Safety of new food contact materials: Migration and sorption studies based on Tenax, powdered milk, baby cereal and oat flakes

1. Introduction

The global environmental crisis associated with the ubiquity of landfills has given rise to pro-environmental measures, which include the “European Green Deal” climate pact, the “fit for 55” package (European Commission, 2021) and the Plastics Directive (European Commission, 2019). The main requirements of the above measures are the energy transformation of industry, the spread of renewable energy sources and the implementation of a more sustainable and environmentally friendly economy (closed loop economy). According to modern environmental requirements, non-biodegradable materials must be replaced by biodegradable or recyclable ones (according to the 3R principle: reduce, reuse, recycle). The global demand for green materials has influenced the search for raw materials and solutions to reduce production costs (Fu et al., 2023) and improve functional properties (Guivier et al., 2024). Global plastics production in 2021 was about 390.7 million tonnes, including 5.9 % bio-based materials (from biosolids or biowaste) (Plastics Europe, 2022). The largest segment of global plastics use is packaging (about 44 % in 2022).

Biodegradable and recyclable materials that are widely used in the production of food packaging and vessels can be called food contact materials (FCMs) (Geueke, Groh, & Muncke, 2018). FCMs must comply with basic food safety requirements (European Commission, 2004, European Commission, 2011). The material cannot affect the quality and sensory properties of the food. However, previous migration studies clearly demonstrate that packaging and vessel materials are not passive to food and can be one of the sources of food contamination with chemical compounds. The intensity of migration processes can depend on various factors, which include the physical properties of the migrant (molecular weight and diffusion coefficient, octanol/water partition coefficient, vapor pressure), the type of FCM, the type of food, the time and temperature of contact between the material and the food and the way of cooking food (use conventional or microwave heating) (Dąbrowska, Borcz, & Nawrocki, 2003; Vera, Aznar, Mercea & Úbeda et al., 2017; Ji et al., 2019; Otoukesh, Vera, Wrona, Nerin, & Es’haghi, Z., 2020; Elizalde, Aparicio, & Rincón, 2020; Bronczyk, Dabrowska, & Majcher, 2023). Therefore, the global production of plant- and bio-based plastics FCMs could become a serious food contamination problem.

The currently observed global environmental pollution (air, water, soil) may influence the appearance of wide groups of chemical compounds in served/packed food, migrating from FCMs. Popular plant-based materials require a particularly thorough safety assessment in this respect, because this type of FCM may contain various environmental pollutants. The natural ability of plants to biodegrade, accumulate and inactivate environmental contaminants means that dangerous chemical compounds can be stored in their tissues. Undesirable environmental pollutants that can contaminate plant-based FCMs include polycyclic aromatic hydrocarbons (PAHs), pesticides (especially those with long degradation times in the environment, such as organochlorine pesticides) and low molecular weight carbonyl compounds. PAHs are by-products of reactions of incomplete combustion processes of organic matter. PAHs can easily adsorb to the surface of solids due to their poor solubility in water, low vapor pressure and aromatic nature (Kubiak, 2013). Plastic waste, especially polystyrene, can be a source of PAHs pollution in the aquatic environment (Si-Qu, Hong-Gang, & Hui, 2017). Plants can sorb PAHs from contaminated environment through roots and tubers. The presence of PAHs in food is controversial because they exhibit mutagenic, genotoxic and carcinogenic effects on the health (Bansal & Kim, 2015; Sampaio et al., 2021). Some PAHs may be degraded to carbonyl compounds in the environment, which can be absorbed by plants (William, Pangzhen, Danyang, & Zhongxiang, 2023). Aldehydes (especially low-molecular-weight compounds) belong to the group of odor-active compounds and can create a specific plant odor, especially when present in mixtures. Previous work has shown that some carbonyl compounds can migrate from plant-based FCMs into coffee and alter its desired sensory properties (Bronczyk et al., 2023). Other aldehydes, especially formaldehyde (FA), acetaldehyde (AA), glyoxal (GLY) and methylglyoxal (MGLY), were determined in bottled mineral waters, but their presence in foods is controversial due to their carcinogenic properties (Dąbrowska et al., 2003; World Health Organization, 2011).

FCMs can also contain contaminants from the production line. Sometimes additives are added intentionally to improve the functionality and desired characteristics of the packaging and vessel materials. Cross-contamination can also occur during the manufacturing process stage. Particularly undesirable contaminants of FCMs from this source include bisphenols: bisphenol-A (BPA) and bisphenol-S (BPS), benzophenone derivatives: 2,4-dihydroxybenzophenone (2,4-DHBP), 2,2,4,4′-tetrahydroxybenzophenone (2,2,4,4’-THBP), 2-hydroxy-4-methoxybenzophenone (2-H-4-MBP) and phthalates: dibutyl phthalate (DBP), diisobutyl phthalate (DiBP), 2-di(ethyl hexyl) phthalate (DEHP). BPA and BPS are commonly used as epoxy resins and polycarbonate precursors to improve strength, hardness, thermal stability, and grease and oil resistance of packaging and vessel materials (Ma et al., 2019). Benzophenones are UV stabilizers and ingredients of inks, paints and printed FCMs. In turn, phthalates are added to packaging and vessel materials to improve their functional properties (flexibility, softness and elasticity). They are also used in the production of varnishes and prints and as additives that improve adhesion to surfaces (Moraes da Costa et al., 2023).

Material additives should be used in moderation. Some of them (e.g. BPA/BPS) exhibit pro-estrogenic effects and are defined as endocrine disrupting compounds (EDCs). BPA can interact with various biological receptors such as estrogen receptor (ER), androgen receptor (AR) and thyroid hormone receptor (THR) (Ma et al., 2019). These disruptions can lead to diseases of the reproductive, nervous and cardiovascular systems, metabolic and immune functions, as well as the growth and development of offspring (Prueitt et al., 2023; Sawadogo et al., 2023). Substitutes, including BPS, are often used to reduce BPA content. However,

in vivo and in vitro studies have shown the effect of BPS on endocrine disorders, which means that commonly used BPA analogues may also be hazardous food contaminants (Heindel et al., 2022). 2,4-DHBP, 2,2,4,4’-THBP and 2-H-4-MBP, as ECDs, may have a toxic effect on hormonally controlled processes, including fertility, development of the nervous system and sexual differentiation. They can interact with enzymes, leading to digestive disorders and also adversely affect the proliferation and migration of cancer cells (Ma et al., 2023). Moreover, exposure to phthalates (DBP, DiBP and DEHP) and their metabolites, are particularly dangerous for pregnant women and teenagers (Topdas, 2023; Tsochatzis et al., 2023). These compounds can cause oxidative stress, which leads to premature birth (Ferguson et al., 2016) and pancreatic β-cell dysfunction. Phthalates as ECDs may lead to the development of obesity and asthma, especially in adolescents (Dong et al., 2022; Zhu et al., 2024). Chronic exposure to phthalate metabolites may influence behavioral disorders in children, such as the development of attention deficit hyperactivity disorder (Hu et al., 2017).

The undesirable properties of substances that may be contained in packaging and vessel materials make it necessary to control their concentration in food. A specific migration limit (SML) has been established for intentionally added substances (IAS), which is included in the list of Regulations (European Commission, 2011; European Commission, 2018; European Commission, 2020). SML is the maximum amount of a substance that can migrate from FCMs into one g of food (Table 1). Some substances in food may be added unintentionally and then are called non-intentionally added substances (NIAS), such as degradation products, impurities, etc.

Table 1. Analyzed pollutants with characteristics: abbreviation used in this study, chemical structure, FCM substance number, molecular weight, IAS/NIAS division, SML parameter (based on the Commission Regulations No. 10/2011 and No. 2018/213).

Migrant		Abbreviation	FCM substance number	Molecular weight (Da)	IAS / NIAS	SML (mg/kg or μg/g of food)
Environmental pollutants	Antracene	ANT	–	178.23	NIAS	–
	Phenanthrene	PHE	–	178.23	NIAS	–
	Formaldehyde	FA	98	30.03	IAS	15
	Acetaldehyde	AA	128	44.05	IAS	6
	Glyoxal	GLY	–	58.04	NIAS	–
	Methylglyoxal	MGLY	–	72.06	NIAS	–
Contaminants from the production line	Bisphenol-A	BPA	151	228.29	IAS	0.05
	Bisphenol-S	BPS	154	250.27	IAS	0.05
	2,4-dihydroxybenzophenone	2,4-DHBP	318	182.22	IAS	6⁎
	2,2,4,4′-tetrahydroxybenzophenone	2,2,4,4’-THBP	–	246.21	NIAS	–
	2-hydroxy-4-methoxybenzophenone	2-H-4-MBP	319	228.24	IAS	6⁎
	Dibutyl phthalate	DBP	157	278.34	IAS	0.3
	Diisobutyl phthalate	DiBP	–	278.35	NIAS	–
	2-di(ethylhexyl) phthalate	DEHP	283	390.56	IAS	1.5

One of the popularly used food simulants in migration studies is Tenax (poly(2,6-diphenyl-p-phenylene oxide)), which according to Commission Regulations No. 10/2011 imitates the dry and frozen foods (Canellas, Vera, & Nerin, 2015; Jung, Simat, Altkofer, & Fugel, 2013; Otoukesh et al., 2020; Suciu, Tiberto, Vasileiadis, Lamastra, & Trevisan, 2013). Tenax is a porous organic polymer that is characterized by high chemical stability (up to 350 °C) (Alfeeli, Taylor, & Agah, 2010). Some studies report disadvantages of Tenax as a food simulant, including the cost of the reagent, the need for long-term regeneration and difficult Tenax management caused by static electricity from friction (Rubio, Valverde-Som, Sarabia, & Ortiz, 2019). The use of Tenax may also lead to overestimated IAS and NIAS concentrations compared to real food samples (Almeida Soares et al., 2023; Baele, Vermeulen, Claes, Ragaert, & De Meulenaer, 2020; Cacho, Campillo, Vinas, & Hernandez-Cordoba, 2012; Elizalde et al., 2020; Rubio et al., 2019). These reports suggest that interpretation of the results of migration studies with Tenax should be cautious. However, it should be noted that the results of such studies can provide an understanding of contaminants that may not necessarily migrate from FCMs into real food. They can be a warning sign for taking early preventive measures.

There are many literature reports on migration studies of contaminants from carton packaging into Tenax (Cai et al., 2017; Rubio et al., 2019; Suciu et al., 2013). However, there is little information about the safety of currently popular plant- and bio-based plastics FCMs (Asensio, Montañés, & Nerín, 2020; Wrona et al., 2023) and especially concerning the migration of various contaminants from such FCMs into Tenax. It is important to investigate the potential for migration of hazardous environmental contaminants, such as PAHs and aldehydes. In addition, different FCMs production technologies can result in different levels of contamination by additives and stabilizers. It is also important to investigate the factors that cause large discrepancies in migration studies of various contaminants between Tenax and real food samples.

In the first part of the work, migration studies of various environmental contaminants (PAHs, aldehydes) and manufacturing additives (BPA, BPS, 2,4-DHBP, 2,2,4,4’-THBP, 2-H-4-MBP, DBP, DiBP and DEHP) from currently popular plant- and bio-based plastics FCMs into Tenax were carried out. Migration studies were conducted under various time and temperature conditions. In the second part of the work, the specific surface area and pore distribution of Tenax and real foods (powdered milk, baby cereal and oat flakes) were determined. The effects of material structure and migrating particle size on the efficiency of migration processes were also compared.

2. Materials and methods

2.1. Reagents and chemicals

Analytical standards of polycyclic aromatic hydrocarbons: ANT and PHE, carbonyl compounds: FA, AA, GLY, MGLY, bisphenols: BPA, BPS, benzophenone derivatives: 2,4-DHBP and 2,2,4,4’-THBP (Table 1) were obtained by Sigma Aldrich (Poland) and 2-H-4-MBP was obtained by Honeywell, Fluka (Germany). All the standards were of a purity higher than 99.9 %. Individual stock solutions of 5 μg/mL were prepared in methanol and stored in amber vials at 4 °C. Working standard solutions containing all the analytes at 1 μg/mL were prepared daily in methanol or acetone and stored at 4 °C. Chromatographic quality methanol, acetonitrile and acetone were purchased from Sigma Aldrich (Poland). Tenax with a particle size of 60–80 mesh and a pore size of 200 nm were purchased from Sigma Aldrich (Poland). Preparation of Tenax for migration studies was performed similarly to procedure described in the literature (Rubio et al., 2019) with the following changes. Five g of Tenax was placed in a cellulose thimble and purified for 6 h with 70 mL methanol in a Soxhlet apparatus before use. Then the food simulant was preheated to 160 °C for 6 h and stored in a desiccator. Tenax was mixed to homogenize the material before migration studies. O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBOA) reagent was purchased from Aldrich-Chemie (Steinheim, Germany) PFBOA was prepared gravimetrically as an aqueous solution at a concentration of 2 mg/mL.

2.2. Sample collection

Commercially available plant- and bio-based plastics FCMs were analyzed. They can be popularly used to serve dry food. A description of the material details and an example of dry food that can be served are presented in Table 2.

Table 2.. List of tested FCMs, abbreviations used, average weight (for three replicates) and type of dry food that can be served in individual vessels.

Analyzed FCMs	Abbreviation of the sample name	Average weight of the vessel (g)	Example of food served on a vessel that determines the use of Tenax in migration tests (in accordance with Regulation No. 10/2011)
Plate of wheat bran	PWB	97.3	Dry pasta, e.g. spaghetti
Bowl of bamboo	BAM	5.1	Shelled or dried nuts
Bowl of plant residues	PLR	10.3	Dried or freeze-dried vegetables
Bowl of wood	WB	1.5	Shelled and roasted almonds
Cup of thermoplastic starch	TS	3.0	Sugar and sugar products in solid form
Cup of polylactide	PLA	4.5	Milk powder
Bowl of expanded polypropylene	EP	12.9	Popcorn, cornflakes
Brown paper cup	PCB	13.2	Cocoa powder with lower fat content

2.3. Migration studies

Four g of Tenax per 1 dm² of surface was used in migration tests, according to the guidelines (Commission Regulation (EU) No 10/2011). The analyzed materials were cut into pieces measuring 5 cm × 5 cm (the surface was 25 cm²), placed on watch glasses and covered with one g of Tenax. The sample was wrapped in aluminum foil (to eliminate the possibility of evaporation) and placed in an oven heated to 70 °C for 2 h or to 40 °C for 10 days, which corresponds to short or long conditions of contact between food and the vessel (Commission Regulation (EU) No 10/2011). In next step, Tenax was extracted twice with 25 mL of an appropriate extractant (acetone to determine PAHs, methanol to determine the others contaminants, such as aldehydes, benzophenone derivatives and bisphenols) within 1 h at ambient temperature. The extracts were concentrated to 4 mL by vacuum evaporation (p = 850 hPa) and placed in amber glass vials. Blank samples were prepared for analysis in the same way as test samples, but without the use of FCMs. Three replicates were performed for each sample and blanks. Satisfactory results were obtained in percent recovery (80 %-105 %) for each analyte. The use of 1 g of Tenax sorbent did not cause overload phenomena.

2.4. Chromatografic analysis

Polycyclic aromatic hydrocarbons were determined by the GC-FID technique (HP 5890II) with autosampler and flame ionization detector (FID). The injection volume was 1 μL, injector and detector temperature were set at 280 °C. The chromatograph was equipped with Rtx 5-W/Integra Guard capillary column (30 m × 0.25 mm × 0.25 μm, Restek, USA). The analysis was performed using helium as carrier gas at a flow rate of 1.75 mL/min. The initial column temperature was 90 °C (hold for 3 min) and then ramped at 10 °C/min to 270 °C. The total analysis time was 21 min.

Low-molecular weight carbonyl compounds were analyzed using Fisons Instruments 8000 equipped with ⁶³Ni electron capture detector (GC-ECD). The samples were prepared in several steps: derivatization with PFBOA reagent (reaction added in Supplementary Material as Fig. S1), liquid-liquid extraction (LLE) with hexane and purification, as described in previous work (Bronczyk et al., 2023). Injections of 0.5 μL of the extract were introduced via „on column” injector into chromatographic column. A Rtx-5MS (Restek, USA) fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness) was employed for analysis, and a Rtx-1301 (Restek, USA) fused silica capillary column (30 m × 0.32 mm i.d. × 0.5 μm film thickness) was used as a confirmation column. Injector temperature was set at 80 °C. Gas flow was set at 80 kPa. Helium was used as carrier gas and nitrogen was used as make-up gas for the detector. Analysis was carried out on a temperature program starting at 80 °C for 4 min, then increasing the temperature to 240 °C with an increase of 7 °C/min, and then to 290 °C with an increase of 20 °C/min. DataApex, Clarity 6.2, Czech Republic software was used to collect and process chromatographic data.

Bisphenols and benzophenone derivatites were determined by the HPLC-DAD technique (Agilent 1100 Series HPLC System with Diode Array Detector). The injection volume was 1 μL. The chromatograph was equipped with an Ultra AQ C18 column (5 μm, 250 mm × 4.6 mm; Restek, USA). A mixture of acetonitrile and water in a volume ratio of 70:30 was used as the mobile phase in the analysis of BPA and BPS and methanol (100 %) was used in the analysis of 2,4-DHBP, 2,2,4,4’-THBP and 2-H-4-MBP, respectively. The mobile phase flow rate in both analyzes was 1 mL/min in isocratic mode. The total analysis time was 3 min for bisphenols, and 4 min for benzophenone derivatives, respectively. Spectra were collected at a wavelength of λ = 254 nm.

Quantification of migrated contaminants was carried out using an external standard calibration curve, which was prepared by plotting concentration against peak area or, in the case of isomeric peaks, combined areas (e.g. duplet for acetaldehyde). All standards were prepared gravimetrically in concentration ranges: for ANT and PHE, 1–10 μg/L; for carbonyl compounds, 1–30 μg/L; and for BPA, BPS, and benzophenone derivatives, 0.05–10 μg/L, respectively. The chromatograms are available in the Supplementary Materials as Figs. S2 and S3. Limit of detection (LOD) and quantification (LOQ) were determined for each analyte using “Regression Statistics”. The precision of the method was evaluated in terms of repeatability and expressed as relative standard deviation (RSD%). The RSD% was obtained by analyzing the samples in optimized conditions, using three replicates and three points of calibration curve for each analyte. The analytical parameters of all migrants analyzed are shown in Table 3.

Table 3. List of tested migrants and chromatographic data: retention time (min), standard curve equation (includes measurement errors of parameters a and b and expressed in the form y = a(±SE)x + b(±SE)), limit of detection (LOD), limit of quantification (LOQ) and relative standard deviation (RSD).

Chromatografic analysis	Migrant	Retention time (min)	Standard curve equation	LOD (μg/L)	LOQ (μg/L)	RSD (%)
GC-FID	PHE ANT	14.67 14.79	y = 2.70(±0.15)x − 1.60(±0.69) y = 1.51(±0.07)x − 0.91(±0.34)	0.88 0.77	2.70 2.30	8.5 9.3
GC-ECD	FA	5.82	y = 38(±2)x + 2011(±185)	0.003	0.009	1.7
	AA	7.39; 7.54	y = 57(±4)x + 1343(±162)	0.005	0.015	4.1
	GLY	22.21; 22.38	y = 67(±5)x + 623(±60)	0.015	0.045	6.7
	MGLY	22.76; 22.93	y = 92(±7)x + 169(±18)	0.015	0.045	7.5
HPLC-DAD	BPA	2.89	y = 1.07(±0.02)x + 4.05(±0.57)	0.12	0.36	1.8
	BPS	2.33	y = 1.27(±0.09)x + 2.49(±0.04)	0.13	0.38	1.7
	2,4-DHBP	3.19	y = 3.55(±0.05)x + 0.14(±0.10)	0.13	0.38	2.3
	2,2,4,4’-THBP	2.80	y = 1.84(±0.11)x + 0.09(±0.02)	0.52	1.60	9.7
	2-H-4-MBP	3.74	y = 4.02(±0.16)x + 0.33(±0.03)	0.35	1.10	5.9

The results of the target analysis were confirmed in parallel measurement using GC/MS (Varian GC–MS 4000), observing fragmentation peaks typical of the analytes (Table 4). Moreover, untargeted analysis demonstrates the presence of other contaminants, including phthalates. Compounds were determined using a BP5 MS capillary column coated with 5 % phenylpolysilphenylene-siloxane (30 m × 250 μm i.d., 0.25 μm film thickness, SGE Analytical Science). One microliter of derivatives was injected in splitless mode, and the solvent delay time was set to 3.5 min. The initial oven temperature was held at 40 °C for 3 min, ramped to 280 °C at a rate of 15 °C/min and held for 10 min. Helium was used as a carrier gas at a constant flow rate of 1 mL/min through the column. The temperatures of the front inlet, transfer line and electron impact (EI) ion source were set at 250, 280 and 230 °C, respectively. The ionization energy was 70 eV. The mass spectral data was collected in a full scan mode (m/z 30–600) and in SIM (selected ion monitoring) mode. Mass spectra of identified contaminants standards obtained in the SIM mode (monitoring of selected ions) are presented in Table 4.

Table 4. Monitored SIM ions for the analyzed contaminants in the GC–MS system.

Migrant	Monitored Ions (SIM)
FA AA GLY MGLY BPA BPS 2,4-DHBP 2,2,4,4’-THBP 2-H-4-MBP DBP DiBP DEHP	181; 195; 161 181; 195; 161 181; 117; 161 15; 29; 43; 45; 72 39; 65; 91; 119; 213; 228 39; 65; 93; 110; 141; 250 39; 65; 93; 121; 214 69; 81; 110; 137; 229; 246 51;77; 105; 108; 151; 227 104; 149; 205; 223 104; 149; 205; 223 57; 149; 167, 279

2.5. Evaluation of the effect of food structure and physical properties of molecules on migration intensity

The Brunauer-Emmett-Teller (BET) gas adsorption method has become the most widely used standard procedure for determining the surface area of fine-grained and porous materials from adsorption data. This method is based on the physical adsorption of a vapor or gas onto the surface of a solid. The specific surface area and pore size (BET isotherm) of Tenax and food samples (powdered milk, baby cereal, oat flakes) were examined, to evaluate the influence of the structure of simulated and real foods and the properties of contaminants on the intensity of migration processes. The composition of the food analyzed, according to the product label, is summarized in Table 5.

Table 5. Description of the composition of the food samples (expressed in g/100 g of product), according to the manufacturer’s data (product label).

Food sample	Content of
Food sample	Fat (including saturated fatty acids)	Carbohydrates (including sugars)	Fiber	Protein	Salt	Mineral components
Granulated, non-fat powdered milk	0.8	51	NS*	35	1.2	Calcium (1.404) Phosphorus (1.012)
Baby cereal	1.4	87	2.1	7.6	0.02	Sodium (6.5)
Whole grain oat flakes	6.9	60	9.8	12	<0.01	NS*

NS*: not specified by the manufacturer.

High-purity nitrogen (> 99.999 %) was used as adsorbate. The powdered materials were pre-gassed at 100 °C for 24 h. The selection of pre-degassing parameters took into account resistance to elevated temperatures, including susceptibility to changes in pore structure. The sample was then filled with nitrogen and weighed to determine the real (dry) weight of the sample. The pre-gassed sample vial and the weighing vial (empty) were sealed in an Autosorb iQ Station 1 port transducer, Quantachrome® ASiQwin™ Automatic Gas Sorption Data Acquisition and Reduction ©1994–2013, Quantachrome Instruments version 3.01., and then immersed in liquid nitrogen at 77.35 K. Measurement of gas adsorption on the test material consisted of gradually filling the volume of two vials with the same amount of nitrogen in the relative pressure range from 0.01 to 0.99 p/p₀.

In the next experiment, the adsorption capacity of Tenax and food samples (powdered milk; infant cereal and oatmeal) and the influence of the physical properties of migrant compounds on the intensity of migration processes were evaluated, as recovery test. The spiking experiment was conducted as follows: 1 mL of standard solutions of the tested migrating compounds (2,4-DHBP, 2,2′,4,4’-THBP; 2-H-4-MBP; PHE and ANT) containing analytes at appropriate concentrations were applied to a glass Petri dish to obtain a final concentration of 3 μg/L for 2,4-DHBP, 2,2′,4,4’-THBP and 2-H-4-MBP and 5 μg/L for ANT and PHE. These chemical compounds were chosen because of their similar structure but different molecular weights.

One g of Tenax or food (powdered milk; baby cereal and oat flakes) was applied to the materials, then wrapped in aluminum foil and placed in an oven heated to 70 °C for 2 h. The remaining steps of sample preparation for chromatographic analysis in this study are the same as in Section 2.3 Migration studies. To assess the statistical significance of the results obtained in both experiments, the student’s t-test in Statistica software was used, taking the criterion p < 0.05 as a sign of statistical significance.

April 13, 2025 at 04:18PM
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