubricant (Stearic Acid, Gotalube), thermal stabilizer (Ca-Zn, Gotalube GL-4522®), and optical brightener (Hostalux KCD®) were donated by the Federal Institute of Rio Grande do Sul (IFRS).

2.2 Epoxidized Trimethylolpropane Trioleate Synthesis (EPO) Trimethylolpropane trioleate, OTMP, (30.92 g, 0.10 mol double bond) was mixed with formic acid (7.55 mL, 0.2 mol) in a round-bottomed tritubulated flask under mechanical stirring followed by the addition of an aqueous solution of 30% hydrogen peroxide (204 mL, 2 mol) drop by drop for one hour (maintaining a molar ratio of 1:20:2 double bond/hydrogen peroxide/formic acid). This addition was performed at room temperature, and the reaction proceeded for 4 h at 65 ºC. Thereafter 100 mL of a saturated solution of sodium bisulfite was added and the mixture was stirred for 15 min. The mixture was placed in a separation funnel and washed with 130 mL diethyl ether. The organic phase was separated and washed with 200 mL deionized water and 200 mL brine, and the pH of the solution should be around 7. The aqueous solution was discarded, the organic phase dried with anhydrous sodium sulfate, filtered, and concentrated in a rotary evaporator. The isolated product was characterized by 1H-NMR, TGA, GPC, and Brookfield Viscosity. Yield: 55% OTMP= 1H NMR (400 MHz, CDCl3) δ (ppm): 5.33 (He, m, 5.97H), 4.01 (Ha, s, 6H), 2.29 (Hb, t, 6.10H), 1.99 (Hd and Hf, m, 11H), 1.60 (Hc, m, 6.28H), 1.29 (CH2, m, 60H), 0.87 (CH3, t, 12H). (Figure S1 – Supplementary Material). EPO = 1H NMR (400 MHz, CDCl3) δ (ppm): 3.99 (Ha, s, 6H), 2.87 (He, m, 5.76H), 2.28 (Hb, t, 6.57H), 1.59 (Hd,f, m, 6.24H), 1.47 (Hc, m, 20.97 H), 1.30 - 1.25 (CH2, m, 40H), 0.86 (CH3, t, 12H). (Figure S2 – Supplementary Material).

2.3 PVC compound preparation 100g of PVC resin (sp1000) was mixed in an intensive mixer, developed by IFRS, with external lubricant (Stearic Acid, 0.5 PHR), thermal stabilizer (Ca-Zn, 3 PHR), and an Polímeros, 31(2), e2021020, 2021

PVC plasticizer from trimethylolpropane trioleate: synthesis, properties, and application optical brightener (Hostalux, 0.2 PHR) until a temperature of 80 ºC was reached, by shearing with 7200 rpm. At this temperature, the plasticizers EPO and/or DOCH (50 PHR) were added, the temperature was increased to 95 ± 1 ºC, and kept stirring for 30 minutes to obtain the dry blend. After this time, the dry blend obtained was calendered in calender with dimensions 149 x 92 x 59 cm (H x W x D, MH150C, MH Equipaments®) and two horizontal stainless steel rolls (Ø 11 x 23 cm, 25 rpm) at 130 ºC, forming rectangular films with 22 x 33 cm length versus width, and 1 mm thickness. The films were cut into 11x11 cm squares, stacked, and pressed at 150 °C with 8 tons of pressure for 20 min. The pressed squares were cut according to type IV specimen [ASTM D638-02], with 4 ± 1 mm thick and 11 cm in total length, and stored at 25 °C. The samples were labeled 50DOCH, 4010, 2525, 1040, and 50EPO according to Table 1.

2.4 Characterization of EPO and PVC compounds 2.4.1 1H NMR measurements H NMR spectra were performed in equipment Bruker 400 MHz. Samples were dissolved in deuterated chloroform (CDCl3), using the chloroform signal at 7.26 ppm for calibration. 1

2.4.2 Infrared spectroscopy (FTIR) The Infrared spectra (IR) were recorded on a PerkinElmer spectrometer model Frontier in the ATR (attenuated total reﬂectance) mode, the spectra expressed by the wavenumber ratio (cm-1) being the sample analyzed from 6000 to 400 cm-1. 2.4.3 Gel Permeation Chromatography (GPC) Analyzes were performed on Viscotek chromatograph with GPCmax module (VE2001) equipped with detector TDA402 and Shodex columns (806M, 805L, 804L, and 803L). The samples were solubilized in THF and filtered with a Chromafil Xtra PVDF - 45/25 filter with a pore size of 0.45 μm before injection. For calibration standard monodisperse polystyrene samples were used.

and relative humidity of 50 ± 10% using a Shore A durometer (Woltest®, 0-100 Shore A). The samples were at least 5.0 mm thick and the analysis was performed at five different positions, 5 mm apart. The durometer readings were taken after ten seconds of penetration of the durometer tip into the sample. 2.4.7 Tensile properties To determine the tensile properties of PVC samples the universal testing machine (DL2000, EMIC, Brazil) was used, the test was based on ASTM D638-14 at a speed of 50 mm.min-1 with a load cell of 500 Kgf. Five samples were conditioned in a standard laboratory atmosphere. A 25 mm strain gauge was used, and the samples were 6 mm wide in the working area and 5 ± 1 mm thick. 2.4.8 Thermogravimetric analyzer TGA measurements were performed in a Perkin Elmer TGA 4000, based on ASTM E1131-08. The analysis parameters used were a heating ramp from 30 °C to 900 °C at a heating rate of 20 °C.min-1 under an inert atmosphere (nitrogen). The initial mass of the samples was 9.500 mg ± 1 mg. 2.4.9 Migration testing To evaluate the migration of polymeric matrix plasticizers tests were performed in n-heptane according to ASTM D1239-14[27] and resolution n°105 of ANVISA (National Health Surveillance Agency, Brazil)[28]. This test consists of weighing the materials and after submerging seven (7) days in the n-heptane, and after this time the samples are weighed again.

3 Results and Discussion 3.1 EPO synthesis

According to ASTM E1640, the DMA analysis was performed on a Perkin Elmer DMA 8000 equipment at a heating rate of 2 °C.min-1 using a strain amplitude of 0.010 mm and a frequency of 1 Hz.

Since the 1950s, conventional epoxidation, also called Prilezhaev epoxidation, can be used to epoxidize vegetable oils. This conventional epoxidation method uses peracids, with performic, peracetic, and m-chloroperbenzoic being the most common[29]. Even though benzoic peracids have high activity in epoxidation, they are characterized by high cost and difficulty in separating the oil and the aqueous phase. Among the other two peracids, performic acid is preferable to peracetic acid, because the latter requires the addition of a strong and corrosive acid as a catalyst (e.g. HNO3, HCl, H2SO4) and higher temperatures, which can cause hydrolysis of the oxirane ring formed[30,31]. Therefore, when performing reactions on larger scales, it is preferable to use the in situ generated performic acid and for this reason, this method was chosen for the epoxidation of trimethylolpropane trioleate (OTMP) to obtain EPO from the epoxidized product as shown in Figure 1.

2.4.6 Hardness Test (Shore A)

3.2 NMR analysis

Based on ASTM D2240-15, the test was performed in a standard laboratory atmosphere at a temperature of 23 ± 2 ºC

The following signals are observed in the OTMP spectrum (Figure 2A): at 5.3 ppm for oleate fraction of

2.4.4 Brookfield viscosity The viscosity of the solutions was measured at 60°C on a DVII + PRO digital viscometer (Brookfield) and the results were expressed as dynamic viscosity (cP). 2.4.5 Mechanical dynamic analysis

Table 1. Plasticizer content in each formulation (PHR). DOCH EPO

50DOCH 50 0

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4010 40 10

2525 25 25

1040 10 40

50EPO 0 50

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Souza L. A., Francisquetti E. L., Dalagnol R. D., Roman Junior C., Schanz M. T. G., Maier M. E., & Petzhold, C. L.

Figure 1. Epoxidation of OTMP to obtain EPO.

Figure 2. 1H-NMR spectra of (A) OTMP and (B) EPO (400 MHz, CDCl3)[1].

vinyl hydrogens; at 4.0 ppm, the methylene hydrogens CH2-O; at 2.3 ppm, the methylene hydrogens CH2-CO; at 2.0 ppm, allylic methylene hydrogens CH2-CH = CH, at 1.6 ppm, the methylene hydrogens CH2-CH2-CO, at 1.3 ppm, the aliphatic methylene hydrogens and 0.9 ppm methyl hydrogens -CH3. The lower intensity signals at 3.5 and 2.8 ppm refer to hydrogens derived from linoleate present in the starting reagent[32]. The EPO NMR spectrum (Figure 2B) was confirmed the complete consumption of the double bonds (signal disappearance at 5.0 and 2.0 ppm) and the appearance of the signal at 2.9 ppm, corresponding to the hydrogens of the oxirane ring and at 1.5 ppm of the methylene hydrogens adjacent to the oxirane. The product was obtained with a conversion of 100% of the double bonds and approximately 81% epoxy selectivity (calculated by Equation 1 using the integration obtained through 1H NMR spectrum), giving a functionality of 2.3 epoxy groups.mol-1. As it is known from literature[33] excess formic acid and long reaction times can lead to opening reactions of the epoxy group resulting in OH/ formiate groups. 4/10

Selectivity =

H epoxide *100 (1) H doublebond

3.3 FTIR analysis The FTIR spectra of OTMP and EPO are shown in Figure 3. In the OTMP spectrum was observed the double bond signal at 3014 cm -1 (=C-H stretch due to cis unsaturated fatty acid), which disappears in EPO IR-spectrum and a new absorption band at 810 cm-1 attributed to the C-O-C asymmetric bending of the epoxy group is observed[34].

3.4 GPC and brookfield analysis Table 2 shows the number average molar mass and Brookfield viscosity of the OTMP, EPO, and the commercial PVC plasticizer, DOCH. The number average molar mass was determined by GPC using a calibration curve with standard monodisperse polystyrene samples and THF as eluent. Therefore, the obtained Mn is a relative value and depends on the hydrodynamic volume of the molecule in Polímeros, 31(2), e2021020, 2021

PVC plasticizer from trimethylolpropane trioleate: synthesis, properties, and application solution. The compounds based on trimethylolpropane esters showed higher viscosity and higher molar mass than DOCH. Although EPO and OTMP have similar molar masses, EPO showed a higher viscosity than OTMP, probably due to the presence of stronger intermolecular interactions due to the presence of epoxy groups. However, etheriﬁcation and esteriﬁcation of the epoxide groups of EPO generating compounds with higher molar mass cannot be ruled out as observed in GPC curves of the plasticizers in Figure S3 (Supplementary Material).

3.5 TGA analysis TGA/DTG curves of the plasticizers performed under an inert atmosphere are presented in Figure 4, and initial decomposition Tonset, and maximum degradation rate temperature (Tmax) are summarized in Table 3. The compounds derived from vegetable oil (OTMP and EPO) showed higher thermal stability than DOCH, which presents a degradation process with Tonset at 204 ºC and Tmax at 315 ºC. While OTMP presented only one process of weight loss with Tonset at 342 ºC and Tmax at 441 ºC, EPO degraded in two processes: from 200 to 400 oC, due to the elimination of formiate groups attached to the opened oxirane rings corresponding to 11% weight loss and from 400 to 500 oC, relative to the main carbon chains degradation. As expected, the epoxidation of double bonds increases the thermal stability of the compounds[35,36]. None of the three compounds presented residue.

3.6 Characterization of PVC compounds Characterization was performed on the specimens produced according to the formulation in Table 1. It was not possible to obtain a specimen as pure PVC, because degradation occurs during calendering, and the OTMP cannot plasticize PVC.

Figure 3. FTIR spectra of OTMP and EPO.

Figure 4. TGA and DTG curves of the plasticizers DOCH, OTMP, and EPO (N2, 20oC.min-1).

3.6.1 DSC analysis An essential parameter of the plasticizer efficiency is the reduction of the glass transition temperature (Tg) compared to pure PVC. The Tg of the PVC resin used is 86°C (determined by DSC in Figure S4 - Supplementary Material). The DSC heating curve of pure EPO showed a melting peak around -10 °C (Figure 5) which was not

Table 2. Number average molar mass and Brookfield viscosity of PVC plasticizers. DOCH OTMP EPO

Mn (g.mol-1) 373 1325 1317

Viscosity (cP) 9.49 22.35 52.85

Figure 5. DSC heating curves of EPO and PVC 50EPO (20 °C/min, N2 atmosphere).

Table 3. Thermal degradation temperatures of DOCH, OTMP, and EPO.

First Process

Second Process

Tonset (ºC) Tmax (ºC) Loss Weight (%) Tonset (ºC) Tmax (ºC) Loss Weight (%)

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DOCH 204 315 100 ----

OTMP 342 441 100 ----

EPO 219 279 11 364 453 89

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Souza L. A., Francisquetti E. L., Dalagnol R. D., Roman Junior C., Schanz M. T. G., Maier M. E., & Petzhold, C. L. observed in the plasticized PVC, indicating compatibility between the PVC and the plasticizer. As observed in Table 4, the addition of EPO to PVC decreased the glass transition temperature by about 30 ºC compared to resin, as there is a weakening of intermolecular interactions and an increase in the free volume between the PVC chains resulting in higher overall mobility of the main chain[37-40]. The polar ester and epoxy groups of the plasticizers interact with the polar part of the PVC polymer chain, spacing out the PVC molecules reducing their friction, and increasing the compatibility[1]. Furthermore, the long non-polar alkyl chain can increase the free volume between polymer molecules, resulting in a reduction of Tg[41]. 3.6.2 Mechanical properties The compatibility and plasticized effect of EPO in PVC were investigated by DMA measurements. The Tg of the PVC compounds was obtained by the maximum peak of the tan δ. Figure 6 shows tan δ versus temperature curves of plasticized PVC with DOCH and EPO mixtures. All compounds presented only one symmetrical tan δ peak, indicating that the materials are uniform, in other words, the plasticizers are compatible with PVC[6,42]. Therefore, the sample 50DOCH presents a lower glass temperature than EPO plasticized samples, since EPO has a molar mass three times higher than DOCH. Similar results were observed by Omrani et al.[43] using a bio-based plasticizer from oleic acid. However, in the work of Ma et al.[44], the addition of a compound with a high molar mass causes an increase in the free volume of the molecule and, consequently, a decrease in the glass transition temperature. This effect was related to the dendritic structure of the molecule, which promotes the separation of PVC molecules. Tan et al.[41] also observed that the incorporation of flexible oxyethyl units in the plasticizer can efficiently reduce the Tg of the plasticized PVC, although the higher molar mass. The storage modulus of the PVC compounds (Figure 7) increases as the amount of EPO increases, indicating greater resistance to deformation. These data corroborate with the high Tg and Shore A hardness values (Table 4), because the higher the energy storage, the lower the free volume, which leads to increased stiffness of the PVC compound. Similar results were reported by Feng et al.[45] when using cooking oil modified with an alkyl diacid as a bio plasticizer. The efficiency of the plasticizer can also be described by hardness measurements because it is associated with the free volume in the PVC matrix[46]. The lower the hardness of the material, the higher the mobility of the PVC chains, consequently, the better is the plasticization efficiency. The addition of EPO to the PVC matrix has slightly increased the hardness of the material regarding the formulation with the commercial DOCH (Table 4), which should be associated with its higher molar mass in comparison with DOCH.

Omrani et al. also obtained similar Shore A hardness for PVC plasticized with a bio-based plasticizer synthesized from the oleic acid and thioglycolic acid[42]. Mehta et al.[47] prepared PVC plasticized with a blend containing 26 PHR of DOP and 26 PHR of an epoxy acylated ricinoleic acid binary alcohol ether ester and obtained a compound with a hardness of 95 Shore A. This value is much higher when compared with the hardness of 50EPO (89 Shore A), using 50 PHR of vegetable triester. Table 5 shows the mechanical properties of PVC compounds. The addition of EPO increases the elastic modulus and the elongation at break of the material; however, no significant variation of tensile strength was observed indicating that EPO is a potential plasticizer to substitute the petrochemical DOCH. The elongation at break reached the highest value (24% higher than 50DOCH) when equal weight proportions of DOCH and EPO are presented (2525). Similar results were

Figure 6. Tan δ versus temperature for PVC compounds.

Figure 7. Storage modulus versus temperature of PVC compounds.

Table 4. Properties of PVC compounds. Tg (ºC) Hardness (Shore A)

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50DOCH 37 82

4010 38 83

2525 40 86

1040 48 88

50EPO 46 89

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PVC plasticizer from trimethylolpropane trioleate: synthesis, properties, and application Table 5. Mechanical properties of PVC compounds. Sample 50DOCH 4010 2525 1040 50EPO

Tensile strength at break (MPa) 14.4 ± 0.8 14.1 ± 1.0 14.8 ± 0.2 14.3 ± 0.1 14.2 ± 0.6

Young’s Modulus (MPa)

Tensile strength (MPa)

Elongation at break (%)

31.5 ± 4.8 32.3 ± 2.9 34.7 ± 2.4 69.7 ± 0.7 64.2 ± 2.5

2.3 ± 0.1 2.5 ± 0.2 2.4 ± 0.1 2.4 ± 0.1 2.6 ± 0.1

215 ± 22 221 ± 74 268 ± 20 231 ± 04 227 ± 26

Table 6. TGA data of PVC resin and compounds. PVC-Resin 50DOCH 4010 2525 1040 50EPO

Tonset (oC) 238 230 257 264 276 278

T50 (oC) 346 325 333 347 358 379

TM1 (oC) 318 319 312 329 340 341

TM2 (oC) 477 479 481 483 477 479

Residue (%) 4.9 3.9 5.6 6.1 5.8 5.4

observed for the plasticizing effect of epoxy soybean oil compared with dioctyl phthalate petrochemical plasticizer[48]. Feng et al. also obtained a much higher elongation at break using a natural origin plasticizer[45]. 3.6.3 TGA analysis The thermal stability of the PVC compounds was investigated by thermogravimetric analysis (TGA) as showed in Figure 8. The thermal data as initial decomposition and 50% loss weight temperatures (Tonset and T50) and, the maximum degradation rate temperature corresponding to each process (TM1 and TM2) were summarized in Table 6. All PVC compounds showed the characteristic two-loss weight processes. The first one corresponds to the PVC dehydrochlorination and the second to the formation of aromatic and allyl compounds followed by degradation of hydrocarbons chains. After the hydrogen chloride loss and breakage of double bonds, cross-linking occurs between polymer chains originating carbon black, which explains the residue of 4 to 6% regarding the initial weight of the PVC resin[49,50]. While for the formulation with commercial plasticizer DOCH (50DOCH) the thermal stability decreased, all specimens formulated with EPO improved the thermal stability. The initial degradation temperature, T50, and TM1 increase as the content of EPO increases. Comparing pure PVC resin with 50EPO, an increase of 40 ºC in the Tonset was observed, better than the results obtained for plasticizers based on epoxidized soybean oil[45,48]. Only TM2 remains very close to PVC resin confirming that the plasticizer acts directly on the loss of hydrochloric acid from PVC. As described in the literature the epoxy groups can absorb hydrogen chloride degraded by light and heat, preventing the continuous decomposition of PVC and prolonging its lifetime[1]. 3.6.4 Migration test The migration stability of the EPO plasticizer in PVC blends was preliminarily investigated by leaching test using n-heptane, which is a good solvent for the bio plasticizers and Polímeros, 31(2), e2021020, 2021

Figure 8. TGA and DTG curves of PVC compounds (N2, 20 °C.min-1).

a fatty food simulant[27,28]. The plasticizers presented lower leaching resistance in n-heptane (weight loss higher than 10%) since they are organic compounds. Unsurprisingly the extraction of a high molar mass plasticizer EPO was lower than the low molar mass plasticizer DOCH, improving the resistance to exudation[51]. The weight loss results (average value among five samples) were 19% for 50DOCH, 13% for 50EPO, and 10% for 2525.

4. Conclusions Epoxidized trimethylolpropane trioleate (EPO), an acid oleic derivate, was easily synthesized and used as PVC environment-friendly plasticizer in substitution to commercial di(2-ethylhexyl) 1,2-cyclohexanoate (DOCH). Blends of DOCH/EPO (50 PHR) were added to PVC and their properties investigated. The presence of the epoxidized renewable source compound had a direct influence on the degradation temperature of PVC, making the polymer matrix more stable for all formulations compared to the commercial plasticizer DOCH. EPO demonstrated good compatibility with PVC, which is evidenced by the presence of only one tan δ peak 7/10

Souza L. A., Francisquetti E. L., Dalagnol R. D., Roman Junior C., Schanz M. T. G., Maier M. E., & Petzhold, C. L. in the DMA analysis and the improvement of mechanical characteristics of PVC compounds. Thus, the tensile strength, Young modulus, and elongation at break increase achieving the best effect for a mixture containing equal amounts of DOCH/EPO. Therefore, it can be stated that EPO is a potential bio plasticizer that can replace DOCH, a plasticizer of fossil origin, and by replacing DOCH with 50% EPO significantly improved the mechanical and thermal properties of the PVC compound.

5. Acknowledgments The authors acknowledge CAPES and DAAD for the financial support in the partnership program PROBRAL (project nr. 88887.144057/2017-00). L. Souza thanks CAPES and Federal Institute of Rio Grande do Sul (IFRSCampus Farroupilha).

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polyvinyl chloride materials. Polymer International, 68(3), 456-463. http://dx.doi.org/10.1002/pi.5730. 39. Bocqué, M., Voirin, C., Lapinte, V., Caillol, S., & Robin, J. J. (2016). Petro‐based and bio‐based plasticizers: chemical structures to plasticizing properties. Journal of Polymer Science, Part A: Polymer Science, 54(1), 11-33. http://dx.doi. org/10.1002/pola.27917. 40. Ang, D. T. C., Khong, Y. K., & Gan, S. N. (2016). Palm oil‐ based compound as environmentally friendly plasticizer for poly (vinyl chloride). Journal of Vinyl & Additive Technology, 22(1), 80-87. http://dx.doi.org/10.1002/vnl.21434. 41. Tan, J., Liu, B., Fu, Q., Wang, L., Xin, J., & Zhu, X. (2019). Role of the oxethyl unit in the structure of vegetable oilbased plasticizer for PVC: an efficient strategy to enhance compatibility and plasticization. Polymers, 11(5), 779. http:// dx.doi.org/10.3390/polym11050779. PMid:31052451. 42. Jia, P., Zhang, M., Hu, L., Feng, G., Bo, C., & Zhou, Y. (2015). Synthesis and application of environmental castor oil based polyol ester plasticizers for poly (vinyl chloride). ACS Sustainable Chemistry & Engineering, 3(9), 2187-2193. http://dx.doi.org/10.1021/acssuschemeng.5b00449. 43. Omrani, I., Ahmadi, A., Farhadian, A., Shendi, H. K., Babanejad, N., & Nabid, M. R. (2016). Synthesis of a bio-based plasticizer from oleic acid and its evaluation in PVC formulations. Polymer Testing, 56, 237-244. http://dx.doi.org/10.1016/j. polymertesting.2016.10.027. 44. Ma, Y., Song, F., Hu, Y., Kong, Q., Liu, C., Rahman, M. A., Zhou, Y., & Jia, P. (2020). Highly branched and nontoxic plasticizers based on natural cashew shell oil by a facile and sustainable way. Journal of Cleaner Production, 252, 119597. http://dx.doi.org/10.1016/j.jclepro.2019.119597. 45. Feng, G., Ma, Y., Zhang, M., Jia, P., Liu, C., & Zhou, Y. (2019). Synthesis of bio-base plasticizer using waste cooking oil and its performance testing in soft poly (vinyl chloride) films. Journal of Bioresources and Bioprodcts, 4(2), 99-110. http:// dx.doi.org/10.21967/jbb.v4i2.214. 46. Schiller, M. (2015). PVC additives: performance, chemistry, developments, and sustainability. Munich: Carl Hanser Verlag. http://dx.doi.org/10.3139/9781569905449. 47. Mehta, B., Kathalewar, M., & Sabnis, A. (2014). Diester based on castor oil fatty acid as plasticizer for poly (vinyl chloride). Journal of Applied Polymer Science, 131(11), 40354. http:// dx.doi.org/10.1002/app.40354. 48. Feng, G., Hu, L., Ma, Y., Jia, P., Hu, Y., Zhang, M., Liu, C., & Zhou, Y. (2018). An efficient bio-based plasticizer for poly (vinyl chloride) from waste cooking oil and citric acid: synthesis and evaluation in PVC films. Journal of Cleaner Production, 189, 334-343. http://dx.doi.org/10.1016/j.jclepro.2018.04.085. 49. Yu, J., Sun, L., Ma, C., Qiao, Y., & Yao, H. (2016). Thermal degradation of PVC: a review. Waste Management (New York, N.Y.), 48, 300-314. http://dx.doi.org/10.1016/j.wasman.2015.11.041. PMid:26687228. 50. Marongiu, A., Faravelli, T., Bozzano, G., Dente, M., & Ranzi, E. (2003). Thermal degradation of poly (vinyl chloride). Journal of Analytical and Applied Pyrolysis, 70(2), 519-553. http://dx.doi.org/10.1016/S0165-2370(03)00024-X. 51. Wang, Y., Nie, X., & Li, X. (2016). Synthesis and characterization of novel pentaerythritol ester as PVC plasticizer. Journal of Applied Polymer Science, 133(47), 44227. http://dx.doi. org/10.1002/app.44227. Received: Dec. 17, 2020 Revised: June 15, 2021 Accepted: Aug. 09, 2021

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Supplementary Material Supplementary material accompanies this paper. Figure S1: 1H-NMR spectrum of OTMP (CDCl3, 400 MHz). Figure S2: 1H-NMR spectrum of EPO (CDCl3, 400 MHz). Figure S3: GPC curves of plasticizers DOCH, OTMP, and EPO. Figure S4: Second heating DSC curve of PVC resin (Norvic®, sp1000). This material is available as part of the online article from https://www.scielo.br/j/po

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Polímeros, 31(2), e2021020, 2021

ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.20210017

Development of electrically conductive polymer nanocomposites for the automotive cable industry Miguel Guerreiro1 , Joana Rompante1 , André Costa Leite2* , Luís Paulo Fernandes2 , Rosa Maria Santos2 , Maria Conceição Paiva3  and José António Covas3  Polo de Inovação em Engenharia de Polímeros, Universidade do Minho, Campus de Azurém, Guimarães, Portugal 2 Companhia de Fios e Cabo, Lda. – Coficab, Guarda, Portugal 3 Instituto de Polímeros e Compósitos, Universidade do Minho, Campus de Azurém, Guimarães, Portugal 1

cleiteandre@gmail.com

Abstract Environmental concerns and the urgent need for reduction of fossil fuel consumption motivate materials research towards increased transportation efficiency. This work investigates the possibility of reducing the weight of electrical cables in automotive applications by replacing part of the metallic screen with electrically conductive polymer/carbon nanotube (CNT) nanocomposites. PP and PA12 were tested as possible matrices and the melt processability of the composites prepared by melt mixing was assessed for compositions up to 4 CNT wt. %. The tensile and flexural mechanical properties, the electrical conductivity, as well as the electromagnetic shielding effectiveness were evaluated. The performance of PA12/CNT composites was much higher than that of PP/CNT equivalents, due to better dispersion. It was demonstrated that, at industrial production scale, these materials could achieve a reduction of 4-20 weigth % relative to a standard automotive cable. Keywords: high voltage cables, carbon nanotubes, nanocomposites, electrical conductivity, wire insulation. How to cite: Guerreiro, M., Rompante, J., Leite, A. C., Fernandes, L. P., Santos, R. M., Paiva, M. C., & Covas, J. V. (2021). Development of electrically conductive polymer nanocomposites for the automotive cable industry. Polímeros: Ciência e Tecnologia, 31(2), e2021021. https://doi.org/10.1590/0104-1428.20210017

1. Introduction The automotive industry faces the need to develop new technological solutions to meet increasing societal demands, one of them being the overall reduction of the weight of vehicles[1-3]. This is directly related to the optimization of energy efficiency, through the decrease of fuel consumption and the maximization of battery performance, ideally associated with the simplification of manufacturing processes[4-9]. Thermoplastic polymers used at the industrial scale are typically electrical insulators, however their electrical response can be modified by mixing them with electrically conductive materials[10]. In this context, the emergence of nanotechnology brought the potential of material modification by incorporation of nanoparticles with specific properties, allowing the development of composites with diverse properties at very low nanoparticle loads[11,12]. Some of the solutions explored are based on the use of carbon nanoparticles such as graphene and single-wall (SWCNT) or multi-wall carbon nanotubes (MWCNT), dispersed in a polymeric matrix at a concentration that allows the formation of a percolation network[13-15], producing an electrically conductive nanocomposite[16-19]. The advantage of using nanoparticles is the small concentration required to modify the electrical response of the composites from insulating to conductive, the scientific community reporting concentrations ranging from

Polímeros, 31(2), e2021021, 2021

0,002% to 5% (w/w)[20-25] depending on the morphology and properties of the CNT, and on the preparation method (the lower concentration range is usually attained when using solution mixing, whilst higher concentrations are required when adopting melt mixing). In order to achieve an electrically conductive polymer composite at a low carbon nanoparticle content, a sufficiently good nanoparticle dispersion and distribution in the polymer is necessary[26-28]. Of the different methods that may be used to produce electrically conductive polymer composites, the more advantageous for industrial application is based on melt mixing by co-rotating twin screw extrusion[22,28], as the control of parameters such as residence time and stress levels through proper selection of operating conditions and screw profile allow to obtain good nanoparticle dispersion[27]. Several studies reported the mechanical and electrical properties of nanocomposites containing CNT. For example, the addition of 1, 2 and 3 wt.% of CNT by melt compounding with a polypropylene (PP) copolymer through the dilution of a masterbatch containing 20 wt.% of CNT, and the comparison with direct compounding of 1, 2 and 3 wt.% CNT into PP, was investigated[29]. An increase in electrical conductivity was obtained by both methods, however due to poorer dispersion of CNT achieved by masterbatch dilution,

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Guerreiro, M., Rompante, J., Leite, A. C., Fernandes, L. P., Santos, R. M., Paiva, M. C., & Covas, J. V. these composites showed comparitively lower conductivity than those produced by direct CNT compounding into the PP matrix. Polyamide 12 (PA12)/CNT composites were produced by melt mixing in a microcompounder, and the influence of parameters such as screw rotation and CNT concentration was studied[30]. Optimal volume conductivity was observed at intermediate screw speeds. The present work investigates the possibility of reducing the weight of a standard high voltage cable used in the automotive industry by replacing the conventional metallic screen with an electrically conductive polymer/ carbon nanotube (CNT) nanocomposite. Polypropylene and polyamide 12 were tested as possible matrices, and compositions of up to 4 weight % CNT were prepared by melt mixing and characterized, as higher concentrations would compromise the processability. The best performing nanocomposite (based on PA12) was used for the manufacture of cables under industrial conditions, which were subsequently characterized according to the relevant standards. A weight reduction in the range 4 to 20% was achieved.

2. Materials and Methods 2.1 Materials A Polypropylene cable compound (PP) was supplied by Cabopol – Polymer Compounds (Porto de Mós, Portugal), with a melt flow index of 2.5 g/10 min-1 (230 °C, 2.16 kg) and a density of 0.9 g cm-3. A Polyamide 12 cable compound (PA12) was supplied by Evonik (Essen, Germany), with a melting temperature of 178 °C and a density of 1.01 g cm3 . The MWCNT used as received (i.e., without subsequent functionalization) were NC7000 from Nanocyl, Belgium, characterized by a surface area of 250 to 300 m2 g-1, average length of 1.5 μm and average diameter of 9.5 nm, according to the manufacturer.

2.2 Composite and sample preparation Nanocomposites containing 1, 2, 3 and 4 wt. % of CNT were prepared with each polymer compound. The PA12 compound was dried at 80 °C during 8 h prior to processing. The nanocomposites were prepared by melt mixing using a co-rotating intermeshing twin screw extruder (Coperion ZSK 26 Mc) with a screw diameter of 25 mm and a length to diameter ratio (L/D) of 40. The screws encompassed five mixing zones consisting of kneading disks staggered at 30, 45 and 90°, which were separated by conveying zones. The polymers were fed in the main hopper, while the CNT where added using a side feeder located at L/D of 24, after melting the polymer. The barrel and die were set to 200 °C, the screw speed was 180 min-1 and the feed rate was 6 kg h-1. Most of the extruded strands were pelletized in-line after cooling, using a Pelltec SPP30 cutter (Coperion). Also, a few extruded strands were collected directly in order to measure their electrical conductivity. Plaques with 160 × 130 × 3 mm were produced for all the tested compounds and controls by hot compression molding. For the PP and PP/CNT nanocomposites, the plaques were obtained at 210 °C under 80 bar, pressed between two polytetrafluorethylene (PTFE) films. The PA12 and PA12/ 2/8

CNT nanocomposites were previously dried at 80 °C during 8 h, and compression molded at 220 °C under 80 bar. All the materials were cooled under pressure until reaching 80 °C and then removed from the press. The plates where then cut into standardized specimens, according to the respective testing standards.

2.3 Cable design and cable manufacture Figure 1a illustrates the typical design of a standard high voltage shielded cable. It comprises the metal conductor (1) that is insulated by an extruded polymer layer (2), which is encircled by a screen (a metallic braid) (3) and finally by an extruded jacket (4). In the present work, two alternative designs were considered. In a first attempt, the metallic braid was replaced by a nanocomposite tubing (Figure 1b). Subsquently (Figure 1c), a hybrid solution was also tested, in which both a ligher mettalic screen and a nanocomposite tubing were inserted between the insulation layer and the jacket. The industrial extrusion line (supplied by Siebe Engineering, Germany) able to process either PP or PA12-based nancomposites, included a pay-off (to supply the insulated wire, which was produced beforehand by a conventional extrusion wire-coating operation), a single screw extruder equipped with a barrier-type screw, an extrusion head and die with adjustable wire-guide and die diameter (set to 230 °C), two water cooling throughs approximately 5 and 100 meters long and set to 60 °C and 23 °C, respectively, an on-line diameter sensor and a winder. The line operated at a speed of 60 m min-1. The jacket was applied in a subsequent conventional extrusion coating operation.

2.4 Characterization The melt-volume flow rate (MVR) was measured with a Gottfert MI-3 tester according to method B of the ISO 1133:2005 standard, using a load of 2.16 kg at 230 °C. PA12 and its nanocomposites were dried at 80 °C during 8 h and kept in a desiccator until tested. Calcination tests were carried out by heating approximately 2 g of polymer and composite samples at 600 °C during 2 h in air on a ThermoScientific M110 oven. The samples and residues were weighted on an Ohaus Pionner PA214 microbalance. Thermogravimetric analysis (TGA) was carried out on a TA Instruments Q800, heating the polymers and composites

Figure 1. Schematic designs of a high voltage shielded cable: (a) standard cable design; (b) total substitution design; (c) hybrid solution design. Polímeros, 31(2), e2021021, 2021

Development of electrically conductive polymer nanocomposites for the automotive cable industry under inert atmosphere (Ar) from 40 °C to 800 °C at a heating rate of 10 °C min-1. Tensile testing was carried out on a Shimadzu AG-X universal testing equipment. The tensile strength was measured following the ISO 527-1/-2 standard with 5A shape dumbell bars, a load cell of 1 kN and a displacement rate of 50 mm min-1. The three-point bending test was performed according to the ISO 178 standard, with a load cell of 50 kN, dumbell bars with 40 × 25 × 2 mm, a distance of 32 mm between supports, at 2 mm min-1. Scanning electron microscopy (SEM) images were obtained with a JSM-6010LV SEM (JEOL, Japan), operating at 15 kV accelerating voltage. Samples were cryo-fractured in liquid nitrogen and the resulting surfaces coated with Au to observe the transversal section of filaments. A Keithley 2635B single channel sourcemeter was used to measure the volume electrical conductivity of the extruded strands, varying the potential from -10 V to 10 V and measuring the corresponding current intensity. The average diameter of the PP and PA12 nanocomposite strands produced was 2.2 mm and 1.9 mm, respectively. Cable components (1) and (3) (see Figure 1) were characterized electrically using a Wheatstone bridge. The shielding effectiveness of the cables, expressed as attenuation (dB), was measured according to the IEC 62153 standard. The set-up consisted of an electrical network analyser (Keysight E5071C, Keysight Technologies, USA) which included generator and receiver and a 3 meter long Rosenberger Bedea tube (Berkenhoff & Drebes GmbH, Germany) that contained the test sample. Measurements were made at the most important frequencies (100 MHz, 500 MHz, 1 GHz, 2 GHz, 3 GHz). Characterization of the cable heat resistance was carried out after exposure to 175 °C during 240 h. The electrical insulation along its length and the mechanical force required to bend the cable after exposure to heat were measured following the Original Equipment Manufacturer (OEM) standards.

3. Results and Discussion The effective CNT content of the nanocomposites was assessed by TGA under inert atmosphere. Due to the very low apparent density of the CNT, small variations in their feed rate during composite production could affect the CNT concentration in the nanocomposite. Calcination tests

were carried out to evaluate the content of inorganic fillers in the polymer grades used. Under calcination conditions, oxidative reactions develop leading to polymer (and additives) decomposition[31]. CNT decomposition temperatures in air are usually in the range 390-730 °C, depending mostly on their length and chemical structure[32], and the residue is mostly formed by oxides of the CNT metal catalyst. Table 1 presents the weight % of the residue obtained in the calcination tests of all materials, as well as the residual weight obtained in the TGA tests, measured at 800 °C. The results presented in Table 1 show that the residual weight obtained by calcination is very low for both polymers and their composites: below 0.08 wt% for the plain polymer and below 0.8 wt% for the composites with higher CNT content. Thus, the overall inorganic content is quite low. The differences observed may be induced by experimental factors such as the heterogeneous distribution of CNT along the extrudate, leading to a small shift in wt%. Figure 2 shows the thermogravimetric plots obtained for the matrices and respective nanocomposites with highest CNT loading, i.e., 4 wt%. Above 400 °C a large weight loss is observed for both polymer compounds, the composites presenting slightly higher thermal stability compared to the corresponding matrices[33,34]. For all samples, the polymer matrices registed higher weight loss than any of the nanocomposites, the larger residual weight obtained by TGA being associated to the CNT content. Since the CNT are stable under inert atmosphere up to temperatures near 3000 °C, the residual weigth measured correlates to the nominal CNT incorporation levels. Therefore, these results validate the procedure adopted in this work for the preparation of the nanocomposites. The dispersion of the CNT in the polymers during melt mixing is expected to induce an increase of the melt viscosity[26,28]. This is consistent with the increase observed in the melt pressure measured at the entrance of the shaping die during the compounding stage, presented in Figure 3. However, while for PP the incorporation of up to 4 wt% of CNT raises the melt pressure by approximately 9%, for PA12 the raise in melt pressure is 62%. This difference may relate to the CNT dispersion: at constant CNT content, higher dispersion of individual CNT throughout the composite induce a larger increase in the polymer melt viscosity. Thermo-mechanical degradation of PP and PA12 would lead to chain scission, and thus it would contribute to a decrease in viscosity, and thus in melt pressure. Since the effect is the opposite, the

Table 1. Residual weight % of the polymers and nanocomposites after calcination and thermogravimetric analysis under inert atmosphere. Matrix PP

PA12

CNT (wt. %) 0 1 2 3 4 0 1 2 3 4

Polímeros, 31(2), e2021021, 2021

Calcination residual weight (%) 0.08 ± 0.01 0.14 ± 0.03 0.25 ± 0.05 0.50 ± 0.03 0.82 ± 0.01 0.07 ± 0.04 0.22 ± 0.02 0.26 ± 0.03 0.43 ± 0.07 0.48 ± 0.04

TGA residual weight at 800 °C (%) 0.22 1.21 2.22 3.24 4.21 0.18 1.06 2.09 3.07 4.11

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Figure 2. TGA plots for the polymers and their nanocomposites containing approximately 4 wt.% CNT.

Figure 3. Melt pressure at the entrance of the die: (a) PP and PP/CNT nanocomposites; (b) PA12 and PA12/CNT nanocomposites.

effects observed should be essentially associated with the effective dispersion of the nanofiller. Similar trends are observed for the MVR data (Figure 4). Interestingly, as the CNT content increases up to 4 wt.%, the MVR decreases linearly for PP and its nanocomposites and logarithmically for PA12 at is nanocomposites. Equivalent drops in MVR have been reported[33]. The effect of the addition of CNT on the tensile properties of the composites is depicted in Figure 5 and Table 2. As frequently reported in the literature[26,27], the incorporation of CNT in a polymer matrix may not affect significantly the yield stress, but decreases the ductility. While the yield stress of the PA12/CNT nanocomposites tends to slightly increase relative to the polymer, the reverse is observed for the PP/CNT nanocomposites. This may result from differences in filler dispersion, as mentioned above, i.e., a less effective dispersion of CNT in the PP matrix resulting in the presence of larger CNT agglomerates. Similar differences in behavior are observed for the flexural properties (Figure 6). The addition of CNT to PP has little effect on the flexural modulus and flexural strength 4/8

Figure 4. Effect of the addition of CNT to PP and PA12 on the MVR of the nanocomposites.

of PP, while for PA12 those properties increase up to 45% and 35%, respectively, for composites with 4 wt.% CNT. SEM images of the composites cross-section are presented in Figure 7 at two magnifications. The PP composites depict large CNT agglomerates at lower magnification. At higher magnification, the entangled CNT are observed, as well as Polímeros, 31(2), e2021021, 2021

Development of electrically conductive polymer nanocomposites for the automotive cable industry the CNT agglomerate/polymer boundary, evidencing the absence of dispersed CNT beyond that boundary. Thus, the CNT form isolated islands of agglomerates in PP. Conversely, the CNT dispersed in PA12 may form a few small agglomerates (as depicted for PA12/3 wt% CNT at low magnification), however at higher magnification the individual CNT are observed across the whole matrix, forming a continuum. These observations confirm the good CNT dispersion achieved in PA12, contrasting with the presence of large agglomerates in PP. Figure 8 presents the correlation between electrical conductivity and CNT concentration for the nanocomposites studied in this work. For PA12 electrical percolation was attained at CNT concentrations of approximately 3 wt.%,

Figure 5. Tensile behaviour: stress-strain of (a) PP/CNT and (b) PA12/CNT nanocomposites.

which agrees well with the values reported in the literature[15,35]. The electrical conductivity increased from 9.4 x 10-7 S m-1 for the polymer to 3.52 x 10-3 S m-1 for the PA12/4 wt.% CNT nanocomposite. Conversely, no electrical percolation was reached for PP nanocomposites within the range of CNT addition studied. This result is in line with the previous considerations about the limited dispersion achieved. Considering the results obtained for the electrical conductivity and mechanical reinforcement, the PA12/4 wt.% CNT nanocomposite was selected as a potential candidate to replace the metallic braid integrated in standard high voltage shielded cables. The new cable (Figure 1b) was produced and its properties were compared with those of an equivalent standard cable. A weight reduction of 20% and an increase in production speed of one order of magnitude relative to the standard cable were attained. These are paramount competitiveness factors in the cable industry. Also, the thermal resistance of the two cables, expressed as the bending force after thermal ageing, was similar (7 N). However, while the electrical resistance of the conductor was the same in both cases, the resistance of the screen of the new cable was higher and could not be measured with the standard testing equipment, thus making it unsuitable to most automotive applications. Alternatively, a cable containing a hybrid screen, i.e., consisting of an inner nanocomposite tube enclosed in a loose metallic braid, was also manufactured (Figure 1c). This solution yielded a weight saving of 4% and a higher bending force (11 N) relative to the conventional cable. Figure 9 presents the shield effectiveness of the three types of cables manufactured in this study (Figure 1). While the standard cable exhibits an essentially frequency independent effectiveness, the new solutions show a tendency for a decrease with increasing frequency. However, the hybrid solution exhibits the highest effectiveness throughout the entire frequency range, which makes it appropriate for the applications under study. It is difficult to confront these results with data reported in the literature, since the latter were obtained at a higher frequency range (8-13 GHz in comparison to 0.001-3GHz adopted in this work) and for much thicker samples[36-38]. Anyway, for nanocpmposites containing 5 wt% of CNT, the measured values of shield efficiency were of the order of |30| dB[37].

Table 2. Tensile properties of PP, PP/CNT, PA12 and PA12/CNT nanocomposites. Matrix

(% CNT)

E (MPa)

σS (MPa)

εS (%)

σB (MPa)

εB (%)

0 1 2 3 4 0 1 2 3 4

656 ± 84 633 ± 29 600 ± 37 588 ± 26 617 ± 42 672 ± 41 713 ± 71 744 ± 32 758 ± 57 787 ± 72

23 ± 0.6 22 ± 0.5 19 ± 0.3 20 ± 0.1 22 ± 0.3 43 ± 0.5 47 ± 0.6 48 ± 1.4 -

12 ± 1.9 10 ± 2.5 13 ± 1.2 13 ± 1.1 9 ± 0.9 19 ± 1.2 18 ± 1.5 16 ± 1.1 -

-* 22 ± 0.7 20 ± 0.5 39 ± 0.3 42 ± 0.4 46 ± 2.3 40 ± 3.1 38 ± 1.2 41 ± 0.9 48 ± 1.2

-* 62 ± 9 75 ± 5 64 ± 5 52 ± 8 386 ± 8 46 ± 4 30 ± 5 21 ± 3 18 ± 4

PA12

*No break at 800%.

Polímeros, 31(2), e2021021, 2021

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Figure 6. Flexural properties: (a) flexural modulus and (b) flexural strength of PP and PA12 composites.

Figure 7. SEM images of PP and PA12 composites cross section. On the left column the scale bar represents 10 µm and on the right column 1 µm. 6/8

Polímeros, 31(2), e2021021, 2021

Development of electrically conductive polymer nanocomposites for the automotive cable industry applications. Therefore, an alternative design of a cable containg a screen consisting of a nanocomposite tube and a loose metallic braid was tested. This novel solution showed excellent shielding effectiveness. Therefore, this investigation demonstrated that it is possible and practical to replace the metallic screen of conventional automotive high voltage cables by a nanocomposite solution, or a hybrid nanocomposite/metal solution, depending on the specific requirements of the application, enabling weight savings of the order of 4 to 20%. Figure 8. Electrical conductivity of PP and PA12 and corresponding nanocomposites.

5. Acknowledgements Funding by Operational Programme for Competitiveness and Internationalisation (COMPETE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) to the NanoCCAb project is gratefully acknowledged. MCP and JAC thank the support of the Portuguese Foundation for Science and Technology (FCT) through the National Funds References UIDB/05256/2020 and UIDP/05256/2020.

6. References

Figure 9. Shielding effectiveness (at 5 different frequencies) of the standard cable, cable with nanocomposite screen and cable with hybrid screen.

4. Conclusions This work investigated the possibility of replacing the metallic screen of a high voltage automotive cable with a polymer/carbon nanotube (CNT) nanocomposite, in order to save weight. The study was carried out employing commercial materials, using conventional processing equipment in a production environment, and following the relevant international standards. Polypropylene and polyamide 12 compounds were tested as possible matrices, and compositions up to 4 CNT weight % were prepared and characterized. The PA12/CNT nanocomposites presented better performance, due to the poor dispersion of the CNT in the PP matrix as evidenced by SEM analysis of fractured cross-sections. The addition of CNT to PA12 caused a steady increase in melt viscosity, as inferred from melt pressure at the die and MVR data, but without affecting its processability. A moderate mechanical reinforcement effect was also observed in terms of tensile and flexural resistance. Electrical percolation was attained at CNT concentrations around 3 wt.%, which is an excellent result when taking into consideration that compounding was performed by melt mixing and under practical production conditions. Consequently, a high voltage cable was manufactured with a screen made of a PA12/4 wt.% CNT nanocomposite. This cable evidenced good mechanical/thermal performance, but insufficient electrical conductivity to most automotive Polímeros, 31(2), e2021021, 2021

1. International Cable Federation – ICF. (2015). Trends in automotive wiring. Austria: ICF. 2. WardsAuto. (2017). Auto engineers see future CAFE rules easing. Southfield, MI: WardsAuto Group. 3. Community Research and Development Information Service – CORDIS. (2017). Ultra conductive copper-carbon nanotube wire. Cambridge, UK: CORDIS. 4. Advanced Industries. (2012). Lightweight, heavy impact. USA: McKinsey & Company. 5. Berylls Strategy Advisors. (2016). Automotive lightweight: heavy impact. In Economic Symposium. Austria. 6. European Comission. (2014). Strategy for reducing Heavy-Duty Vehicles fuel consumption and CO2 emissions - COM(2014)285. Belgium: European Comission. 7. International Council On Clean Transportat – ICCT. (2016). 2020-2030 CO2 standards for new cars and light-commercial vehicles in the European Union. Brussels: ICCT. 8. International Council On Clean Transportat – ICCT. (2017). Lightweighting technology developments. USA: ICCT. 9. European Environment Agency – EEA. (2020). Monitoring CO2 emissions from passenger cars and vans in 2018. Luxembourg: EEA. 10. Lekawa-Raus, A., Patmore, J., Kurzepa, L., Bulmer, J., & Koziol, K. (2014). Electrical properties of carbon nanotube based fibers and their future use in electrical wiring. Advanced Functional Materials, 24(24), 3661-3682. http://dx.doi. org/10.1002/adfm.201303716. 11. Mittal, V., Kim, J. K., & Pal, K. (2011). Recent advances in elastomeric nanocomposites. London: Springer. http://dx.doi. org/10.1007/978-3-642-15787-5. 12. Popov, V. N. (2004). Carbon nanotubes: properties and application. Materials Science and Engineering R Reports, 43(3), 61-102. http://dx.doi.org/10.1016/j.mser.2003.10.001. 13. Ali, M. N., Alamri, H., & Wahab, A. (2015). Conductive nanocomposite fabrication by graphene enriched polypropylene master batch. International Journal of Engineering Development and Research, 3(4), 979-990. Retrieved in 2021, February 15, from https://www.ijedr.org/papers/IJEDR1504172.pdf 14. Du, F., Fischer, J. E., & Winey, K. I. (2005). Effect of nanotube alignment on percolation conductivity in carbon nanotube/ 7/8

Guerreiro, M., Rompante, J., Leite, A. C., Fernandes, L. P., Santos, R. M., Paiva, M. C., & Covas, J. V. polymer composites. American Physical Society, 72(12), 1-4. http://dx.doi.org/10.1103/PhysRevB.72.121404. 15. Bauhofer, W., & Kovacs, J. Z. (2009). A review and analysis of electrical percolation in carbon nanotube polymer composites. Composites Science and Technology, 69(10), 1486-1498. http:// dx.doi.org/10.1016/j.compscitech.2008.06.018. 16. Bhattacharya, M. (2016). Polymer nanocomposites: a comparison between carbon nanotubes, graphene, and clay as nanofillers. Materials, 9(4), 262. http://dx.doi.org/10.3390/ma9040262. PMid:28773388. 17. Tanaka, K., & Iijima, S. (2014). Carbon nanotubes and graphene. Kidlington: Elsevier. 18. Ma, P.-C., & Kim, J.-K. (2011). Carbon nanotubes for polymer reinforcement. USA: CRC Press Taylor & Francis Group. http://dx.doi.org/10.1201/b10795. 19. Das, D., & Rahaman, H. (2015). Carbon nanotube and graphene nanoribbon interconnects. USA: CRC Press Taylor & Francis Group. 20. Li, J., Ma, P. C., Sze, C. W., Kai, T. C., Tang, B. Z., & Kim, J.-K. (2007). Percolation threshold of polymer nanocomposites containing graphite nanoplatelets and carbon nanotubes. In 16th International Conference on Composite Materials. Koyoto: International Conference On Composite Materials. Retrieved in 2021, February 15, from https://www.iccm-central.org/ Proceedings/ICCM16proceedings/contents/pdf/FriG/FrGM107ge_lij223410p.pdf 21. Socher, R., Krause, B., & Pötschke, P. (2017). Effect of additives on MWCNT dispersion and electrical percolation in polyamide 12 composites. In AIP Conference Proceedings. Lyon: Polymer Processing Society. http://dx.doi.org/10.1063/1.5016703. 22. Paiva, M. C., & Covas, J. A. (2016). Carbon nanofibres and nanotubes for composite applications. In S. Rana & R. Fanguiro (Eds.), Fibrous and textile materials for composite applications (pp. 231-260). Singapore: Springer Nature. http:// dx.doi.org/10.1007/978-981-10-0234-2_7. 23. Hocke, H., & Vitovsky, J. (2014). EP2810977A1. Munich: European Patent Office. Retrieved in 2021, February 15, from https://data.epo.org/gpi/EP2810977A1 24. Yan, D., Zhang, H. B., Jia, Y., Hu, J., Qi, X. Y., Zhang, Z., & Yu, Z. Z. (2012). Improved electrical conductivity of polyamide 12/ graphene nanocomposites with maleated polyethylene-octene rubber prepared by melt compounding. ACS Applied Materials & Interfaces, 4(9), 4740-4745. http://dx.doi.org/10.1021/ am301119b. PMid:22889067. 25. Socher, R., Krause, B., Hermasch, S., Wursche, R., & Pötschke, P. (2011). Electrical and thermal properties of polyamide 12 composites with hybrid fillers systems of multiwalled carbon nanotubes and carbon black. Composites Science and Technology, 71(8), 1053-1059. http://dx.doi.org/10.1016/j. compscitech.2011.03.004. 26. Jamali, S., Paiva, M. C., & Covas, J. A. (2013). Dispersion and re-agglomeration phenomena during melt mixing of polypropylene with multi-wall carbon nanotubes. Polymer Testing, 32(4), 701707. http://dx.doi.org/10.1016/j.polymertesting.2013.03.005. 27. Vilaverde, C., Santos, R. M., Paiva, M. C., & Covas, J. A. (2015). Dispersion and re-agglomeration of graphite nanoplates in polypropylene melts under controlled flow conditions. Composites. Part A, Applied Science and Manufacturing, 78, 143-151. http://dx.doi.org/10.1016/j.compositesa.2015.08.010.

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Polímeros, 31(2), e2021021, 2021

ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.20210041

Increasing the physical and combustion performance of Oriental beech by impregnating borates and coating liquid glass Yilmaz Anil Gunbekler1 , Hilmi Toker1 , Caglar Altay2 , Mustafa Kucuktuvek3*  and Ergun Baysal1  Department of Wood Science and Technology, Faculty of Technology, Muğla Sıtkı Koçman University, Muğla, Turkey 2 Department of Interior Design, Aydın Vocational School, Aydın Adnan Menderes University, Aydın, Turkey 3 Department of Interior Architecture and Environmental Design, Faculty of Architecture, Alanya Hamdullah Emin Paşa University, Alanya, Antalya, Turkey 1

*mkucuktuvek@gazi.edu.tr

Abstract This study was designed to investigate physical properties such as color changes after weathering and water absorption (WA) levels, and combustion performance of borates-impregnated and LG-coated Oriental beech wood. Results showed that borates impregnated and LG-coated Oriental beech wood showed positive lightness stability after weathering. The best color stabilization was obtained with LG-coated Oriental beech. Except for the 1 h WA period, LG did not show a water repellent effect after the water absorption test. Borates impregnation before LG-coating caused to decrease in weight loss of Oriental beech after the combustion test. Moreover, the lowest weight losses were obtained in borate impregnated Oriental beech wood. Keywords: physical properties, combustion properties, borates, liquid glass, Oriental beech wood. How to cite: Gunbekler, Y. A., Toker, H., Altay, C., Kucuktuvek, M., & Baysal, E. (2021). Increasing the physical and combustion performance of Oriental beech by impregnating borates and coating liquid glass. Polímeros: Ciência e Tecnologia, 31(2), e2021022. https://doi.org/10.1590/0104-1428.20210041

1. Introduction Architects and civil engineers demand high-performance wooden materials for their building designs. Besides the durability of the material, its performance against weathering plays an important role in material selection in the construction and furniture industry. On the other hand, users are expecting environmentally friendly, sustainable, aesthetic, and durable wooden materials for building proposes. Wood material, which has been used for various purposes since the appearance of mankind in history, is one of the most important raw materials. With the rapid development of technology, the usage areas of wood material have diversified and the amount used has increased. This increase in the use of wood material causes it to be among the decreasing natural resources today[1]. It has a wide range due to its lightness, easy processing, continuous production, and superior physical and mechanical properties in various areas of use, compared to concrete, iron, aluminum, PVC, and other various building materials. It also finds uses in construction techniques, paper and cellulose, cardboard, furniture, and many other industries[2]. The weathering process was primarily linked to sunlight irradiation and its rate was increased by higher air temperature, moisture, and other pollutants in the outdoor environment[3]. Accordingly, ultraviolet (UV) radiation and moisture

Polímeros, 31(2), e2021022, 2021

were identified to be the main causes of deterioration and discoloration of wood surfaces in weather conditions[4,5]. Since the wood material has a hygroscopic structure, it exchanges moisture with the surrounding air to adapt to the temperature and relative humidity of the environment, and if this change occurs below the fiber saturation point, it changes dimensionally. Therefore, it is not resistant to biotic and abiotic factors[6]. Traditional wood preservatives are used in the wood preservation industry to prevent the degradation of wood materials by biological organisms. Many traditional wood preservatives such as copper chromium arsenate (CCA) are prohibited due to their harmful environmental effects. However, using preservatives, environmentally friendly wood preservatives have been developed in recent years to extend the life of the wood material. Natural extracts are among the most environmentally friendly preservatives used to protect wood material. It has been seen in many scientific studies that natural plant extracts are beneficial in wood preservation[7]. Boron compounds have gained importance for wood preservatives due to their high efficiency against biological pests. Other important properties of the compounds are their ability to be easily applied by dissolving with water, their diffusion ability to wood, being cheap and easy to supply compared to other preservatives, being

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O O O O O O O O O O O O O O O O

Gunbekler, Y. A., Toker, H., Altay, C., Kucuktuvek, M., & Baysal, E. environmentally compatible and significantly increasing the fire resistance of wood material.

tetrahydrate (DOT) were prepared to impregnate the Oriental beech wood test specimens.

Various paints, varnishes, and coatings are used to improve the natural appearance of the wood and to protect it against external weather conditions[8]. Coating of the wood surface may be considered as a solution to reduce the effects of weather conditions on material deterioration and to preserve the aesthetic properties of the material exposed outdoor in different cardinal directions or varying design details[9]. If the wooden surface is not protected, it can quickly lose its aesthetic feature. Opaque coatings or transparent protective coatings are among the solutions used to protect the wood. Transparent finishes reveal the natural texture of the wood, but sunlight can damage the wood surface and cause the coating to peel off[10]. Inorganic fillers, such as silicon dioxide, aluminum oxide or carbonates, silicates, and sulfates of various metals, are mainly used in wood coatings and it can be considered that liquid glass (LG) is in this group[11].

The impregnation of the Oriental beech wood specimens was carried out, according to ASTM standards[18]. Test specimens were impregnated with a pre-vacuum of 760 mm Hg for 30 minutes. The specimens were then allowed to diffuse in solution at atmospheric pressure for 30 minutes. The borates retention of Oriental beech was calculated from the following Equation 1:

LG is a two-component product used for waterproofing wooden surfaces requiring high chemical and physical resistance. It protects the surfaces against UV light radiation and outdoor weather conditions[12]. The wood material is flammable and easily combustible. In addition to protecting the wood against fungus and insects, the impregnating agents also have a flame-retardant feature. The impregnation materials decompose below the decomposition temperature of the wood material and rapidly transform cellulose into charcoal and water. Thus, the formation of volatile and flammable substances at higher temperatures is prevented. Therefore, wood burns less and flames are prevented from spreading around[13]. The main components of wood appear to have quite different thermal degradation properties. It is understood that wood components decompose in different temperature ranges to release volatile substances. For example; cellulose 240-350 °C, hemicellulose 200-260 °C, lignin 280-500 °C[14].

= R

(

)

G.C × 103 kg / m3 (1) V

Where: G = T2 -T1;

T2 = Specimen weight after impregnation (kg);

T1 = Specimen weight before impregnation (kg); V = Specimen volume (m3);

C = Solution concentration (%). After borates impregnation, Oriental beech wood specimens were oven-dried at 103 ± 2 ºC until the specimens reached the constant weight. Then similar to the impregnation process of borates, the wood specimens were coated with LG with a pre-vacuum of 760 mm Hg for 30 minutes. The specimens were then allowed to diffuse in solution at atmospheric pressure for 30 minutes.

2.3 Natural weathering

Borates are recognized as easily applicable, inexpensive, fire-retardant, and more importantly environmentally-friendly wood preservatives[15-17]. This study aimed to investigate some physical properties such as color changes after weathering and water absorption levels, and combustion performance of borates-impregnated and LG-coated Oriental beech wood, according to market expectations.

The site where the test samples are subjected to natural weathering is within the boundaries of Mugla Sitki Kocman University. The geographical location of the university (37° 09 ‘N and 28° 22’ E, 670 m above sea level) in Mugla, Southern Aegean Region of Turkey. Weather conditions of Mugla city were taken from the directorate of meteorology. Weather conditions during the weathering process are shown in Table 1. Wood specimens, according to ASTM Standards[20] were prepared for weathering exposure. The experimental specimens were subjected to natural weathering during the three months spanning September, October, and November 2020. The exposure rack is adjusted to the south at an angle of 45°. Wood specimens were prepared for natural weathering, according to ASTM Standards[21]. The color change on the surface of the test specimens after natural weathering was investigated.

2. Materials and Methods

2.4 Color changes

2.1 Preparation of test specimens Oriental beech (Fagus orientalis L.) wood specimens were prepared with dimensions 10x100x150 mm, 20x20x20 mm, and 9x19x1016 mm (radial, tangential, and longitudinal) dimensions for color, water absorption, and combustion tests, respectively.

2.2 Impregnation procedure 3% aqueous solutions of boric acid (BA), borax (BX), ammonium pentaborate(APB), and disodium octaborate 2/8

After the natural weathering process, the color factors L*, a*, and b* were determined. These factors are determined by the CIEL*a*b* technique and the L* axis signify the lightness, whereas a* and b* are the chromaticity coordinates. In this technique, the +a* factors characterize the red color, and -a* factors characterize the green color and the +b* factors characterize the yellow color, whereas -b* represents the blue color. The L* value can differ from 100 (white) to zero (black)[22]. The colors of the specimens were measured by a colorimeter before and after weathering. The colorimeter is X-Rite SP Series Spectrophotometer, X-ride Pantone, MI, USA. The determining spot was adjusted to be equivalent Polímeros, 31(2), e2021022, 2021

Increasing the physical and combustion performance of Oriental beech by impregnating borates and coating liquid glass Table 1. Weather conditions of Mugla during the weathering period[19]. Months (2020) Average temperature (°C) Highest temperature (°C) Lowest temperature (°C) Humidity (%) Average wind speed (m/sn) Total rainfall per month (mm=kg. m-2)

September 24.93 33.30 18.43 49.56 0.97 3.50

or not more than one-third of the distance from the center of this area to the receptor field stops. The total color difference, (ΔE*) was determined for each test specimen, according to ASTM standards[23]. The color changes were calculated using Equations 2 to 5: ∆a* = af * – ai *

∆b * = bf * – bi *

(2)

∆L * = Lf * – Li *

( ∆E *)

(3)

 ∆a * 2 + ) (

(4) 1/2

( ∆b *)2 + ( ∆L *)2 

(5)

Where Δa*, Δb*, and ΔL* represent the changes between the initial and final interval values. Five replicates were made for each treatment group. Color measurements were made in parallel to the fibers.

2.5 Water absorption test For the water absorption test, Oriental beech wood specimens were kept in distilled water for 1, 8, 24, 48, 72, 96, 120, 144, and, 168 hours under 20 ± 2 oC water temperature. At the end of each soaking period, specimens were removed from the water, dried with paper, and immediately weighed. Thus, the amount of water absorption by each specimen was calculated with the help of Equation 6. WA

(

 M – M f 0i 

)

/ M 0i  × 100 

(6)

Where: WA= Water absorption (%); Mf= Specimen weight after water absorption (g); Moi= Oven dry weight after impregnation (g).

2.6 Combustion test Combustion test specimens were prepared in 9x19x1016 mm dimensions, according to ASTM standards[24] principles. The specimens continue for a total of 10 minutes in the computer-controlled combustion testing device, with 4 minutes of flame-induced and 6 minutes of non-flame burning for each specimen. The weight loss rate as a result of the combustion was calculated with the help of the following Equation 7. = LW

BC − AC × 100 AC

Polímeros, 31(2), e2021022, 2021

(7)

October 18.12 26.53 11.33 63.17 0.54 5.93

November 10.93 18.90 5.11 63.21 0.92 6.20

In here: LW= Loss of weight (%); BC= Specimen weight before combustion test (g); AC= Specimen weight after combustion test (g);

2.7 Statistical evaluation The test results were obtained as a result of the measurements and they were analyzed with SPSS statistical software program. Test results were uploaded to the computer and variance analysis was performed. Duncan test was applied at 95% statistical confidence level. The homogeneity groups (HG) of the experimental results were used for statistical evaluation. Different letters in HG indicate the difference that can be considered statistically significant.

3. Results and Discussions 3.1 Color changes Weathering is defined as the slow decomposition and decay of materials exposed to weather factors[25]. Weathering effects can be observed depending on the type of wood or process. Abiotic factors affecting the original color and natural appearance of the wood are weathering. Ultraviolet (UV) radiation causes some changes in the wood surface. As a result, the wood turn gray in color. The fibers of the wood surface exposed to continuous radiation weaken due to two reasons. One of them is the depolymerization of lignin and the other carbohydrates in the cell wall. During the natural weathering wood materials are exposed to sunlight and as a result, lignin absorbs UV lights. This is the reason for the color changes in wooden materials. The growth of fungi may also occur on the wooden surface after weathering as well as warping, checking, and splitting[26,27]. In order to eliminate the negative effects of outdoor weather conditions and seasonal weather changes on wood material; preservatives such as impregnation, paint, and varnish applications are applied for wood surfaces. However, the surfaces of wooden materials with top surface treatments still deteriorate over time and the upper surface appearance and strength cannot reach the desired level. In wood exposed to various biological, chemical and physical effects, it is not easy to measure the performance of surface treatment materials in outdoor conditions. Table 2 shows L*, a*, and b* values of un-impregnated and non-coated (control) group, solely coated, and impregnated and coated Oriental beech specimens and also illustrates the values of change for all three-color parameters (ΔL*, Δa*, and Δb*), as well as the total color changes (ΔE*) 3/8

Gunbekler, Y. A., Toker, H., Altay, C., Kucuktuvek, M., & Baysal, E. Table 2. Color change values of Oriental beech wood specimens before and after 3 months of natural weathering. Chemicals Control LG BX BX+LG BA BA+LG DOT DOT+LG APB APB+LG

Retention (Kg/m3) 16.32 17.06 19.85 20.16 18.96 18.61 16.53 15.88

Color values before natural weathering

Color values after natural weathering

Total color changes

𝜟L*

𝜟a*

𝜟b*

𝜟E*

72.30 61.46 62.49 57.95 66.56 60.71 67.85 54.46 63.97 54.55

9.28 8.81 10.33 8.77 11.08 8.08 9.43 6.41 9.66 7.67

20.19 22.01 19.52 15.47 21.31 20.50 19.91 15.49 18.97 15.30

-15.36 -2.22 -2.98 8.13 -3.73 3.77 -7.34 6.36 -6.13 9.81

-1.78 -0.40 -1.26 -0.64 -1.62 -0.95 -0.30 -1.43 -0.40 -0.19

5.09 4.56 6.23 9.92 4.77 2.56 5.83 8.21 7.77 8.86

16.27 5.08 6.36 12.84 6.26 8.15 9.37 10.48 7.80 13.22

Homogeneity Groups A FG FG BC FG G DE CD EF B

Note: LG: Liquid glass; BX: Borax; BA: Boric acid; APB: Ammonium pentaborate; DOT: Disodium octaborate tetrahydrate.

of the Oriental beech wood specimens after 3 months of weathering. The retention values of Oriental beech wood specimens impregnated with borates were varied from 15.88 to 20.16 kg. m-3. Before weathering, all of the treatment groups were shown a decrease in L* values than that of the control group. While the L* value of the control group was 72.30, it varied from 54.46 to 67.85 for the impregnated and LG-coated Oriental beech wood. The decrease in the L* value of Oriental beech wood specimens showed that the specimens became darker after the borates impregnation and LG-coating. These results are in good agreement with those of Ustun et al.[28], Baysal[29], and Simsek and Baysal[30] studied the effects of some impregnation chemicals on the color changes of wood. The experimental results showed that borates impregnation before LG-coating caused to decrease L* values of Oriental beech wood specimens than that of only LG-coated Oriental beech. Moreover, L* values of the borates impregnated Oriental beech wood was higher than that of LG-coated Oriental beech wood. According to the experimental results, the a* and b* values of the control group were 9.28 and 20.19, while a* and b* values were 8.81 and 22.01, respectively for LG-coated Oriental beech wood. Impregnation with borates before LG-coating caused to decrease a* and b* values of Oriental beech before weathering. Except for borates impregnated and LG-coated Oriental beech wood, the negative lightness stability (ΔL*) values for other treatment groups occurred. Therefore, the wood surface became rougher and darker after weathering. The darkening of wood might be have been due to the degradation of lignin and other non-cellulosic polysaccharides[31-33]. After weathering, Δa* and Δb* values of the control group were found to be -1.78 and 5.09, respectively. The control group and all treatment groups gave negative Δa* and positive Δb* values after weathering. Negative Δa* and positive Δb* values show that wood specimens’ surfaces maintained greenish and yellowish tones. Impregnation with borates and LG-coating improved the color stability of Oriental beech wood compared to the untreated (control) group. While total color change (ΔE*) was 16.27 for the control group, it varied from 5.08 to 13.22 for all treatment groups after weathering. Color change values showed that the best 4/8

color stability was observed with LG-coated Oriental beech. However, borates impregnation before LG-coating caused to increase ΔE* of Oriental beech. There was a statistical difference in ΔE* values between borates impregnated and LG-coated Oriental beech and the control group (p≤0.05).

3.2 Water absorption Water absorption (WA) levels of Oriental beech wood impregnated with borates and coated with LG are given in Table 3. Especially in the first 8 hours, WA values are increasing fast and these data are consistent with the previous scientific study[34]. While the control specimens absorbed nearly 51.82% of its weight of water; the LG-coated wood absorbed 14.55% water after 1 h. However, except for 1h, WA levels of LG-coated Oriental beech wood were highly increased other all WA periods. The experimental results showed that the highest WA levels were obtained for APB impregnated Oriental beech wood for all WA periods. While the WA level of APB impregnated Oriental beech was 63.05% after 1 h, it was 103.52% for APB impregnated Oriental beech wood after 336 h. As a result, APB impregnated Oriental beech received more than half of the total water in 1 hour of the period. Except for the 1 h WA period, WA levels of borate impregnated and LG-coated Oriental beech was lower than that of only LGcoated Oriental beech. Baysal et al.[35] reported that WA of a mixture of styrene and methylmetacrilate (1:1; v/v) treated wood could be reduced as much as 8 times when compared with untreated wood. Baysal et al.[36] investigated water absorption levels of heaven wood treated with various boron compounds and coated with some vinyl monomers. Boric acid (BA), borax (BX), and a mixture of BA and BX (7:3; weight: weight) were used as boron compounds. Methyl methacrylate (MMA), styrene (ST), a mixture of ST and MMA (7.3; volume: volume), and isocyanate were used as vinyl monomers. According to the results, while the vinyl monomers used in the study, provide a significant reduction in the water absorption rate of the specimens; the monomer treatment applied on boron compounds also showed a similar effect. In our study, while LG showed a water repellent effect only 1 h WA period, for other WA periods it showed the same effect with borates and control group. Polímeros, 31(2), e2021022, 2021

Polímeros, 31(2), e2021022, 2021

51.82 14.55 48.76 15.84 30.53 15.01 63.05 16.07 53.06 16.02

Control LG BX BX+LG BA BA+LG APB APB+LG DOT DOT+LG

AB D B D C D A D AB D

H.G.

72.72 49.48 69.75 44.86 40.88 38.20 78.94 43.28 76.47 42.50

After 8 hours

A BC A BC C AB A C A C

H.G.

74.20 52.24 73.30 49.34 58.75 46.54 77.53 49.46 76.50 47.10

After 24 hours A BC A BC B C A BC A C

H.G. 83.06 63.36 84.64 61.72 68.12 55.76 87.31 60.87 87.58 60.41

After 48 hours A B A B B B A B A B

H.G. 88.49 70.16 88.08 68.96 71.88 61.57 93.05 66.55 92.00 67.55

After 72 hours A B A B B B A B A B

H.G. 91.34 74.70 88.66 69.18 73.72 65.94 94.79 71.15 93.58 72.48

After 96 hours

Water absorption rates (%)

A B AB B B B A B A B

H.G.

After 120 hours 95.43 79.82 93.26 75.13 75.97 70.21 98.18 73.68 96.40 78.27

Note: LG: Liquid glass; BX: Borax; BA: Boric acid; APB: Ammonium pentaborate; DOT: Disodium octaborate tetrahydrate; H.G.: Homogenity group.

After 1 hour

Chemicals

Table 3. Water absorption levels of Oriental beech wood.

A C AB BC C C A C A C

H.G.

After 144 hours 99.64 82.53 95.71 81.55 77.12 75.85 100.48 78.61 97.93 82.10 A BC AB BC C C A C AB C

H.G.

After 168 hours 103.05 88.51 96.75 86.13 81.47 79.46 103.57 84.63 99.42 85.42 A ABCD ABC ABCD CD D A BCD AB ABCD

H.G.

Increasing the physical and combustion performance of Oriental beech by impregnating borates and coating liquid glass

5/8

Gunbekler, Y. A., Toker, H., Altay, C., Kucuktuvek, M., & Baysal, E. 3.3 Combustion properties

4. Conclusions

Weight loss of Oriental beech impregnated with borates and coated with LG after combustion test is given in Table 4. While the lowest weight loss was obtained as 69.10% for DOT impregnated Oriental beech wood, the highest weight loss was measured as 83.70% for the control group. Weight loss of LG-coated Oriental beech was lower than that of the control group. But, there was no statistically significant difference in weight loss between the control group and LG-coated Oriental beech wood. Borates impregnation significantly decreased weight loss of Oriental beech compared to un-coated (control) group.

Some physical and combustion properties of Oriental beech wood impregnated with borates and coated with liquid glass were studied.

Experimental results showed that borates impregnation before LG-coating caused lower weight loss values than that of only LG-coated Oriental beech wood display the protective consequence of borates in combustion. These conclusions are consistent with the former report on the weight losses of un-treated and boron–vinyl monomer combination-treated wood[35]. Our results showed that except for BX impregnated and LG-coated Oriental beech, there were statistically significant differences in weight loss between borates impregnated and LG-coated Oriental beech and only LG-coated Oriental beech wood. Yalinkilic et al.[37] investigated fire properties of borates and some water repellent chemicals impregnated Calabrian pine wood (Pinus brutia Ten.). They found that boron compounds increased the fire resistance of wood. Also, it was found that the increasing effect of water repellent chemicals on combustion is prevented with borates to some extent. Baysal et al.[36] investigated combustion characteristics of heaven wood treated with various boron compounds and some vinyl monomers. Boric acid, borax, and a mixture of boric and borax (7:3; weight: weight) were used as boron compounds. Methylmethacrylate, styrene, a mixture of styrene and methylmethacrylate (7:3; volume: volume), and isocyanate were used as vinyl monomers. In terms of weight loss caused by combustion, a mixture of boric acid and borax (7:3; weight: weight) mixture gave the most favorable result with a 63% weight loss ratio. The experimental results were compatible with these researchers’ findings. Table 4. Weight loss of Oriental beech wood specimens after combustion test. Chemicals Control LG BX BX+LG BA BA+LG DOT DOT+LG APB APB+LG

Mean 83.70 81.40 80.02 80.93 71.64 72.78 69.10 69.27 69.57 71.81

Weight loss (%) Standard Homogeneity deviation group 1.03 A 1.79 AB 1.03 B 3.46 AB 2.05 CD 1.31 C 1.37 D 3.74 CD 3.55 CD 2.52 CD

Note: LG: Liquid glass; BX: Borax; BA: Boric acid; APB: Ammonium pentaborate; DOT: Disodium octaborate tetrahydrate.

6/8

Before weathering, all of the treatment groups were shown a decrease in L* values than that of the control group. After 3 months of natural weathering test, negative lightness stability (ΔL*) values were occurred on the control specimens and on the specimens treated only LG, BX, BA, DOT, APB. Positive lightness stability (ΔL*) values were obtained from the specimens treated BX+LG, BA+LG, DOT+LG, APB+LG. The color stability of LGcoated Oriental beech wood was higher than those of other treatment groups. The liquid glass showed a water repellency effect of only 1 h WA period. Except for 1 h WA periods, WA levels of borates impregnated and LG-coated Oriental beech was lower than only LG-coated Oriental beech. Weight loss was the highest for the control group after the combustion test. Borates impregnation before LG-coating caused to decrease in weight loss of Oriental beech after combustion test. Borates impregnated Oriental beech gave the best results in terms of combustion properties. Boron compounds have a low environmental impact compared to other conventional impregnation materials and cause acute toxicity in very small amounts. In addition, it is colorless and odorless, has no corrosive properties, and is resistant to fire. LG possesses excellent water resistance and can be produced as a transparent or color product that is resistant to atmospheric conditions. LG has high performance against UV light radiation and outdoor weather conditions. As a result; It may be beneficial to use liquid glass and boron compounds for waterproofing the wood material and to increase the resistance of the wood material against UV light, radiation, and outdoor weather conditions, and to increase its combustion performance.

5. Acknowledgements This study was taken from some of the data of the master’s thesis of Yilmaz Anil Gunbekler, who is continuing his master’s degree at Mugla Sıtkı Kocman University Institute of Science. This study also was supported by a scientific project of the Scientific Research Unit of Mugla Sıtkı Kocman University. The project number is 20/099/02/2.

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Polímeros, 31(2), e2021022, 2021

ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.20210039

O Silane-coupled kenaf fiber filled thermoplastic elastomer O based on recycled high density polyethylene/natural rubber O blends O Cao Xuan Viet , Hanafi Ismail , Abdulhakim Masa and Nabil Hayeemasae *  O Department of Polymer Materials, Faculty of Materials Technology, Ho Chi Minh City University of Technology – HCMUT, Ho Chi Minh City, Vietnam O School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Engineering Campus, Nibong Tebal, Penang, Malaysia O Rubber Engineering and Technology Program, International College, Prince of Songkla University, Hat Yai, Songkhla, Thailand O Department of Rubber Technology and Polymer Science, Faculty of Science and Technology, Prince of Songkla University, Pattani Campus, Pattani, Thailand O O Abstract O A silane coupling agent, namely γ-aminopropyltriethoxysilane (APS), was used to modify kenaf powder (KP). It was then used as filler in a thermoplastic elastomer (TPE) based on Recycled High Density Polyethylene/Natural Rubber Blends (rHDPE/NR). The attachment of silane onto KP was verified by Fourier Transform Infrared Spectroscopy O (FTIR), while the performance of the TPE was assessed in terms of mechanical and thermal properties. The results revealed that specific functional groups of APS were efficiently grafted onto the KP. Tensile strength was improved by O the modification with silane and this also affected Young’s modulus of the TPE. Also, improved thermal stability was confirmed by thermogravimetric analysis (TGA), as the degradation temperature increased upon inclusion of silane. O These effects are attributed to improved compatibility of the KP and rHDPE/NR blend. Such compatibilizing effect was confirmed by Differential Scanning Calorimetry (DSC) that indicated significantly increased crystallinity after the O modification with silane. Keywords: recycled polyethylene, natural rubber, Kenaf powder, silane coupling agent. O 1

*nabil.h@psu.ac.th

How to cite: Viet, C. X., Ismail, H., Masa, A., & Hayeemasae, N. (2021). Silane-coupled kenaf fiber filled thermoplastic elastomer based on recycled high density polyethylene/natural rubber blends. Polímeros: Ciência e Tecnologia, 31(2), e2021023. https://doi.org/10.1590/0104-1428.20210039

1. Introduction The consumption of plastic materials in our daily life has been constantly increasing because of numerous consumer goods made of plastics. This consumption also creates problems and concerns regarding disposal and environmental impacts. This is because most plastics are not biodegradable, taking a long time to degrade in the nature, possibly even several centuries. With more and more plastic products, the landfill volume of plastics waste is a growing concern, interest in recycling post-consumer polymers has gained attention. The largest fraction of polymer wastes consists of polyolefins, such as polyethylene (PE) and polypropylene (PP)[1,2], and recycling is an alternative destination for these materials. Thermoplastic elastomers (TPE) are the materials of choice to widen the use of recycled plastics. TPE is a special class of viscoelastic materials that contain properties of thermoplastics and elastomers[3-5]. The incorporation of various types of filler into a TPE is done either to improve the properties or to reduce the raw

Polímeros, 31(2), e2021023, 2021

material costs. These cover many types of available fillers. Natural fiber has been one of efficient fillers to reinforce the polymer matrix. For examples, sisal fibers have been used to reinforce Polyurethane composite, it has shown an excellent thermal stability and mechanical properties[6]. Fibers from other plants were also used to enhance several composites’ properties[7,8]. It can be clearly seen that the use of natural fiber in the thermoplastic composites is still considered a filler of choice in plastic composite. One of the interesting source of fiber is from Kenaf. Kenaf gains a lot of interest in the context of making composites as a filler in polymer matrix[9]. Kenaf stem composes of two distinct fiber types, in bark and core. On average the stem has 65% woody core and 35% of bark by weight[10]. The abundance of kenaf core combined with its easy processability are the main reason that make this material attractive characteristics to substitute commercial fillers which are potentially toxic.

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Viet, C. X., Ismail, H., Masa, A., & Hayeemasae, N. Although natural fillers can offer many advantages in composites, the polarity in nature of these fillers have considerably less compatible with non-polar polymer matrices, such as cis-polyisoprene (NR) and polyethylene (PE). Such disadvantage causes a bad dispersion of filler and gives poor interfacial adhesion between these two phases, leading to composites with rather poor durability and thermal stability[11]. Hence, the surface treatment of natural fibers is the best solution to overcome this drawback. One of the most successful treatment is through the use of silane coupling agent, which has lately demonstrated the efficacy of various silane coupling agents in the modification of natural fibers[11,12]. Silane interfacial coupling agents are widely applied on cellulosic fillers to form stable bonds with the polymer matrix[13]. Like other natural fibers, Kenaf powder (KP) also requires surface modification in order to increase its compatibility to the non-polar matrix. The use of silane coupling agent is believed to modify the surface functionality of the KP and subsequently enables KP to bond chemically to the rubber matrix. The aim of this research was to use a silane coupling agent in the composite that has KP filler in recycled high density polyethylene (rHDPE)/natural rubber (NR). Apart from that, dicumyl peroxide (DCP) was also introduced, DCP is considered to be suitable crosslinker for both PE and NR[14]. It was expected to promote cocrosslinking between rHDPE and NR. To date, no prior report has been published on the potential of this approach. This work focused on the mechanical properties and the morphology of the composites as well as their thermal stability. The results obtained from this present study will improve scientific understanding of how a silane coupling agent and DCP could influence the properties of KP filled rHDPE/NR blends.

2. Materials and Methods

2.2 Preparation of silane-treated KP The treatment of KP was done by immersing KP in NaOH solution (5% w/v) for 2 h at room temperature. Then KP was washed with distilled water containing a few drops of acetic acid washed again with distilled water and dried. Next, silane treatment was carried out in a mixture of water/ethanol (30/70 v/v) for the pretreated KP. The pH of the solution was adjusted to 4 with acetic acid and stirred for 1 h. Then, 10 g of KP was soaked in the solution and continuously stirred for 3 h. The silane-treated KP was filtered and dried in a vacuum oven at 80 °C for 24 h prior to compounding

2.3 Preparation of the blends Table 1 lists the formulations used for preparing the TPE based on KP filled rHDPE/NR composites. There are three alternative types of formulations, namely untreated KP filled rHDPE/NR composites (KP), silane-treated KP filled rHDPE/ NR composites (KP-APS) and silane-treated KP rHDPE/NR composites in the presence of DCP (KP-APS-DCP). These three types of composites were compared in order to select ingredients for achieving the best composite. The mixing was done in an internal mixer (Haake Rheomix Polydrive R600/610 mixer) with a rotor speed of 50 rpm at 165 °C. The rHDPE was firstly charged into the mixer and molten, followed by NR. After this, the steps in compounding depend on the method of preparation whereby the APS and/or APS/ DCP were added prior to introduce KP at the final step. The total mixing time was kept constant at 12 min for all composites. Samples collected from the internal mixer were compression molded in an electrically heated hydraulic press, model GT-7014-A30C. The molding involved preheating the samples for 8 min at 165 °C, followed by compressing at 100 kg/cm2 for 3 min at similar temperature, and subsequently cooling down under pressure for another 2 min.

2.4 Mixing torque

2.1 Materials Recycled high density polyethylene (rHDPE) was obtained from Zarm Scientific and Supplies Sdn Bhd, Penang, Malaysia. The melt flow index of rHDPE was 0.237 g at 10 min. The SMR 20 grade was supplied by Mardec Berhad, Selangor, Malaysia., γ-aminopropyltriethoxysilane (APS) and dicumyl peroxide (DCP) were supplied by SigmaAldrich (M) Sdn Bhd, Selangor, Malaysia. Kenaf powder was obtained from core part and it was ground using a table-type pulverizing machine until getting the size ranging from 32 to 150 µm was received.

The torque was computationally recorded from Haake Rheomix Polydrive R 600/610 mixer at 10 sec intervals while mixing. The development of torque over mixing time were assessed, while the torque at the end of processing was considered a stabilized torque.

2.5 Tensile properties All the composites were tested under tension force at a cross-head speed of 50 mm/min using an Instron 3366 machine according to ASTM D412. The specimens

Table 1. Formulations used to prepare KP filled rHDPE/NR composites. Material

Amount (php or phr) KP

KP-APS

KP-APS-DCP

rHPDEa

SMR 20a

KPa

0, 10, 20, 30 & 40

APS

DCP

0.05

Remarks: a refers to parts per hundred of polymers (php) and b refers to parts per hundred of rubber (phr).

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Polímeros, 31(2), e2021023, 2021

Silane-coupled kenaf fiber filled thermoplastic elastomer based on recycled high density polyethylene/natural rubber blends were in dog-bone shape at 1 mm thickness. Prior to the test, the thickness were measured whereby the results were recorded in terms of tensile strength, Young’s modulus and elongation at break.

2.6 Scanning Electron Microscopy (SEM) The samples from tensile fractured surfaces were used to observe the morphological characteristics of the composites. It was captured using a Supra-35VP field emission scanning electron microscope (SEM). All the samples were sputter coated with gold/palladium using Bio-Rad Polaron Division device prior to scanning to prevent electrostatic charging during imaging.

2.7 Thermogravimetric analysis (TGA) The thermal stability of the composites was determined using using Perkin Elmer TG-6 Analyzer. The sample was heated from 30 °C to 600 °C at a heating rate of 10 °C/min in nitrogen atmosphere. The temperatures at various weight loss and char residue were recorded and further discussed to interpret the thermal stability of the composites.

2.8 Differential Scanning Calorimetry (DSC) The melting and crystallization characteristics of the composites were evaluated using Perkin-Elmer DSC-7 Analyzer. The samples were heated from 30 °C to 200 °C at a heating rate of 10 °C/min in nitrogen atmosphere. The crystallinity of composite (XCOM) and crystallinity in rHDPE fraction (XRHDPE) before and after surface treatment were determined by using the Equations 1 and 2:

X= com

∆H f

∆H 0f

= X rHDPE

× 100 (1)

Xc × 100 (2) Wf(rHDPE)

where ΔHf is the heat of fusion of sample analyzed, ΔH0F (PE) = 293 J/g[15] is used for 100% crystalline HDPE homopolymer, and WF(RHDPE) is the weight fraction of rHDPE in the composite.

3. Results and Discussions 3.1 FTIR FTIR spectra of KP and KP-APS are displayed in Figure 1A. The chemical modification of KP led to changes in a few peaks due to molecular interactions. For example, a peak at 1726 cm-1 was observed for the KP due to the unconjugated C=O groups in hemicellulose. This peak fully disappeared after pretreatment with NaOH. In addition, for the KP-APS, bands were observed at 1267 cm-1 and 710 cm-1 which correspond to Si-C stretching and Si-O-Si stretching, respectively[16]. Another peak at 465 cm-1 is associated with Si-O-C asymmetric bending. These bands are simply due to the silanization reactions between APS and KP. The changes in these bands can also due to plausible interactions as illustrated in Figure 1B. The presence of Si-O-Si might also be detected as a shoulder in the peak located at 1060 cm-1[15]. Figure 2 shows FTIR spectra of KP and KP-APSDCP. It can be seen that the introduction of APS led to the appearance of two new peaks at 1726 cm-1 and 1267 cm-1, corresponding to the carbonyl group of ester or acids[17] and Si-C stretching of APS, respectively. A slight increase in peaks in the 1000-1080 cm-1 region should also be highlighted. The strengthening of these bands could be associated to the asymmetric stretching of Si-O-Si or SiO-C bonds[16]. The OH stretching vibrations in the region 3100-3500 cm-1 were observed at a lower intensity for the treated composite. This is evidence of the interactions between the hydroxyl groups in KP and the functional groups in APS. The double band at 1726 cm-1 might be related to the C=O vibrations of amide since carboxyl groups formed during the processing of rHDPE/NR, which could react with the amino functionality of APS[18]. After addition of DCP and APS, the absorption peak of the C=C group of NR (1662 cm-1) was significantly reduced, while the vinyl group of rHDPE did not appear in either untreated or treated samples. This indicates that the NR was more prone to crosslinking/grafting reactions than rHDPE since NR has a large number of C=C groups. The above results confirm the chemical reactions between APS and KP and the existence of a crosslinked network, with possible bonds between the filler and the polymer matrix through grafting and crosslinking.

Figure 1. FTIR spectra of KP and KP-APS (A) and Plausible interaction between cellulose in KP and APS coupling agent (B). Polímeros, 31(2), e2021023, 2021

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Viet, C. X., Ismail, H., Masa, A., & Hayeemasae, N. 3.2 Mixing torque The mixing torque of KP, KP-APS and KP-APS-DCP is shown in Figure 3A. All the curves have three distinct peaks corresponding to the materials input at certain stage. Basically, the torque decreased over time and reached a stable torque at the end of mixing cycle. In the case of KPAPS and KP-APS-DCP, a decrease in torque was observed before 6 min of mixing due to the lubricant action of the APS added. The decreasing slope for KP-APS-DCP sample was smaller than that of the others. This might be related to the crosslinking reactions taking place in the matrices and the stronger polymer-filler interactions in the presence

Figure 2. FTIR spectra of KP and KP-APS-DCP.

of APS and DCP. Figure 3B illustrates effects of treatment type on stable torque of KP filled rHDPE/NR composites. It can be observed that the stable torques of KP-APS and KP-APS-DCP were higher than that with KP. The treatment of KP with silane may lead to an increase in melt viscosity of the composites due to stronger interactions between filler and polymer. Meanwhile, the addition of APS and DCP into the composites led to a greater increase in stable torque as a result of crosslinking.

3.3 Tensile properties Figure 4 displays typical stress-strain curves of KP, KP-APS and KP-APS-DCP. Generally speaking, both KP-APS and KP-APS-DCP cases had increased tensile strength and tensile modulus, and the KP-APS-DCP cases had distinctly positive effect on tensile strength. This is particularly at a low KP content (10 phr), a highest tensile strength was observed for KP-APS-DCP. There are two main factors affecting such improvement; one being due to an improve in polymer-filler interaction promoted by silane coupling agent another was simply due to the role of DCP itself which helps to promote co-crosslinking between rHDPE and NR. This has brougth to an increase in ultimate tensile strength of the composite. Tensile strength elongation at break and Young’s modulus of the composites are listed in Table 2. A better tensile strength was found in the composites with APS pretreatment. This is simply because of an improved filler dispersion in the matrix and a better degree of adhesion at the interfaces[19,20]. Also,

Figure 3. Time profiles of mixing torque (A) and stabilization torque (B) of KP, KP-APS and KP-APS-DCP. Table 2. Tensile strength, elongation at break and Young’s modulus of KP, KP-APS and KP-APS-DCP. Sample 10KP 10KP-APS 10KP-APS-DCP 20KP 20KP-APS 20KP-APS-DCP 30KP 30KP-APS 30KP-APS-DCP 40KP 40KP-APS 40KP-APS-DCP

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Tensile strength (Mpa) 12.11 ± 0.21 12.75 ± 0.23 14.80 ± 0.50 11.92 ± 0.41 12.32 ± 0.25 14.05 ± 0.73 11.60 ± 0.15 12.11 ± 0.31 13.20 ± 0.41 10.92 ± 0.62 11.61 ± 0.40 13.40 ± 0.25

Elongation at break (%) 115.51 ± 13.1 62.50 ± 10.0 360.15 ± 27.2 20.12 ± 10.0 21.22 ± 8.2 58.17 ± 10.3 12.50 ± 5.0 9.21 ± 3.1 26.09 ± 4.9 12.05 ± 4.7 10.12 ± 2.1 25.06 ± 2.3

Young’s modulus (GPa) 0.57 ± 0.025 0.63 ± 0.012 0.68 ± 0.013 0.62 ± 0.022 0.63 ± 0.013 0.74 ± 0.012 0.66 ± 0.005 0.78 ± 0.021 0.81 ± 0.013 0.71 ± 0.007 0.79 ± 0.014 0.83 ± 0.013

Polímeros, 31(2), e2021023, 2021

Silane-coupled kenaf fiber filled thermoplastic elastomer based on recycled high density polyethylene/natural rubber blends

Figure 4. Stress-strain curves of KP, KP-APS and KP-APS-DCP.

thanks to a previous alkaline treatment that could remove impurities and wax from the fiber surfaces and create a rougher topography. Thus, physical interaction could be promoted and better adhesion was further enhanced by silane treatment[16]. The interaction between phases was expected to improve since the KP surfaces became less hydrophilic due to chemical bonding between APS and the OH groups[21]. In contrast, when APS was used in combination with DCP (KP-APS-DCP), the tensile strength of this composite increased significantly with a maximum improvement of 23.7%. This was possible through the following hypothetical mechanism of interaction between APS, KP and rHDPE/NR matrix. Firstly, the alkoxy groups in APS underwent hydrolysis. Moisture for the hydrolysis might come from the surface humidity of KP. Next, the attachment of silane to hydroxyl groups of fiber was accomplished through hydrogen bonds or through ether linkages (-O-)[22]. This accounted for the interface between KP and APS. The N-H group of APS might also react with OH groups of KP or other silane molecules. Thermal treatment is commonly applied to ensure that coupling agents adhere to the surface of cellulosic fibers[12,21]. In this case, it was strongly believed that the grafting reaction of APS on the surfaces of KP truly occurred during the mixing at 165 °C (as confirmed by FTIR results). Regarding non-polar polymers like PE and NR, they have very little intrinsic reactivity since they do not contain any reactive groups. Thus, DCP was introduced to attack their molecular chains creating reactive sites for bonding. At an elevated temperature, DCP was firstly decomposed to produce an oxy radicals, these radicals had the potential to abstract hydrogen from rHDPE, NR or polymer parts of KP. Such polymer radicals are then free to interact each other through free-radical reaction[14]. Radical combinations might lead to the co-crosslinking of rHDPE and NR matrix as well as to grafting between KP and polymer matrix. Table 2 also illustrates the effects of silane coupling agent on the elongation at break (Eb) of KP, KP-APS and KP-APS-DCP. Clearly the KP-APS treatment exhibited an adverse effect on Eb, while KP-APS-DCP showed the opposite trend. Lower Eb found in KP-APS was associated with the enhanced adhesion between filler and matrix. Better adhesion restricts the deformation capacity of a composite; thus catastrophic failure occurs after a comparatively small strain. In contrast, the elongation of KP-APS-DCP was Polímeros, 31(2), e2021023, 2021

greatly improved compared to the KP sample. It can be seen that the Eb at 10 phr of KP was nearly 200% higher than that of the untreated composites. This is simply due to the crosslinking in the presence of DCP. As for the tensile modulus or Young’s modulus of the composites, there was an increase in tensile modulus with KP content due to this contributing a rigid phase. When considered at similar filler loadings, the moduli were higher for KP-APS-DCP than those for KP-APS and KP. This is quite surprising, since a reduction in tensile modulus is generally observed in both crosslinked PE and crosslinked PE composites filled with natural fiber as a result of decreased crystallinity of the matrix[23]. This indicates that the crosslinking degree of rHDPE was relatively low. The improved modulus could be related to better adhesion between the fiber and polymer matrix raised by silane coupling interaction. Better adhesion led to more restricted deformation capacity of the matrix in the elastic zone, and increased the modulus. It needs to be noted that the NR phase was also crosslinked, increasing stiffness of the composites. Elongation, tensile strength, and modulus followed similar trends, suggesting that the toughness of the resultant composites was improved by addition of APS. According to Oksman and Clemons[24], the toughness of a filled polymer composite can be responsible to several ways e.g., an increment in matrix toughness, an improve in interaction of filler and polymer matrix, or filler related properties such as filler content, filler particle size, and filler dispersion. In this work, crosslinking of rHDPE/NR in the presence of APS and DCP improved toughness of the polymer matrix. There was also indication of improved adhesion between KP filler and the rHDPE/NR matrix in the treated composites as discussed earlier. Therefore, a possible explanation for the improvement in all tensile properties of the KP-APS-DCP could be toughening of the matrix and better adhesion between filler and matrix.

3.4 Morphological study The SEM micrographs of KP, KP-APS and KP-APS-DCP are shown in Figure 5. At a low filler content (10 phr), strong adhesion between the components was clearly detected. Indeed, the deformation of rHDPE matrix was prominent for composites treated with APS and DCP (KP-APS-DCP). A number of fibers attached on the matrix are visible, and less fiber pull-out is observed on the fractured surface. This indicates that the level of adhesion between filler and matrix was greatly improved. This behavior is related to the crosslinking in rHDPE and NR phases. This could be an explanation for the improved tensile strength and elongation at break of KP-APS-DCP at a low filler loading. At a higher filler content (40 phr), the fractured surface showed less plastic flow or deformation of matrix compared to the composites with 10 phr of KP. However, the polymer-filler interactions of KP-APS-DCP were stronger than in the untreated composites (KP). The failure surface seemed to be uniform and both matrix and fiber appeared to fail simultaneously. Some fibers were firmly imbedded and coated in the matrix which indicates good wetting by the polymer. In addition, breakage of the fibers was also visible. This was further evidence supporting the concept 5/8

Viet, C. X., Ismail, H., Masa, A., & Hayeemasae, N.

Figure 5. SEM micrographs of KP, KP-APS and KP-APS-DCP (200× magnification).

Figure 6. Typical TG and DTG curves of KP, KP-APS and KPAPS-DCP.

that APS and DCP improved interfacial adhesion between KP and polymer matrix.

3.5 Thermogravimetric analysis (TGA) Figure 6 shows the TG profiles of KP, KP-APS and KP-APS-DCP. Summary of key temperatures read from the TGA and DTG curves including temperatures at 5% (T5%), 50% (T50%) and maximum weight loss (Td) and weight residue (W) is presented in Table 3. Generally, the silane treatments improved thermal stability of the composites to some extent, particularly at a low filler content. This is clearly seen from the shifts in degradation temperature. Tserki et al.[25], suggested that lignin and hemicelluloses exhibit poor thermal stability, while reversely effect was found for the fiber with a higher cellulose content. For the treatment of KP by silane, KP was firsly washed with NaOH, this process has reduced the hemicelluloses and lignin to a considerable extent[14], thus improving the thermal stability at this temperature range (disappearance of hemicellulose peak from the DTG curve). However, lignin seems to be more stable than cellulose and hemicellulose at high 6/8

Figure 7. DSC thermograms of KP, KP-APS and KP-APS-DCP.

temperatures, hence the lower lignin content resulted in poorer thermal resistance at high temperatures. Alkaline pretreatment decreases the char yield since it removed a portion of the cell structure (hemicellulose or lignin) and eliminated some inorganic matter[26]. The KP-APS-DCP slightly modified the thermal stability of the composites. It appeared to be more thermally stable with Td temperature shifting from 487.3 °C to 491 °C. This could be due to enhanced thermal degradation of PE and NR chains in the presence of DCP[27]. APS and DCP may induce grafting between the matrix and the filler, which improved thermal stability of the composite.

3.6 Differential scanning calorimetry (DSC) DSC curves of KP, KP-APS and KP-APS-DCP are shown in Figure 7 and the thermal behavior of these composites is summarized in Table 4. The melting temperature of rHDPE in the modified samples was slightly lower than in the unmodified samples due to crosslinking of the matrix as well as grafting of PE chains to the fiber[22]. However, the temperature decrease was marginal. Tm values of treated Polímeros, 31(2), e2021023, 2021

Silane-coupled kenaf fiber filled thermoplastic elastomer based on recycled high density polyethylene/natural rubber blends Table 3. TGA data of KP, KP-APS and KP-APS-DCP. Sample 10KP 10KP-APS 10KP-APS-DCP 40KP 40KP-APS 40KP-APS-DCP

T5% (°C) 304.7 331.0 325.3 277.4 289.1 282.4

T50% (°C) 464.3 470.0 475.2 463.0 461.6 465.5

Td (°C) 487.3 488.6 491.0 488.0 488.2 490.5

W (%) 0.17 0.98 1.46 7.3 5.92 6.44

∆HF(COM) (J/g) 93.35 99.65 98.51 75.49 84.28 85.5

XCOM (% crystalline) 31.86 34.0 33.62 25.76 28.76 29.18

XRHDPE (%) 50.07 53.43 52.83 51.52 57.52 58.36

Table 4. DSC parameters of KP, KP-APS and KP-APS-DCP. Sample 10KP 10KP-APS 10KP (APS-DCP) 40KP 40 KP-APS 40KP (APS-DCP)

Tm (°C) 132.95 131.89 131.51 132.58 132.46 132.29

composites increased with the amount of filler, while untreated composites showed the opposite trend. In the case of KP-APS, the crystallinity increased significantly, which could be attributed to the good compatibility and improved interfacial adhesion between filler and matrix caused by fiber surface modification[28]. It is also noted that the removal of hemicelluloses contributed to higher crystallinity of the fibers, and in the composites. This result is in agreement with the improved tensile strength and modulus, as discussed earlier. Similar results were also obtained for KP-APS-DCP, and DCP probably initiated crosslinking in NR or in the amorphous phase of PE, hence the extent of crystallization did not change in this phase. In addition to this, this composite also showed lower melting temperatures compared to the KP, while the degree of crystallinity rose significantly. This indicates that DCP possibly initiated crosslinking in the NR phase or grafting of the polymer chains onto the kenaf fiber[29]. This observation is in accordance with the increased tensile modulus of KP-APS-DCP.

4. Conclusion Surface modification of the KP with silane coupling agent (KP-APS) showed a better effect on tensile strength but the increment was not significant. It also improved the tensile modulus and reduced the elongation at break of the composites. The interactions between matrix and filler were expected to improve since the KP surfaces became less hydrophilic due to chemical bonding of silane to the hydroxyl groups on the surfaces. When APS was used in combination with DCP, all the tensile properties were significantly improved. This was attributed to the toughening of the matrix and better adhesion between filler and matrix induced by crosslinking/grafting reactions. The silane treatment also enhanced the thermal stability of the composites to some extent, particularly at a low filler content. DSC results revealed an increase in crystallinity of the treated biocomposites in both cases. The use of APS in combination with DCP to blend rHDPE and NR provided Polímeros, 31(2), e2021023, 2021

good physical and thermal stability. Hence, this formulation was clearly successful, and is highly recommended when the strength and thermal stability are of primary concern. This system can also be considered when there the blends are exposed to elevated temperatures during production or use

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Viet, C. X., Ismail, H., Masa, A., & Hayeemasae, N. 8. Ismail, M. R., Yassen, A. A. M., & Afify, M. S. (2011). Mechanical properties of rice straw fiber-reinforced polymer composites. Fibers and Polymers, 12(5), 648-656. http://dx.doi. org/10.1007/s12221-011-0648-5. 9. Schneider, J. P., Myers, G. E., Clemons, C. M., & English, B. W. (1995). Biofibers as reinforcing fillers in thermoplastic composites. Journal of Vinyl and Additive Technology, 1(2), 103-108. http://dx.doi.org/10.1002/vnl.730010212.n. 10. Webber, C. L., 3rd, Whitworth, J., & Dole, J. (1999). Kenaf (Hibiscus cannabinus L.) core as a containerized growth medium component. Industrial Crops and Products, 10(2), 97-105. http://dx.doi.org/10.1016/S0926-6690(99)00014-X. 11. Raj, R. G., Kokta, B. V., Dembele, F., & Sanschagrain, B. (1989). Compounding of cellulose fibers with polypropylene: effect of fiber treatment on dispersion in the polymer matirx. Journal of Applied Polymer Science, 38(11), 1987-1996. http:// dx.doi.org/10.1002/app.1989.070381103. 12. Abdelmouleh, M., Boufi, S., Belgacem, M. N., & Dufresne, A. (2007). Short naturalfibre reinforced polyethylene and natural rubber composites: effect of silane coupling agents and fibres loading. Composites Science and Technology, 67(7-8), 16271639. http://dx.doi.org/10.1016/j.compscitech.2006.07.003. 13. Kim, T.-W., Lee, S.-Y., Chun, S.-J., Doh, G.-H., & Paik, K.-H. (2011). Effect of silane coupling on the fundamental properties of wood flour reinforced polypropylene composites. Journal of Composite Materials, 45(15), 1595-1605. http://dx.doi. org/10.1177/0021998310385589. 14. Tanrattanakul, V., & Udomkichdecha, W. (2001). Development of novel elastomeric blends containing natural rubber and ultra‐low‐density polyethylene. Journal of Applied Polymer Science, 82(3), 650-660. http://dx.doi.org/10.1002/app.1893. 15. Alix, S., Philippe, E., Bessadok, A., Lebrun, L., Morvan, C., & Marais, S. (2009). Effect of chemical treatments on water sorption and mechanical properties of flax fibres. Bioresource Technology, 100(20), 4742-4749. http://dx.doi.org/10.1016/j. biortech.2009.04.067. PMid:19477120. 16. Pereira, P. H. F., Rosa, M. F., Cioffi, M. O. H., Benini, K. C. C. C., Milanese, A. C., Voorwald, H. J. C., & Mulinari, D. R. (2015). Vegetal fibers in polymeric composites: a review. Polímeros: Ciência e Tecnologia, 25(1), 9-22. http://dx.doi. org/10.1590/0104-1428.1722. 17. Pretsch, E., Bühlmann, P., & Badertscher, M. (2009). Structure determination of organic compounds: tables of spectral data. Germany: Springer-Verlag. http://dx.doi.org/10.1007/978-3540-93810-1. 18. Demjén, Z., Pukánszky, B., & Nagy, J., Jr. (1999). Possible coupling reactions of functional silanes and polypropylene. Polymer, 40(7), 1763-1773. http://dx.doi.org/10.1016/S00323861(98)00396-6. 19. Natarajan, S., Rathanasamy, R., Palaniappan, S. K., Velayudham, S., Subburamamurthy, H. B., & Pal, K. (2020). Comparison of MA-g-PP effectiveness through mechanical performance of functionalised graphene reinforced polypropylene.

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Polímeros: Ciência e Tecnologia, 30(3), e2020035. http:// dx.doi.org/10.1590/0104-1428.05620. 20. Cui, Y., Lee, S., Noruziaan, B., Cheung, M., & Tao, J. (2008). Fabrication and interfacial modification of wood/recycled plastic composite materials. Composites. Part A, Applied Science and Manufacturing, 39(4), 655-661. http://dx.doi. org/10.1016/j.compositesa.2007.10.017. 21. Castellano, M., Gandini, A., Fabbri, P., & Belgacem, M. N. (2004). Modification of cellulose fibres with organosilanes: under what conditions does coupling occur? Journal of Colloid and Interface Science, 273(2), 505-511. http://dx.doi. org/10.1016/j.jcis.2003.09.044. PMid:15082387. 22. Xie, Y., Hill, C. A. S., Xiao, Z., Militz, H., & Mai, C. (2010). Silane coupling agents used for natural fiber/polymer composites: a review. Composites. Part A, Applied Science and Manufacturing, 41(7), 806-819. http://dx.doi.org/10.1016/j.compositesa.2010.03.005. 23. Grubbström, G., Holmgren, A., & Oksman, K. (2010). Silane-crosslinking of recycled low-density polyethylene/ wood composites. Composites. Part A, Applied Science and Manufacturing, 41(5), 678-683. http://dx.doi.org/10.1016/j. compositesa.2010.01.018. 24. Oksman, K., & Clemons, C. (1998). Mechanical properties and morphology of impact modified polypropylene–wood flour composites. Journal of Applied Polymer Science, 67(9), 1503-1513. http://dx.doi.org/10.1002/(SICI)10974628(19980228)67:9<1503::AID-APP1>3.0.CO;2-H. 25. Tserki, V., Zafeiropoulos, N. E., Simon, F., & Panayiotou, C. (2005). A study of the effect of acetylation and propionylation surface treatments on natural fibres. Composites. Part A, Applied Science and Manufacturing, 36(8), 1110-1118. http://dx.doi. org/10.1016/j.compositesa.2005.01.004. 26. Shebani, A. N., van Reenen, A. J., & Meincken, M. (2008). The effect of wood extractives on the thermal stability of different wood species. Thermochimica Acta, 471(1-2), 43-50. http:// dx.doi.org/10.1016/j.tca.2008.02.020. 27. Marcovich, N. E., & Villar, M. A. (2003). Thermal and mechanical characterization of linear low-density polyethylene/ wood flour composites. Journal of Applied Polymer Science, 90(10), 2775-2784. http://dx.doi.org/10.1002/app.12934. 28. Bouza, R., Lasagabaster, A., Abad, M. J., & Barral, L. (2008). Effects of vinyltrimethoxy silane on thermal properties and dynamic mechanical properties of polypropylene-wood flour composites. Journal of Applied Polymer Science, 109(2), 1197-1204. http://dx.doi.org/10.1002/app.28159. 29. Khonakdar, H. A., Morshedian, J., Wagenknecht, U., & Jafari, S. H. (2003). An investigation of chemical crosslinking effect on properties of high-density polyethylene. Polymer, 44(15), 4301-4309. http://dx.doi.org/10.1016/S0032-3861(03)00363-X. Received: May 06, 2021 Revised: Aug. 21, 2021 Accepted: Aug. 23, 2021

Polímeros, 31(2), e2021023, 2021

ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.20210013

Modification of poly(lactic acid) filament with expandable graphite for additive manufacturing using fused filament fabrication (FFF): effect on thermal and mechanical properties João Miguel Ayres Melillo1 , Iaci Miranda Pereira2 , Artur Caron Mottin3*  and Fernando Gabriel da Silva Araujo1  Rede Temática em Engenharia de Materiais – REDEMAT, Universidade Federal de Ouro Preto – UFOP, Ouro Preto, MG, Brasil 2 Centro Tecnológico do Exército – CTEx, Rio de Janeiro, RJ, Brasil 3 Departamento de Mecânica, Centro Federal de Educação Tecnológica de Minas Gerais – CEFET-MG, Belo Horizonte, MG, Brasil

*mottindesign@gmail.com

Abstract Fused Filament Fabrication, better known as Fused Deposition Modeling®, is currently the most widespread 3D Printing Technology. There has been a significant demand for developing flame-retardant filaments. Thereby enabling them, for example, in electronics and automotive applications. In this study, commercial PLA filament was modified by the addition of 1, 3 and 5% (%wt.) of expandable graphite. The composites were reprocessed, via extrusion, into filaments for Fused Filament Fabrication. Thermal properties of the filament composites were evaluated by thermogravimetric analysis and differential scanning calorimetry. Mechanical properties of thermo-pressed specimens indicated that no strong adhesion was promoted between the filler and matrix. This is a challenge with expandable graphite reported by many authors. All composites with expandable graphite achieved the V-2 rating of UL-94 flammability test. In spite of this, the results indicated that flammability of the PLA was reduced. All composite filaments were printable and prototypes were successfully 3D printed. Keywords: Fused Filament Fabrication (FFF), PLA, expandable graphite, prototypes. How to cite: Melillo, J. M. A., Pereira, I. M., Mottin, A. C., Araujo, F. G. S. (2021). Modification of poly(lactic acid) filament with expandable graphite for additive manufacturing using fused filament fabrication (FFF): effect on thermal and mechanical properties. Polímeros: Ciência e Tecnologia, 31(2), e2021024. https://doi.org/10.1590/0104-1428.20210013.

1. Introduction Additive manufacturing (AM) alludes to adding raw materials during manufacturing, and includes several assembly and rapid prototyping processes[1]. Among the various AM technologies, material extrusion technology is currently the most popular[2,3]. Material extrusion technology was developed by Scott Crump in 1989 and patented as fused deposition modeling (FDM)[1]. Thus, FDM is the patented acronym and FFF is the “open-source” acronym for machines with the same principle, and stands for Fused Filament Fabrication. In FFF-type 3D printing a thermoplastic polymer filament undergoes melting-solidifying cycles before it forms a desirable shape, that is, only simple physical-state processes are involved[4]. Thus, it allows the user to print products of any dimension and complexity. In addition, the prototypes can be produced faster and customizable[4]. The most used thermoplastic polymers in FFF-type 3D printing are acrylonitrile butadiene styrene (ABS), poly(lactic acid) (PLA), high impact polystyrene (HIPS), thermoplastic polyurethane

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(TPU) and aliphatic polyamides (nylon)[5]. PLA is a biobased polymer, that is, PLA is obtained from natural and sustainable raw material, such as cornstarch. Moreover, it degrades in soil by microorganisms under certain conditions of temperature and humidity[6]. In addition to these ecological benefits, PLA also offers reasonable performance in technical applications related to its mechanical properties[6]. However, its high ignitability is a drawback, since it limits the usage, for example, in electronics and automotive applications[7]. Therefore, there is a need to develop FFF materials with low flammability and density. Wang et al.[8], incorporated DOPO (9,10-dihydro9-oxa-10-phosphaphenanthrene10-oxide) aminated derivative into PLA to improve the flame resistance. In spite of good results, higher content of DOPO-NH2 presented uneven dispersion in PLA matrix, with decreasing in the mechanical properties. Xue et al. [9] developed a flame retardant poly(lactic acid) (PLA) composite with the addition of only 2 wt% of ammonium polyphosphate (APP)

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Melillo, J. M. A., Pereira, I. M., Mottin, A. C. & Araujo, F. G. S. and 0.12 wt% of resorcinol bis(diphenyl phosphate) (RDP). The composite achieved the V-0 rating of UL-94 test, with mechanical properties which enabled it to be drawn into filaments for FFF 3D printing. The use of natural graphite as flame retardant in polymers is limited by the difficulty of incorporation viscous polymers. Hence, for this usage it has been replaced with graphite treated with intercalation reagents, known as expandable graphite (EG)[10]. When EG composites are exposed to high temperature, EG expands and produces a voluminous protective layer, thus providing flame retardancy[10]. Wei et al.[11] used EG to produce fire retardant PLA. Their results indicated significant reduction at the rate of combustion due to the protective intumescent char created on the material surface. In this context, the main aim of this study was to modify 3D printing PLA filament with different contents of EG. The purpose of this is imparting flame-retardant property to the PLA filament. Moreover, prototypes made from the composite filaments were successfully 3D printed, which demonstrated that, in spite of modification, the filament kept printable.

2. Experimental 2.1 Materials

microscope (SEM, Bruker D2-phaser) with electron beam operating at 5kV. 2.4.2 Thermogravimetric analysis (TGA) Thermogravimetric analysis was carried out in a DTA60 thermoanalyzer (Shimatzu) under synthetic air atmosphere (flow = 50 mL min-1). Five milligrams of each filament were placed in aluminum crucibles and the experiments were conducted from room temperature to 750 °C, using a heating rate of 10 °C min-1. 2.4.3 Differential scanning calorimetry (DSC) DSC analyses were performed in a differential scanning calorimeter PerkinElmer® DSC800 equipped with a PerkinElmer® Intracooler II standard and calibrated with high purity indium. Approximately two milligrams of each filament were placed in aluminum crucibles and the experiments were carried out under nitrogen atmosphere (flow = 20 mL min-1). The following protocol was applied to each sample: (i) isotherm at -40 ºC for 3 min; (ii) heating from -40º C to 190 ºC, using heating rate of 30 ºC min-1; (iii) isotherm at 190º C for 3.0 min; (iv) cooling from 190 ºC to -40 ºC, using cooling rate of 5º C min-1; (v) isotherm at -40 ºC for 3.0 min, and (vi) heating from -40 ºC to 190 ºC, using heating rate of 10 ºC min-1. The degree of crystallinity (χC) was estimated using Equation 1.

The white PLA filament was supplied by 3D LAB (Betim, MG). The expandable graphite (Grafexp 95200-110) was kindly donated by Nacional de Grafite (Itapericica, MG). ∆H m − ∆H cc = × 100 χc Polyethylene glycol (PEG 20,000) was purchased from filler ( % w )  (1) 0  Sigma Aldrich (cod. 81300). All materials were used as ∆H m × 1 −    100   received, without any further purification.

2.2 Modification of PLA filament with EG First the EG was passed through a sieve (100 mesh) and then manually mixed and heated with PEG (Tm = 6366 ºC). After cooling to room temperature, the mixture was ground (analytical mill IKA-A10). Three mixtures PEG/EG were prepared with different PEG: EG ratios (1:1, 1:3 and 1:5). After that, PLA filament was cut into pellets using a granulator (AX Plásticos - SP, Brazil). The pellets were manually premixed with each of the as prepared PEG/EG, in addition with the control PLA + PEG (without EG). Samples were named as PLA-EG0%, PLA-EG1%, PLA-EG3% and PLA-EG5% according to EG content. Composite Filaments with 1.75 mm diameter were obtained by extrusion at 180 °C using a mono-screw extruder (Filmaq3D STD - Brazil).

2.3 Plates production To produce the plates the composite filaments were granulated and hot pressed (AX Plastics-AX P8T), at a temperature of 190 °C for 2 min under pressure of 2 t and an extra 2 min under pressure of 5 t. The plates were cut in specimens for the tensile test (ASTM D638), UL94 flammability assay and colorimetric assessment.

2.4 Characterizations 2.4.1 Scanning electron microscopy (SEM) The morphology of EG before and after the thermal treatment at 900 ºC was observed using a scanning electron 2/9

Where ΔHCC and ΔHM are the enthalpies of cold crystallization and melting (J g-1), respectively, which were calculated from the peaks of cold crystallization and melting in the DSC curves of the second heating. ∆H m0 = 93.1 J g-1 is the fusion enthalpy of 100% crystalline PLA[12], and filler (%w) is the weight percentage of EG. 2.4.4 Tensile test Specimens cut according to ASTM D-638 (2014) were subjected to tensile tests using the universal testing machine (EMIC DL 2000) equipped with a 50 N load cell at a speed of 5 mm min-1. For cutting the specimens, the filaments were pelleted and hot pressed (AX Plastics-AX P8T) at 190 °C for 2 min under 2 t pressure and another 2 min under 5 t pressure. The results presented are the average of at least three specimens with the standard deviation from the mean. 2.4.5 UL-94 vertical test Composites flammability was preliminary assessed by the UL (Underwriter’s Laboratory) 94 vertical burn test. UL‑94 vertical test was carried out on strips measuring 125 mm × 13 mm × 3.0 mm following the ASTM D3801 standard. In this test, the upper part is clamped to a support and strips are ignited from the bottom, while the flame self-extinguishing time is measured (Figure 1). The flame is brought into contact with the specimen for 10 s, after which the burner is removed and the flame self-extinguishing time is measured (T1). The flame is brought once again into contact with the specimen for Polímeros, 31(2), e2021024, 2021

Modification of poly(lactic acid) filament with expandable graphite for additive manufacturing using fused filament fabrication (FFF): effect on thermal and mechanical properties 10 s, and the flame self-extinguishing time is measured (T2). The glow time (T3) is measured after the application of the second flame, and the sum of after flame time and afterglow time is recorded, that is, T2 plus T3. At least five specimens for each sample were tested. The qualitative ranks for evaluating the test results are V-0, V-1, V-2 or non-rating[10,11,13]. Table 1 specifies the classification criteria used in the UL-94 vertical test. From table 8.1 standard UL-94 Underwriters Laboratories Inc. revised July 29, 1997. 2.4.6 Colorimetry assay The color parameters in the CIELab space were measured with the aid of a CM-600D spectrophotometer (Konica Minolta). The operating conditions of the spectrophotometer were: scanning from 360 to 740 nm, illuminating CIE D65 and observer angle of 10º. Five measurements were performed at different points of samples and the data were read by the Spectra Magic NX software. The parameters L*, a*, b*, as well as the color difference (∆E*) in relation to the control (PLA-EG0%) were determined. The results were the average of the five values with the respective standard deviation.

2.5 FFF-type 3D printing The 3D printing prototypes were fabricated using the composite filaments to feed a FFF-type 3D printer (Voolt 3D – Brazil) with a 0.30 mm nozzle. The nozzle and printing bed were heated to 190 °C and 65 °C, respectively. The layer height was set to 0.2 mm and a print speed of 50 mm/min was used. The computer aided design (CAD) was sourced from Thingiverse® (MakerBot Industries, LLC).

3. Results and discussion 3.1 Expandable graphite morphology Expandable graphite (EG) is produced by inserting chemicals, such as sulfuric acid (H2SO4) or nitric acid (HNO3), between the graphite layers[10]. When EG is exposed to high temperature, it expands due to releasing of gaseous products. A voluminous protective layer is produced, thus providing flame retardancy performance to various polymeric matrices[10]. The expansion of EG was carried out at 900 °C in a refractory oven for one minute. From the ratio between density and volume it was possible to verify that, after the heat treatment the graphite density decreased 9 times. Figure 2 shows SEM images of EG before and after the heat treatment.

3.2 Thermal behavior of composite filaments In FDM 3D printers the filament passes it through a high temperature nozzle where it is heated to a soft state. So, it is important a previous knowledge about their thermal properties. 3.2.1 Thermogravimetric analysis (TGA) For all filaments the onset of degradation occurs just above 260 °C (curves not shown). There was no significant difference in thermal stability between them. Yang et al. [14] indicated the temperature of 326 °C as the beginning of PLA degradation. However, these authors also realized that when PEG is added to PLA, the thermal stability was reduced due to the poor thermal stability of PEG. Liu et al. [15] reported that the addition of 5% PEG 6000 shifted the TG curve to lower temperatures compared to that of pristine PLA. The same was observed by us, probably denoting lack of adhesion between phases, with possible PEG segregation. Table 2 shows the values from DTG curves (not shown).

Figure 1. Vertical flame testing set.

The peaks at T1 and T2 were associated with polymer degradation. It can be noted that in the composite filaments the first peak at T1 appeared at temperatures well below that of PLA-EG0%.

Table 1. Ratings of UL 94 vertical test. Criteria conditions Afterflame time for each individual specimen t1 or t2 Total afterflame time for any condition set (t1 plus t2 for the 5 specimens) Afterflame plus afterglow time for each individual specimen after the second flame application (t2+t3) Afterflame or afterglow of any specimen up to the holding clamp Cotton indicator ignited by flaming particles or drops

Polímeros, 31(2), e2021024, 2021

V0 ≤10 s ≤50 s ≤30 s

V1 ≤30 s ≤250 s ≤60 s

V2 ≤30 s ≤250 s ≤60 s

No No

No Yes

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Figure 2. SEM images of EG before (left) and after (right) the heat treatment. Table 2. Results of TGA obtained from DTG curves. Filament

T1max deg (°C)

T2max deg (°C)

T3max deg (°C)

Residue

PLA-EG0% PLA-EG1% PLA-EG3% PLA-EG5%

363 341 343 335

365 369 360

502 502 492 489

1.15

Tmax deg: temperature at which maximum degradation occurs.

Probably, T1 for the composites had suffered interference from EG expansion, starting at 280 °C, in which sulfuric acid is released from confinement with formation of volatiles[11], promoting reduction in the molar mass of PLA[16]. After this, the composites show the same temperature range (T2) of PLA-EG0%. In addition, it is possible to perceive the effect of EG in increasing thermal stability, when the maximum degradation temperature goes from 363 to 365 and then to 369 °C, as the EG content increases. This behavior is the same observed by several authors and is attributed to the barrier effect promoted by EG due to the swelling that occurs after exfoliation[17]. However, the filament with the highest EG content showed the lowest thermal stability, with Tdeg max below PLAEG0%. Uhl et al.[17] found slightly increase in the maximum temperature of degradation at 1% EG, while high expandable graphite contents, 3% and 5%, apparently were detrimental to PA-6 stability. They attributed this to the release of acid degradation products, which could facilitate the degradation of the PA-6. The peak at T3 is due to reaction with oxygen and carbonization of the samples[18]. 3.2.2 Differential scanning calorimetry (DSC) Usually, DSC analysis involves three steps. The first heating to erase the polymer thermal history. The thermal 4/9

history refers to the heating / cooling processes to which the sample was submitted, prior to carrying out the thermal analysis[19]. The cooling to assess the ability of the polymer to crystallize under cooling. And finally, a second heating to check the crystallization under heating, if any, in addition to melting and second order transitions, such as glass transition temperature. It was not possible to observe the crystallization of PLA during cooling in any of the compositions. Athanasoulia et al. [20] did not observe crystallization peak during the cooling (10 °C min-1) of pristine PLA. According to them, this fact is due to the highly amorphous nature of PLA. By using a cooling rate of 5 °C min-1 they were able to visualize a small and large crystallization peak around 95 °C. In our case, even with slow cooling at 5 ºC min-1, it was not possible to visualize any exothermic event corresponding to PLA crystallization. Refaa et al.[21] studied PLA crystallization PLA in detail. According to them, during PLA crystallization the cooling kinetics exceeds the crystallization kinetics. Thus, for cooling rates greater than 2 ºC min-1 the obtained PLA is practically amorphous. Li and Huneault[22] also reported that they did not observe the exothermic peak referring to pristine PLA cooling crystallization (20 ºC min-1). According to these authors, the addition of PEG enhances the mobility of PLA chains Polímeros, 31(2), e2021024, 2021

Modification of poly(lactic acid) filament with expandable graphite for additive manufacturing using fused filament fabrication (FFF): effect on thermal and mechanical properties facilitating crystallization during cooling. However, even with 5% PEG (3350 g mol-1) they were unable to detect the crystallization. Only with PEG content above 10%, such authors reported a wide and weak crystallization exotherm around 80 ºC. In view of this, it is reasonable to think that with a content of 1% PEG, as in our case, it would be hard to visualize the crystallization exotherm during cooling. Figure 3 shows the DSC curves obtained in the second heating. The values represent the average of two samples. Table 3 summarizes the parameters collected from DSC second heating. The glass transition temperature of PLA and the melting temperature of PEG are very close, and can easily overlap[23]. The event that appears around 60 ºC was treated as Tg of the PLA. According to the values in Table 3, Tg slightly increased with EG content up to 3%, but it was not visualized for higher EG content. Possibly due to a reduction in the amount of PLA amorphous phase[20]. In contrast, Mngomezulu et al. [18] observed a slight steadily increase in Tg of PLA for EG content of 5, 10 and 15%. In the case of PLA, which is a predominantly amorphous polymer, but crystallizable, cold crystallization can occur during heating[24]. Conversely, if PEG does not completely crystallize during cooling, it will not crystallize during subsequent heating[25]. Athanasoulia et al.[26] reported that the cold crystallization peak of PLA disappeared when the cooling rate was lowered from 10 °C min-1 to 2 °C min-1. They attributed this to the completion of crystallization process during cooling, due to the low cooling rate used.

Figure 3. DSC curves obtained from second heating: (a) PLA-EG0%, (b) PLA-EG1%, (c) PLA-EG3% and (d) PLA-EG5%.

All DSC curves in Figure 3 exhibited cold crystallization upon heating. In respect to PLA-EG0% (Table 3), cold crystallization temperature (Tcc) was shifted to lower value than Tcc reported for pristine PLA. This behavior is usually attributed to the plasticizing effect of PEG[26]. With the addition of 1% EG Tcc increased of about 10 ºC, but Tcc shifted to lower values for higher EG contents. Barletta et al.[27] reported that better homogeneity and stronger interactions give rise to composites with larger interfacial area between polymer and filler. At this interface, the filler can effectively decrease the free energy of arising new crystalline nuclei and, therefore, increase the trend of polymer to crystallize during heating (Tcc decreases). Murariu et al.[28] attested the nucleating effect of expanded graphite in PLA composites. They observed the shift of Tcc to lower temperatures compared with Tcc of pristine PLA. On the other hand, stronger interactions between matrix and filler might also act in the opposite direction, reducing the mobility of polymer chains and, therefore, their ability in relation to rearrangement in ordered crystalline structures (Tcc increases)[22]. In our case, the behavior of cold crystallization seems to be related to the EG content. Tcc increased in PLA- EG1% and PLA- EG3%, probably due to strong interactions between EG and matrix which would be hindering cold crystallization. Conversely, Tcc decreased in PLA- EG5%, possibly due to EG action as nucleating agent. Regarding the degree of crystallinity (Table 3), all samples containing EG presented higher degree of crystallinity (χc) than PLA-EG0%. However, χc slightly decreased in PLA-EG5% compared with PLA-EG3%. Murariu et al.[28] reported low χc, around 1%, for pristine PLA. According to them, addition of expanded graphite up to 6% resulted in a pronounced increase of χc. For further nanofiller addition authors observed a reduction in χc. They attributed this fact to possible aggregation of the graphite. The curves of Figure 3 show a double melting peak, which is usually associated with the melting of crystals of different sizes and shapes[18]. There are also some studies relating double melting peak with the recrystallization of the melt during the second heating cycle[21]. Androsch et al.[29] described the conditions under disordered PLA α’ crystals could recrystallize into more stable PLA α crystals. However, they reported melting temperature of α crystals around 170-180 ºC, far above melting temperatures found by us. The curves in Figure 3 showed different ratios of areas between the two melting peaks. Refaa et al.[21] related such difference with different heating rates. In our case, there was no variation in the heating rate.

Table 3. Data from DSC second heating. Filament

TgPLA (°C)

TccPLA (°C)

ΔHcc (J/g)

TmPLA 1 (°C)

TmPLA 2 (°C)

ΔHm (J/g)

χc (%)

PLA-EG0% PLA-EG1% PLA-EG3% PLA-EG5%

59.0 60.3 60.5

102.3 112.0 111.1 94.0

-13.26 -11.56 -12.34 -17.52

144.8 146.8 147.1 137.0

151.9 151.8 152.0 148.3

15.71 18.68 20.09 25.02

2.6 7.7 8.6 8.5

PLA TgPLA: PLA glass transition temperature, Tcc : PLA cold crystallization temperature, ΔHcc: cold crystallization enthalpy, TmPLA: PLA melting temperature, ΔHm: melting enthalpy and χc: crystallinity degree.

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Maybe the graphite particles incorporated into the PLA fracture before the sample breaks or have very little adhesion to the matrix and are pulled out of the matrix[33]. Likewise, the elongation at break decreased whereas the modulus increased as EG content increased. This behavior can be justified by the low tensile deformation capacity of graphite, below 0.5%, and high value of the Young’s modulus of graphite (4100 MPa to 27,000 MPa), respectively[33].

drip. Herein, PLA-EG0% had the same behavior and did not obtain classification in the UL-94 test. For composite with 1 wt. % of expansible graphite, combustion time was reduced, but authors reported flaming dripping. They classified the composites as V-2, the same level of ours. However, unlike us, with 5 wt. % of EG, they reached level V-0. Two hypotheses are suggested to justify this disagreement. The first is that the presence of PEG might be impairing the performance of EG as flame retardant. However, It was already demonstrated the synergism between PEG and ammonia polyphosphate (APP) in the system PLA / PEG 20,000 / APP to obtain the V-0 classification[34]. Thus, there is not an objective evidence to support the hypothesis that the presence of PEG may be responsible for PLA-EG5% composite not reaching the V-0 classification. The second hypothesis is that the lack of adhesion between the phases might be responsible for the poor performance of EG. According to Chen et al.[35], the lack of compatibility between the polymer matrix and the expandable graphite impairs the performance of this flame retardant. Mngomezulu et al.[18] observed that graphite layers were still aggregated and with poor filler dispersion in PLA matrix. Conforming to Li et al.[36], the addition of silane coupling agent gave rise to grafted EG (GEG). Both, the dispersion and the compatibility with the matrix of low-density polyethylene were improved. The effect as flame retardant of GEG (UL94 V-0) was achieved with content of approximately 12 and 15 wt. %. Xiong et al.[37] also attributed better thermal stability and flame-resistance in poly (urethane-imide) (PUI)/EG foams to silane coupling agent.

3.4 Vertical burning test (UL-94)

3.5 Colorimetry assay

The results of ranking criteria (see Table 1) is shown in Table 5. Only PLA-EG0% did not achieve classification according to the criteria for UL-94 test. All composites presented burning the cotton by dripping the material in flames or sparks emitted. Otherwise, composites with 1% and 3% graphite would be classified as V-1 and the composite with 1% graphite would be classified as V-0. Wei, Bocchini and Camino[11] reported that pristine PLA was not classified as a flame retardant, since it completely burned with flaming

Color representation systems translate the colors of objects by numbers. The CIELAB space is composed of three axes. The vertical L* axis represents lightness and varies from 100 (white) to zero (black). The a* and b* axes represent chromacity. The a* axis varies from +a* (red) to –a* (green). The b* axis varies from + b* (yellow) to –b* (blue)[38]. The color difference between two stimuli, the standard and the sample, can be quantified in the diagram L* a* b*. The distance between the two positions, that is, the total color change (∆E*), is defined by Equation 2[38].

3.3 Tensile properties The values of mechanical properties derived from the tensile test are shown in Table 4. As can be seen in Table 4, tensile strength diminished as EG content increased. Yang et al.[30] reported that, with 5% of bio-based flame retardant, both tensile strength and elongation at break were negatively affected. According to them, probably due to adverse impacts on the crystallization and molar mass of PLA. In fact, the DSC results indicated that addition of EG might be affecting the crystallization behavior of PLA. On the other hand, Li et al.[31] stated that the poor interfacial compatibility between EG and the polymer results in an outstanding decrease in the polymer mechanical properties. To overcome this shortcoming, such authors suggested decreasing the particle size of EG and adding octene–ethylene (POE). Even though, the tensile strength kept decreasing with increasing of POE content. The increase in Young’s modulus was only noticeable in PLA-EG5%. Similar behavior was also observed for PA 11 composites reinforced with expandable graphite[32].

Table 4. Values of tensile properties. Specimen PLA-EG0% PLA-EG1% PLA-EG3% PLA-EG5%

σ (MPa) 34.03 ± 1.01 29.72 ± 3.94 23.73 ± 0.12 4.70 ± 1.62

ε (%) 20.94 ± 1.29 20.71 ± 1.20 18.59 ± 0.08 16.38 ± 0.86

E (MPa) 727 ± 15.5 731 ± 16.4 733 ± 4.50 830 ± 38.0

σ: tensile strength, ε: elongation at break and E: Young modulus.

Table 5. Classification in the test for flammability (UL-94). Sample

T1+T2

T2+T3

Flaming particles

Classification

PLA-EG0% PLA-EG1% PLA-EG3% PLA-EG5%

<10 <30 <30 <10

*** <30 <30 <10

<250 <50 <250 <50

*** <60 <60 <30

Yes Yes Yes Yes

None V-2 V-2 V-2

T = time in seconds. *** not applicable.

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Modification of poly(lactic acid) filament with expandable graphite for additive manufacturing using fused filament fabrication (FFF): effect on thermal and mechanical properties ∆E* =

( ∆L ) + ( ∆a ) + ( ∆b ) *

(2)

The values of L*, a* and b*, along with the values of ∆E* and A (absorbance), for PLA-EG0% (standard) and their composites with EG are shown in Table 6. The absorbance behaved as expected, that is, it increased steadily with EG content. This trend is in line with the results found by Przekop et al.[33]. The authors’ research involved addition of graphite into PLA to produce filaments. According to criteria reported by these authors, the ∆E* value of PLA-EG1% can already be classified as marked color difference compared to PLA-EG0%. Obviously, ∆E* value increases with the increase in EG content.

4. Prototypes additively manufactured using FFFtype 3D printer In FFF technology, the thermoplastic polymer filament is driven to an extruder that contains a heater to melt it. The filament is pulled inward with the aid of a roller

mechanism in the feeder and extruded the molten polymer through a circular nozzle. The nozzle extrudes semi liquidstate filament and laid it to desired location with the help of the programmed tri-axial actuator. The 3D printed part is formed on a flat surface platform, known as heat bed[4,39]. Figure 4 shows the obtained composite filaments and the FFF-type 3D printer used to additively manufacture of the prototypes (in detail). Since 3D printing process is performed in layers, the part has an evident marking of the layers. To circumvent this effect and reduce the visible “steps”, the layer height might be reduced. This adjustment improves the surface quality of the part, but the time needed to print considerably increases[40]. A manner to let the part with finer finish is to carry out a post treatment. Different methods include the use of chemical solutions, heat, laser and ultrasound. The most common chemical used to reduce the surface roughness is acetone. However, PLA is not so easily dissolved in acetone, which makes it difficult to smooth the layers[41]. Although several challenges and limitations exist, additive manufacturing

Table 6. CIELAB parameters, total color variation (∆E *) and absorbance (A). Sample PLA-EG0% PLA-EG1% PLA-EG3% PLA-EG5%

L* 81.34±2.03 43.13±1.23 31.88±1.07 27.69±0.51

a* -2.15±0.09 -0.72±0.07 -0.29±0.04 -0.14±0.05

b* 4.66±0.32 -2.24±0.23 -1.77±0.25 -1.58±0.22

∆E* 38.85±1.23 49.90±1.04 54.04±0.52

A 0.3±0.01 0.84±0.02 1.11±0.03 1.23±0.01

Absorbance at 420 nm.

Figure 4. Prototypes additively manufactured using FFF-type 3D printer fed with: (a) PLA-EG0%, (b) PLA-EG1%, (c) PLA-EG3% and (d) PLA-EG5%. Polímeros, 31(2), e2021024, 2021

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Melillo, J. M. A., Pereira, I. M., Mottin, A. C. & Araujo, F. G. S. (AM) is expected to revolutionize the fabrication process of engineering components[1].

5. Conclusions The possibility of printing parts with complex shapes, with the exact amount of raw material, is a great advantage of 3D printing. In this study, composite filaments based on PLA and 1, 3 and 5 wt.% of EG were developed for using in FFF-type 3D printing. The effect of EG on thermal and tensile properties of PLA was investigated. Based on the findings of these analyses, it is likely that expandable graphite needs stronger interfacial adhesion with the PLA matrix. The possibility to impart anti-flammability property to PLA filament by modifying it with expansible graphite was evaluated. All composites reached the classification V-2 in the vertical burning test (UL-94). This was not the expected result, and it’s probably due to the lack of adhesion between phases, as pointed out by the mechanical and thermal assays. The obtained composite filaments kept printable, and prototypes were successfully made from all of them. Further research should involve silane as coupling agent and also a flame co-retardant to work in synergism with expansible graphite.

6. Acknowledgements The authors acknowledge the financial support from National Council for Scientific and Technological Development - CNPq and National Council for Scientific and Technological Development Coordination for the Improvement of Higher Education Personnel – CAPES. The authors would also like to acknowledge Nacional de Grafite for kindly supplying expansible graphite.

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