Preprint for investigating aquatic biodegradation of pristine and UV-irradiated microplastics from conventional and biodegradable agricultural plastics using advanced analytical techniques

Preprint for investigating aquatic biodegradation of pristine and UV-irradiated microplastics from 1 conventional and biodegradable agricultural plastics using advanced analytical techniques 2 3 Ula Putar1, Aida Fazlić2, Lukas Brunnbauer3, Janja Novak1, Anita Jemec Kokalj1,4, Jernej Imperl1, Jiří 4 Kučerík5, Petra Procházková2, Stefania Federici6, Rachel Hurley7, Andrijana Sever-Škapin8,9, Pavlína 5 Modlitbová2, Pavel Pořízka2,10, Jozef Kaiser2,10, Andreas Limbeck3, Gabriela Kalčíková1,10* 6 *corresponding author: [email protected] 7 8 1Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, 9 Slovenia 10 2Central European Institute of Technology, Brno University of Technology, Purkyňova 656/123, 61200 11 Brno, Czech Republic 12 3TU Wien, Institute of Chemical Technologies and Analytics, Getreidemarkt 9/164-I²AC, 1060 Vienna, 13 Austria 14 4University of Ljubljana, Biotechnical Faculty, Department of Biology, Jamnikarjeva 101, 1000 15 Ljubljana, Slovenia 16 5Mendel University, Department of Agrochemistry, Zemědělská 1752, 613 00 Brno, Czech Republic 17 6Department of Mechanical and Industrial Engineering, University of Brescia & INSTM RU of Brescia, 18 Via Branze 38, 25123 Brescia, Italy 19 7Norwegian Institute for Water Research, Økernveien 94, 0579, Oslo, Norway 20 8Slovenian National Building and Civil Engineering Institute, Dimičeva ulica 12, 1000 Ljubljana, Slovenia 21 9Faculty of Polymer Technology—FTPO, Ozare 19, 2380 Slovenj Gradec, Slovenia 22 10Faculty of Mechanical Engineering, Brno University of Technology, Technická 2896/2, 61669 Brno, 23 Czech Republic 24 25 26 Abstract 27 There is an increasing tendency to replace conventional agricultural plastic mulching films with 28 biodegradable alternatives. However, while the latter biodegrade well under controlled conditions 29 (e.g. industrial compost), their biodegradation in non-target environments is questioned and poorly 30 understood. Therefore, in this study, microplastics derived from conventional polyethylene (PE) and 31 biodegradable polybutylene adipate terephthalate starch blend (PBAT) mulching films were exposed 32 to UV irradiation and subsequently tested for their ready biodegradability in an aqueous medium. The 33 results showed limited biodegradation for pristine and UV-aged PE: no morphological, surface 34 chemical or internal changes were observed. Pristine PBAT showed signs of initial biodegradation, 35 while UV-aged PBAT biodegraded by up to 57%. New functional groups appeared on the PBAT surface 36 after UV irradiation according to FTIR analysis and crystallinity increased after biodegradation. 37 Elemental analysis revealed a range of metals in PE and PBAT microplastics. No changes in metal 38 distribution analysed in microplastic after UV-aging or biodegradation were found, except that less 39 titanium was present in PBAT after biodegradation indicating potential leaching. None of the PBAT 40 microplastics had ecotoxic effects towards the aquatic plant Lemna minor. Pristine and UV-aged PE 41 showed negative effects on roots, but these were not observed after biodegradation. Low 42 biodegradation of pristine PBAT and possible leaching of metals demonstrated here raise questions 43 about the sustainable use of biodegradable alternatives, especially when they enter non-target 44 environments. 45 46 Keywords: aging, aquatic ecosystems, compostable plastics, degradation, open environment, 47 microplastics 48 49 50 51 52 1. Introduction 53 The production and use of plastics have increased dramatically in recent decades due to their 54 versatility and economic feasibility (PlasticsEurope, 2024). However, this widespread use has come at 55 a significant cost to the environment through extensive pollution of aquatic and terrestrial ecosystems 56 (Ghaffar et al., 2022; Napper and Thompson, 2023; Simul Bhuyan et al., 2021) leading to urgent action 57 to mitigate plastic pollution, with an increasing focus on the development of biodegradable 58 alternatives (Dilshad et al., 2021; Moshood et al., 2022; Rosenboom et al., 2022). 59 In sectors with high plastic consumption, such as agriculture, the transition from conventional plastic 60 to biodegradable is a crucial step in reducing plastic pollution. Unlike conventional plastics, 61 biodegradable alternatives are supposed to be degraded within the soil environment, eliminating the 62 need to collect them after use and minimising agricultural waste. So, the synthesis and production of 63 biodegradable plastics is coupled with testing of their biodegradability in the soil environment 64 (Briassoulis and Dejean, 2010). Many studies have shown that biodegradable plastics may degrade in 65 soil but the degree and rate of this degradation is affected by the composition of the plastics, presence 66 of additives, and the conditions of the surrounding environment such as soil properties, microbial 67 activity, water content, temperature, and also pre-exposure to the UV irradiation (Afshar et al., 2024; 68 Hoshino et al., 2001; Lambert and Wagner, 2017; Liao and Chen, 2021; Mazzon et al., 2022; Oberlintner 69 et al., 2021; Pischedda et al., 2019). A recent study revealed that PBAT mulching film degrades in situ 70 in agricultural soil, but after 16 months of burial in soil, the film degradation was still not complete 71 (approximately 66% of the film degraded) and considerable amount of macroand microplastics was 72 found in soil (Convertino et al., 2024). Field tests tracking the degradation of biodegradable plastics in 73 soil have revealed that the time period for degradation observed in laboratory tests (for certification 74 of biodegradation in soil) are indeed also met in field conditions but only when considering thermal 75 days, as opposed to calendar days (e.g. Griffin-LaHue et al. (2022)). This highlights an important factor 76 when translating laboratory tests to the field context, where there is a likelihood for biodegradable 77 plastic residues to remain in the soil for several years after use and/or incorporation into soils. 78 Despite extensive research on the biodegradation of plastic alternatives used in the soil environment, 79 there are concerns about their fate once they enter the aquatic environment (Kaing et al., 2024). This 80 is because biodegradable mulching films can fragment due to UV exposure during agricultural use 81 (Convertino et al., 2024; Yang et al., 2022), and the resulting microplastics can be then potentially 82 transferred into water bodies (Ren et al., 2021; van Grinsven and Schubert, 2023). Evidence for the 83 propagation of microplastics from soil environments across wider spatial scales and to connected 84 environments has already been observed (Crossman et al., 2020; Han et al., 2022), demonstrating the 85 potential likelihood for biodegradable in soil plastic fragments to be transferred to aquatic 86 environments. This pathway raises critical questions about the actual biodegradability of these newly 87 synthesised materials in aquatic environments, whose dynamics and consequences differ significantly 88 from those in terrestrial environments. 89 In this context, the aim of this study was to compare and evaluate the biodegradation of two materials 90 used as mulching films — conventional polyethylene (PE) and biodegradable polybutylene adipate 91 terephthalate starch blend (PBAT) — in the aquatic environment (i.e. in ready biodegradability test 92 according to OECD guidelines (OECD, 1992)). To increase environmental relevance, the materials were 93 tested both in their pristine form and after exposure to UV-VIS irradiation as UV light is one of the most 94 common abiotic degradation factors in the environment (Li et al., 2023). Traditionally, the 95 biodegradability of plastics is evaluated by measurement of oxygen consumption or evolved CO2 (Pires 96 et al., 2022). However, these methods provide limited insight into the degradation process, and 97 therefore we coupled a conventional biodegradability test with advanced analytical techniques to 98 better understand the initial steps of microplastic biodegradation process. The evaluation focused on 99 i) biodegradability in the aquatic environment, ii) morphological changes, iii) surface chemical 100 alterations, iv) changes in elemental composition, v) internal structure modifications, and vi) the 101 changes in ecotoxicity. Understanding biodegradation in non-target environments is crucial to fully 102 assess the impact on the environment and effectively communicate about sustainable practices. 103 104 2. Material and Methods 105 2.1 Characteristics of microplastics 106 Microplastics were derived from one conventional PE mulch film (internal code M-PEDE-45-black-A0; 107 P6) and one mulch film made of starch and PBAT (internal code M-BIOEL-15-black-A0, P5) within the 108 EU Horizon project PAPILLONS. Both were black and prepared by cryomilling of the virgin mulching 109 films. Extensive properties of the source material mulching films used for the generation of 110 microplastic and their properties were reported by Hurley et al. (2024). The median size of PE and 111 PBAT were 57 ± 38 μm and 147 ± 44 μm, respectively, and they were irregularly shaped fragments 112 (Jemec Kokalj et al., 2024). Organic and inorganic additives were identified in PE and PBAT - these 113 include a series of light stabilisers and antioxidants to slow the degradation of the material, anti-slip 114 agents used for the production of films, and plasticisers to deliver the properties of the mulching film 115 materials (Hurley et al., 2024). 116 A portion of the prepared microplastics was subjected to UV-VIS irradiation to simulate accelerated 117 natural weathering. The samples were carefully placed in glass Petri dishes and shielded by a layer of 118 quartz glass to prevent contamination or possible sample loss during exposure. They were exposed in 119 a Q-SUN Xe-3 UV chamber (Q-Lab, Bolton, UK) for accelerated weathering for 240 hours. The chamber 120 was equipped with three 1800 W Xe lamps calibrated to emit wavelengths corresponding to natural 121 sunlight – xenon arc lamps produce the realistic reproduction of full-spectrum sunlight, including 122 ultraviolet, visible light, and infrared radiation. The samples were irradiated with a power of 40 W/m2 123 at a chamber temperature of 38 °C and 50% of relative humidity, while the black standard was 124 maintained at a temperature of 50 °C. Throughout the exposure period, the samples were thoroughly 125 mixed and homogenised daily to ensure uniformity and consistency of the aging process. The aged 126 microplastics were labelled as UV-PE and UV-PBAT. 127 128 129 130 2.2 Biodegradability in the aquatic environment 131 In order to comprehensively evaluate the biodegradability of PE, UV-PE, PBAT, and UV-PBAT, two 132 experimental set-ups, i.e., open and closed systems, were used. Both systems were operated under 133 comparable conditions (temperature, stirring, content of nutrients and microorganisms, availability of 134 oxygen, etc.). Details are given in subchapters 2.2.1 and 2.2.2. Microplastics from the open system 135 were then analysed using advanced analytical techniques (subchapter 2.3) to assess the changes due 136 to biodegradation. The closed system was set up to monitor biodegradation as a function of oxygen 137 consumption used for the degradation of microplastics by microorganisms as it is common way how 138 to test biodegradability of polymeric substances (ISO, 2019). A similar approach using open and closed 139 system was successfully applied in our previous study on tire wear particles (Klun et al., 2023). 140 141 2.2.1 Open system 142 Artificial freshwater with a low concentration of nutrients and microorganisms (50 mg/L activated 143 sludge) (ISO, 2019) was prepared and 200 mL were added to glass bottles, then 200 mg of microplastics 144 (PE, UV-PE, PBAT or UV-PBAT) were added separately to each bottle to reach the final concentration of 145 1000 mg/L. A blank sample containing only artificial freshwater without the addition of microplastics 146 was also prepared. Two independent replicates of each treatment were performed. The bottles were 147 then placed on a magnetic stirrer, covered with aluminium foil to protect them from light and 148 incubated at 20 ± 2 °C for 28 days. After the test, microplastics (labelled as PE-BIO, PBAT-BIO, UV-PE149 BIO, UV-PBAT-BIO) were recovered by filtration (cellulose filters, pore size 7-12 μm, Macherey-Nagel, 150 Germany) and analysed using the methods described in Section 2.3. 151 152 2.2.2 Closed system 153 The biodegradability test was also performed in a closed system to assess the degradation of the tested 154 microplastics based on oxygen consumption (ISO, 2019). The test was performed at same time and 155 under comparable conditions as the test in open system, with some minor modifications to meet the 156 requirements of the standard procedure (ISO, 2019). Briefly, the same artificial freshwater was used, 157 and 365 mL was filled into the dark glass bottles. The concentration of microplastics was 100 mg/L, as 158 required by the ISO standard, and microplastic particles (PE, UV-PE, PBAT or UV-PBAT) were added 159 separately to each bottle. In addition, a blank treatment was included, and another treatment was 160 prepared to follow the biodegradation of a reference compound (100 mg/L microcrystalline cellulose, 161 Sigma-Aldrich, USA). Three independent replicates of each treatment were performed. Each bottle was 162 then equipped by rubber cap with KOH and sealed with the OxiTop® head (WTW, Germany). The 163 bottles were then placed on a magnetic stirrer in a climate chamber (20 ± 2 °C, dark) and oxygen 164 consumption was measured for 28 days. The degree of biodegradation was calculated according to the 165 standard procedure (ISO, 2019). 166 167 2.3 Advanced analytical techniques 168 Changes in the surface morphology were evaluated using field-emission scanning electron microscopy 169 (FE-SEM) (Section 2.3.1) and surface chemical changes by Attenuated Total Reflectance Fourier170 transform infrared spectroscopy (ATR-FTIR) and Raman spectroscopy (Section 2.3.2). The changes in 171 the elemental composition were studied by laser-ablation inductively coupled plasma mass 172 spectrometry (LA-ICP-MS) and prior to LA-ICP-MS, elemental content in each sample was also analysed 173 by conventional digestion followed by liquid ICP-MS (Section 2.3.3). Differential scanning calorimeter 174 (DSC) was used for evaluation of changes in internal structure (i.e., crystallinity) of microplastics 175 (Section 2.3.4). 176 177 2.3.1 Surface morphology 178 The surface morphology and shape of microplastics were evaluated by FE-SEM Zeiss ULTRA plus (Zeiss, 179 Germany). Prior to the FE-SEM analysis, the samples were coated with a thin Au/Pd layer. 180 181 182 2.3.2 Surface chemical changes 183 To identify surface chemical changes, a FTIR spectrometer (Spectrum Two FT-IR spectrometer, 184 PerkinElmer, USA) with a Universal ATR module was used. The wavenumber ranged from 4000 cm-1 to 185 450 cm-1, with a resolution of 2 cm-1 (10 co-scans). Five repetitions for each sample were recorded and 186 averaged. Background and ATR correction of the spectra were applied. 187 The microplastics were also investigated using Raman spectroscopy. The samples were first fixed onto 188 adhesive tape. For the analysis, a confocal Witec Alpha 300R system (WITec, Germany) was used. The 189 laser employed had a wavelength of 532 nm and operated at a power of 0.5 mW. The integration time 190 for each measurement was set to 1 second. We performed 20 accumulations and collected 10 spectra 191 per sample, then calculated the mean values for spectral analysis. The objective lens used had a 50x 192 magnification. Additionally, we utilized an 1800 g/mm grating. 193 194 2.3.3 Changes in the elemental composition 195 For LA-ICP-MS analysis, individual samples were mounted in acrylic resin (ClaroCit Kit, Struers, Austria) 196 to investigate the lateral distribution of elements. Cross-sections of these embedded samples were 197 prepared by manual sanding using abrasive paper down to a grain size of 5 μm, and then LA-ICP-MS 198 imaging experiments were carried out similarly as described in previous work (Brunnbauer et al., 2024; 199 Pořízka et al., 2023). LA-ICP-MS analysis was carried out using an imageGEO193 laser ablation system 200 (ESL, USA) operating at a wavelength of 193 nm and equipped with a TwoVol3 ablation chamber. Prior 201 to the analysis, a preablation step was applied to remove potential surface contamination from the 202 sample preparation process. 203 The LA system was coupled to an NexION5000 system (PerkinElmer, USA) using the analytical cup with 204 Tygon® tubing with an inner diameter of 1.6 mm. Samples were ablated under a constant stream of 205 helium (0.8 L/min). Argon was used as a make-up gas (0.86 L/min) and mixed with the sample aerosol 206 using a dual concentric injector (DCI) (ESL, USA) right before the ICP. Kinetic energy discrimination 207 (KED) mode (He) was used to avoid the influence of polyatomic interferences on the measurement. 208 ICP-MS data was recorded using Syngistix 3.5 and LA-ICP-MS data evaluation was carried out using 209 Iolite 4.5.7.1. Data were normalised to 13C to compensate for instrumental drifts. Additional 210 measurement parameters are provided in Table S1. 211 Prior to LA-ICP-MS, elemental content in each sample was also analysed by conventional digestion 212 followed by liquid ICP-MS. Briefly, samples were accurately weighed into PTFE vessels (with an 213 approximate mass of 75 mg). Then 4 mL of concentrated HNO3 (67 % (w/w), suprapur, Carlo Erba, Italy) 214 and 1 mL of concentrated H2O2 (30 % (w/w), pro analysi, Fluka, Honeywell, USA) were added. Acid 215 digestion was performed in a microwave system (Ethos UP, Milestone, Italy) with a three-step 216 temperature program: samples were heated to 210 °C (approx. 7.5 °C/min), the set temperature was 217 maintained for 20 min, and then the samples were allowed to cool down to room temperature. 218 Samples were digested in duplicate. The digested solutions were quantitatively transferred to 20 mL 219 volumetric flasks and diluted to the mark with ultrapure water (resistivity >18.2 MΩ/cm, Synergy 220 Water Purification System, Merck Millipore, Germany). Sample solutions were stored in 50 mL 221 polypropylene centrifuge tubes (Sarstedt, Germany). The blank sample was prepared by the same 222 procedure. For the liquid ICP-MS analysis, samples were diluted by a factor of 10 using 1% HNO3. 223 Additionally, 1 µg/L Indium was added as an internal standard. Liquid ICP-MS analysis was carried out 224 using an iCAP Q TQ ICP-MS (ThermoFisher Scientific, Germany) in KED mode (additional measurement 225 parameters are provided in Table S2). The instrument was tuned daily for the maximum 115In signal 226 while keeping 140Ce16O/140Ce below 1.9 % using Tune A solution (ThermoFisher Scientific, Germany). 227 Metal content for (27Al, 56Fe, 39K, 24Mg, 23Na, 60Ni, 208Pb, 47Ti, 68Zn) was determined using a calibration 228 based on a multielement standard (Multi VIII, Certipur®, Merck, Darmstadt, Germany) and a single 229 element standard for Titanium (Certipur®, Merck, Darmstadt, Germany). 230 231 232 233 234 biodegradability test. Interestingly, PBAT contained Pb, but it was stable in the polymer matrix and was 344 not leached out during UV irradiation or after the biodegradation test (Figure 4). 345 346 347 Figure 4. LA-ICP-MS analysis of Ti and Pb in pristine PBAT, UV pre-treated (UV-PBAT) and after 348 biodegradation (PBAT-BIO, UV-PBAT-BIO). 349 350 3.5. Changes in internal structure 351 The changes in the internal structure were evaluated by comparing the crystallinity of the investigated 352 microplastics (Table 1). The crystallinity of PE, UV-PE, PE-BIO and UV-PE-BIO was comparable – none 353 of the treatments changed the internal structure of PE. The crystallinity of pristine PBAT did not change 354 after UV irradiation (UV-PBAT) (p = 0.5037). However, crystallinity was significantly increased after 355 biodegradation, as there were statistically significant differences between PBAT and PBAT-BIO (p ˂ 356 0.0001) and between UV-PBAT and UV-PBAT-BIO (p ˂ 0.0001). 357 Table 1. Crystallinity of investigated pristine microplastics (PE, PBAT), after pre-exposure to UV (UV-PE, 358 UV-PBAT) and after biodegradation (PE-BIO, PBAT-BIO, UV-PE-BIO, UV-PBAT-BIO) (n = 4, mean ± SD). 359 Sample Crystallinity (%) Sample Crystallinity (%) PE 27.4 ± 0.8 PBAT 16.8 ± 1.7 UV-PE 28.5 ± 0.1 UV-PBAT 10.4 ± 0.5 PE-BIO 29.1 ± 1.1 PBAT-BIO 59.4 ± 2.3 UV-PE-BIO 29.4 ± 0.7 UV-PBAT-BIO 37.0 ± 14.6 360 3.6. Changes in ecotoxicity 361 The results of the ecotoxicity tests showed that there were no statistically significant differences in the 362 specific growth rate of duckweed Lemna minor except for pristine PE treatment where specific growth 363 rate was significantly decreased in comparison to control (Figure 5). Similarly, no statistically significant 364 differences were found when comparing the effects of microplastics on chlorophyll a. However, PE and 365 UV-PE significantly decreased root growth, but this was no longer observed after biodegradation (PE366 BIO, UV-PE-BIO). 367 368 369 Figure 5. The effects of pristine microplastics (PE, PBAT), after pre-exposure to UV (UV-PE, UV-PBAT) 370 and after biodegradation (PE-BIO, PBAT-BIO, UV-PE-BIO, UV-PBAT-BIO) on (A) specific growth rate, (B) 371 root growth, and (C) chlorophyll a content. * Significant difference compared to control (p < 0.05). Line 372 – median, square – mean, box – 25‒75% of data, whiskers – range within 1.5 inter quartile range, 373 deltoid - outlier. 374 375 4. Discussion 376 Due to the global plastic crisis, numerous actions have been implemented to mitigate plastic pollution 377 – the most important being the reduction of plastic waste through the widely adopted concepts of 378 reduce, reuse, and recycle (Jia et al., 2019). However, these practices cannot be universally applied, as 379 certain applications require the use of plastics. For example, agriculture relies on the use of plastic 380 mulching films and a shift to the use of biodegradable alternatives such as PBAT has been observed 381 over the previous two decades. Many studies showed that PBAT degrade well in the target 382 environment, i.e., in soil or compost (Kijchavengkul et al., 2010; Liu et al., 2022), but some other 383 studies showed that the degradation in soils is low (Han et al., 2021; Lee et al., 2024) or even reduced 384 when PBAT is blended with other biodegradable polymers e.g. polylactic acid (PLA) (Palsikowski et al., 385 2018). 386 The results of this study showed that the degradation in a not-target aquatic environment did not 387 occur in pristine PE and UV-aged PE. Similarly, biodegradation of PBAT was low (Figure 1), which is in 388 agreement with the results of a study in which no degradation of pristine PBAT in lake sediments was 389 observed over nine months (van Grinsven and Schubert, 2023) and similarly low degradation of PBAT 390 was evaluated in the marine environment over six months (Lee et al., 2024). In general, lower 391 degradation can be expected in the aquatic environment compared to the soil environment, which is 392 linked to lower microbial activity and nutrient availability (Kaing et al., 2024). For example, our previous 393 study showed fast degradation of chitosan plastic films (both with and without added antioxidants) in 394 different soils (Oberlintner et al., 2021), while the same plastic films were only minimally degraded in 395 the aquatic biodegradability test (Ročnik et al., 2020). 396 On the other hand, pre-exposure of PBAT to UV significantly enhanced its further degradation. FTIR 397 analysis revealed a new absorption band appearing in the carbonyl region of PBAT spectra after UV398 aging (see inserted graph in Figure 3B). Additional carbonyl groups are commonly reported in plastic 399 and microplastics after UV-aging and indicate photodegradation (Alavian Petroody et al., 2023; Zidar 400 et al., 2024). In addition, degradation was also visible by SEM as holes appeared on the surface of UV401 PBAT after biodegradation (UV-PBAT-BIO, Figure 2). Therefore, it seems that UV-aging improved the 402 ability of microorganisms to degrade PBAT, as the biodegradation of UV-PBAT increased 3.8-times 403 compared to PBAT (Figure 1). Similarly, increased biodegradation in soil was observed for PBAT films 404 after UV-aging (Convertino et al., 2024). However, changes observed for PBAT following biodegradation 405 may also be linked to the more rapid degradation of thermoplastic starch from the starch-PBAT blend, 406 resulting in a relative enrichment of PBAT (Convertino et al., 2024; Pokhrel et al., 2021; Wang et al., 407 2015). This is relevant as many mulching film applications are expected to be exposed to UV radiation 408 during use on the surface of field soils. But, in regions with low UV intensity, the degradation can be 409 limited as the mulching films contain carbon black, which is used as a photostabiliser (Anunciado et 410 al., 2021). They also contain other stabilisers and antioxidants to slow the degradation of the material 411 (Hurley et al., 2024). Therefore, further work is required to track the progress of degradation of PBAT 412 and its constituent parts, and the corresponding impacts during this biodegradation process. 413 SEM analysis also revealed the presence of some microbial cells on the surface (Figure 2), however, in 414 a smaller amount compared to previous research, where PE fragments were aged in freshwater for 12 415 weeks (Rozman et al., 2023a). This is probably due to the very smooth surface of both PE and PBAT 416 microplastics (Figure 2) as it limits attachment of microorganisms (Rozman et al., 2023b). Agricultural 417 mulch films contains many additives (Hurley et al., 2024) that may also affect biofilm development as 418 they can act toxic limiting the growth of microorganism on the particles surface (Klun et al., 2023). If 419 the biofilm cannot be developed on the plastic surface, the further degradation is likely to be limited 420 (Debroy et al., 2022; Han et al., 2020). 421 Furthermore, LA-ICP-MS revealed alterations in some metals on the PE and PBAT surface. First, it seems 422 that metals within PE are stable, and the only alteration was in the case of Fe which accumulated on 423 the surface of the MPs. On the other hand, the lower content of Ti on PBAT after the biodegradation 424 suggested leaching of Ti into the surrounding environment. Ti is often used as polycondensation 425 catalysts in the production of PBAT so its presence can be expected (Jian et al., 2020). Interestingly, 426 PBAT contained also Pb but the reason or the origin of the Pb is unknown. It has to be noted that the 427 use of highly sensitive LA-ICP-MS helped to reveal the presence of Pb despite the low content 428 measured by to conventional method using acid digestion and liquid ICP-MS (Table S3). 429 The crystallinity of PE and PBAT was not affected by UV irradiation. However, during the biodegradation 430 experiment, whilst the crystallinity of PE remained the same, in PBAT it considerably increased. This 431 may indicate that microorganisms utilise the carbon in amorphous regions which is better accessible 432 (Mohanan et al., 2020) but, in this case, it is also plausible that PBAT was hydrolysed in the aquatic 433 medium during the biodegradation experiment as its hydrolysis was previously reported (Deshoulles 434 et al., 2022). Monitoring changes in crystallinity is particularly important for low-density plastics as an 435 increase in crystallinity also leads to an increase in density, which in turn can affect the distribution of 436 plastics in the aquatic environment (Budhiraja, 2024). 437 Overall, the morphological, surface and internal changes due to UV irradiation and exposure to 438 microorganisms were minimal. Consequently, no significant changes were also observed in the 439 ecotoxicity of PE and PBAT after biodegradation. In some cases, after biodegradation, the effects of PE 440 were even lowered which is usually related to the smoothing of the MPs surface due to the presence 441 of some microbial cells (Jemec Kokalj et al., 2019). However, further ecotoxicity studies using other 442 aquatic species, like algae, crustaceans, and fish models, would be needed to perform complete hazard 443 assessment of biodegradable microplastics after environmental aging. 444 445 446 447 448 449 450 5. Conclusions 451 Plastics are used in our daily lives and in some cases their use cannot be avoided. Therefore, we need 452 to find a sustainable way to deal with plastics or find alternatives that help to reduce plastic pollution 453 and hazard in the environment. In this context, biodegradable plastics seem to be a good alternative, 454 but their biodegradation must occur efficiently, fast, and with minimal impact on the environment. 455 This should not only refer to the environment in which they are intentionally used, but also to relevant 456 non-target environments, as plastics can also enter ecosystems where their presence was not 457 expected. It is also important to reconsider the use of certain additives that enhance the stability of 458 biodegradable plastics and thus increase their potential persistence in the environment. As shown in 459 our study, certified biodegradable materials intended for use in the soil environment were not 460 degraded in the aquatic environment if they were not first pre-exposed to UV. In the future, it would 461 also be useful to combine conventional biodegradation tests with advanced analytical techniques 462 when assessing the biodegradability of plastics, as these can provide important insights into the 463 degradation and surface changes, the presence of a biofilm, the leaching of elements, changes in 464 internal structure, and ecotoxicity. Together, they provide a comprehensive picture of the initial 465 changes that plastics can undergo in the natural environment, their possible effects and their fate. 466 467 Acknowledgements 468 The authors are thankful to Dr. Marija Zupančič for assistance with FTIR measurements and to Amadeja 469 Sajovic Žulovec for her help with preparation of the biodegradability and toxicity test. This work was 470 co-funded by the Slovenian Research and Innovation Agency (ARIS), the Czech Science Foundation 471 (GAČR), and the Austrian Science Fund (FWF) under the PLASTsensing project (J1–4415, 23–13617L, I472 6262-N, https://planterastics.fkkt.uni-lj.si), by Research programs P2–0191 and P2-0273 (ARIS), 473 projects PLAStouch N2–0298 (ARIS), µBioPlast J1-50014 (ARIS), and PLAST-N-cycling (Z1-60166). The 474 authors acknowledge the support of the Centre for Research Infrastructure at the University of 475 Ljubljana, Faculty of Chemistry and Chemical Technology, which is part of the Network of Research and 476 Infrastructural Centres UL (MRIC UL) and is financially supported by the Slovenian Research and 477 Innovation Agency (Infrastructure programme No. I0-0022) and the support of the Faculty of 478 Mechanical Engineering at the Brno University of Technology (no. FSI-S-23-8389). This article was also 479 supported by the project "Mechanical Engineering of Biological and Bio-inspired Systems", funded as 480 project No. CZ.02.01.01/00/22_008/0004634 by Programme Johannes Amos Commenius, call 481 Excellent Research and by the Industry Cooperation funded by Infineon Technologies Austria AG in the 482 course of IPCEI Microelectronics. This article is based upon work from COST Action CA20101 Plastics 483 monitoRIng detectiOn RemedIaTion recoverY - PRIORITY, supported by COST (European Cooperation 484 in Science and Technology, www.cost.eu) and EU Horizon project PAPILLONS (g.a. 101000210). 485 486 Data availability 487 The data that support the findings of this study are openly available on Zenodo under the following 488 link: https://doi.org/10.5281/zenodo.13884524. 489 490 References 491 Afshar, S.V., Boldrin, A., Astrup, T.F., Daugaard, A.E., Hartmann, N.B., 2024. Degradation of 492 biodegradable plastics in waste management systems and the open environment: A critical review. 493 Journal of Cleaner Production 434, 140000. 494 Alavian Petroody, S.S., Hashemi, S.H., Škrlep, L., Mušič, B., van Gestel, C.A.M., Sever Škapin, A., 2023. 495 UV Light Causes Structural Changes in Microplastics Exposed in Bio-Solids, Polymers. 496 Anunciado, M.B., Hayes, D.G., Wadsworth, L.C., English, M.E., Schaeffer, S.M., Sintim, H.Y., Flury, M., 497 2021. Impact of Agricultural Weathering on Physicochemical Properties of Biodegradable Plastic 498 Mulch Films: Comparison of Two Diverse Climates Over Four Successive Years. Journal of Polymers 499 and the Environment 29, 1-16. 500 Boots, B., Green, D.S., Olah-Kovacs, B., De Falco, F., Lupo, E., 2023. Physical and chemical effects of 501 conventional microplastic glitter versus alternative glitter particles on a freshwater plant (Lemnaceae: 502 Lemna minor). Ecotoxicology and Environmental Safety 263, 115291. 503 Briassoulis, D., Dejean, C., 2010. Critical Review of Norms and Standards for Biodegradable 504 Agricultural Plastics Part Ι. Biodegradation in Soil. Journal of Polymers and the Environment 18, 384505 400. 506 Brunnbauer, L., Jirku, M., Quarles, C.D., Limbeck, A., 2024. Capabilities of simultaneous 193 nm - 507 LIBS/LA-ICP-MS imaging for microplastics characterization. Talanta 269, 125500. 508 Budhiraja, V., 2024. Degradation of microplastics in the environment. Doctoral dissertation, 509 University of Nova Gorica, Slovenia. 510 Cai, Y., Lv, J., Feng, J., 2013. Spectral Characterization of Four Kinds of Biodegradable Plastics: Poly 511 (Lactic Acid), Poly (Butylenes Adipate-Co-Terephthalate), Poly (Hydroxybutyrate-Co-Hydroxyvalerate) 512 and Poly (Butylenes Succinate) with FTIR and Raman Spectroscopy. Journal of Polymers and the 513 Environment 21, 108-114. 514 Convertino, F., Carroccio, S.C., Cocca, M.C., Dattilo, S., Dell'Acqua, A.C., Gargiulo, L., Nizzetto, L., 515 Riccobene, P.M., Schettini, E., Vox, G., Zannini, D., Cerruti, P., 2024. The fate of post-use 516 biodegradable PBAT-based mulch films buried in agricultural soil. Science of The Total Environment 517 948, 174697. 518 Crossman, J., Hurley, R.R., Futter, M., Nizzetto, L., 2020. Transfer and transport of microplastics from 519 biosolids to agricultural soils and the wider environment. Science of The Total Environment 724, 520 138334. 521 de Souza, A.G., Komatsu, L.G.H., Barbosa, R.F.S., Parra, D.F., Rosa, D.S., 2022. The effect of ZnO 522 nanoparticles as Ag-carrier in PBAT for antimicrobial films. Polymer Bulletin 79, 4031-4048. 523 Debroy, A., George, N., Mukherjee, G., 2022. Role of biofilms in the degradation of microplastics in 524 aquatic environments. Journal of Chemical Technology & Biotechnology 97, 3271-3282. 525 Deshoulles, Q., Gall, M.L., Benali, S., Raquez, J.M., Dreanno, C., Arhant, M., Priour, D., Cerantola, S., 526 Stoclet, G., Gac, P.Y.L., 2022. Hydrolytic degradation of biodegradable poly(butylene adipate-co527 terephthalate) (PBAT) - Towards an understanding of microplastics fragmentation. Polymer 528 Degradation and Stability 205, 110122. 529 Dilshad, E., Waheed, H., Ali, U., Amin, A., Ahmed, I., 2021. General Structure and Classification of 530 Bioplastics and Biodegradable Plastics, in: Kuddus, M., Roohi (Eds.), Bioplastics for Sustainable 531 Development. Springer Singapore, Singapore, pp. 61-82. 532 Ghaffar, I., Rashid, M., Akmal, M., Hussain, A., 2022. Plastics in the environment as potential threat to 533 life: an overview. Environmental Science and Pollution Research 29, 56928-56947. 534 Griffin-LaHue, D., Ghimire, S., Yu, Y., Scheenstra, E.J., Miles, C.A., Flury, M., 2022. In-field degradation 535 of soil-biodegradable plastic mulch films in a Mediterranean climate. Science of The Total 536 Environment 806, 150238. 537 Han, N., Zhao, Q., Ao, H., Hu, H., Wu, C., 2022. Horizontal transport of macroand microplastics on 538 soil surface by rainfall induced surface runoff as affected by vegetations. Science of The Total 539 Environment 831, 154989. 540 Han, Y., Teng, Y., Wang, X., Ren, W., Wang, X., Luo, Y., Zhang, H., Christie, P., 2021. Soil Type Driven 541 Change in Microbial Community Affects Poly(butylene adipate-co-terephthalate) Degradation 542 Potential. Environmental Science & Technology 55, 4648-4657. 543 Han, Y.-N., Wei, M., Han, F., Fang, C., Wang, D., Zhong, Y.-J., Guo, C.-L., Shi, X.-Y., Xie, Z.-K., Li, F.-M., 544 2020. Greater Biofilm Formation and Increased Biodegradation of Polyethylene Film by a Microbial 545 Consortium of Arthrobacter sp. and Streptomyces sp, Microorganisms. 546 Hoshino, A., Sawada, H., Yokota, M., Tsuji, M., Fukuda, K., Kimura, M., 2001. Influence of weather 547 conditions and soil properties on degradation of biodegradable plastics in soil. Soil Science and Plant 548 Nutrition 47, 35-43. 549 Hurley, R., Binda, G., Briassoulis, D., Carroccio, S.C., Cerruti, P., Convertino, F., Dvořáková, D., 550 Kernchen, S., Laforsch, C., Löder, M.G.L., Pulkrabova, J., Schettini, E., Spanu, D., Tsagkaris, A.S., Vox, 551 G., Nizzetto, L., 2024. Production and characterisation of environmentally relevant microplastic test 552 materials derived from agricultural plastics. Science of The Total Environment 946, 174325. 553 ISO, 2019. ISO 14851:2019 Determination of ultimate aerobic biodegradability of plastic materials in 554 an aquaeous medium - Method by measurement the oxygen demand in a closed respirometer. ISO, 555 Geneva, Switzerland. 556 Jemec Kokalj, A., Dolar, A., Nagode, A., Drobne, D., Kuljanin, A., Kalčíková, G., 2024. Response of 557 terrestrial crustacean Porcellio scaber and mealworm Tenebrio molitor to non-degradable and 558 biodegradable fossil-based mulching film microplastics. Science of The Total Environment 951, 559 175379. 560 Jemec Kokalj, A., Kuehnel, D., Puntar, B., Žgajnar Gotvajn, A., Kalčikova, G., 2019. An exploratory 561 ecotoxicity study of primary microplastics versus aged in natural waters and wastewaters. 562 Environmental Pollution 254, 112980. 563 Jia, L., Evans, S., Linden, S.v.d., 2019. Motivating actions to mitigate plastic pollution. Nature 564 Communications 10, 4582. 565 Jian, J., Xiangbin, Z., Xianbo, H., 2020. An overview on synthesis, properties and applications of 566 poly(butylene-adipate-co-terephthalate)–PBAT. Advanced Industrial and Engineering Polymer 567 Research 3, 19-26. 568 Kaing, V., Guo, Z., Sok, T., Kodikara, D., Breider, F., Yoshimura, C., 2024. Photodegradation of 569 biodegradable plastics in aquatic environments: Current understanding and challenges. Science of 570 The Total Environment 911, 168539. 571 Kalčíková, G., Žgajnar Gotvajn, A., Kladnik, A., Jemec, A., 2017. Impact of polyethylene microbeads on 572 the floating freshwater plant duckweed Lemna minor. Environmental Pollution 230, 1108-1115. 573 Kijchavengkul, T., Auras, R., Rubino, M., Selke, S., Ngouajio, M., Fernandez, R.T., 2010. Biodegradation 574 and hydrolysis rate of aliphatic aromatic polyester. Polymer Degradation and Stability 95, 2641-2647. 575 Klun, B., Rozman, U., Kalčíková, G., 2023. Environmental aging and biodegradation of tire wear 576 microplastics in the aquatic environment. Journal of Environmental Chemical Engineering 11, 577 110604. 578 Krimm, S., Liang, C.Y., Sutherland, G.B.B.M., 1956. Infrared Spectra of High Polymers. II. Polyethylene. 579 The Journal of Chemical Physics 25, 549-562. 580 Lambert, S., Wagner, M., 2017. Environmental performance of bio-based and biodegradable plastics: 581 the road ahead. Chemical Society Reviews 46, 6855-6871. 582 Lee, J., Kim, S., Park, S.B., Shin, M., Kim, S., Kim, M.-S., Shin, G., Kang, T., Kim, H.J., Oh, D.X., Park, J., 583 2024. Mimicking real-field degradation of biodegradable plastics in soil and marine environments: 584 From product utility to end-of-life analysis. Polymer Testing 131, 108338. 585 Li, B., Liu, Q., Yao, Z., Ma, Z., Li, C., 2023. Mulch film: An overlooked diffuse source of organic 586 ultraviolet absorbers in agricultural soil. Environmental Pollution 318, 120935. 587 Liao, J., Chen, Q., 2021. Biodegradable plastics in the air and soil environment: Low degradation rate 588 and high microplastics formation. Journal of Hazardous Materials 418, 126329. 589 Liu, Q., Wang, Y., Liu, J., Liu, X., Dong, Y., Huang, X., Zhen, Z., Lv, J., He, W., 2022. Degradability and 590 Properties of PBAT-Based Biodegradable Mulch Films in Field and Their Effects on Cotton Planting, 591 Polymers. 592 Mateos-Cárdenas, A., Scott, D.T., Seitmaganbetova, G., Frank N.A.M, v.P., John, O.H., Marcel A.K, J., 593 2019. Polyethylene microplastics adhere to Lemna minor (L.), yet have no effects on plant growth or 594 feeding by Gammarus duebeni (Lillj.). Science of The Total Environment 689, 413-421. 595 Mazzon, M., Gioacchini, P., Montecchio, D., Rapisarda, S., Ciavatta, C., Marzadori, C., 2022. 596 Biodegradable plastics: Effects on functionality and fertility of two different soils. Applied Soil Ecology 597 169, 104216. 598 Mohanan, N., Montazer, Z., Sharma, P.K., Levin, D.B., 2020. Microbial and Enzymatic Degradation of 599 Synthetic Plastics. Frontiers in Microbiology 11. 600 Moshood, T.D., Nawanir, G., Mahmud, F., Mohamad, F., Ahmad, M.H., AbdulGhani, A., 2022. 601 Sustainability of biodegradable plastics: New problem or solution to solve the global plastic 602 pollution? Current Research in Green and Sustainable Chemistry 5, 100273. 603 Napper, I.E., Thompson, R.C., 2023. Plastics and the Environment. Annual Review of Environment and 604 Resources 48, 55-79. 605 Niu, B., Chen, J.-B., Chen, J., Ji, X., Zhong, G.-J., Li, Z.-M., 2016. Crystallization of linear low density 606 polyethylene on an in situ oriented isotactic polypropylene substrate manipulated by an extensional 607 flow field. CrystEngComm 18, 77-91. 608 Oberlintner, A., Bajić, M., Kalčíková, G., Likozar, B., Novak, U., 2021. Biodegradability study of active 609 chitosan biopolymer films enriched with Quercus polyphenol extract in different soil types. 610 Environmental Technology & Innovation 21, 101318. 611 OECD, 1992. Test No. 301: Ready biodegradability, OECD Guidelines for the testing of chemicals, 612 section 3, OECD Publishing, Paris. 613 OECD, 2006. Test No. 221: Lemna sp. Growth inhibition test. OECD Guidelines for the testing of 614 chemicals, Section 2, OECD publishing, Paris. 615 Palsikowski, P.A., Kuchnier, C.N., Pinheiro, I.F., Morales, A.R., 2018. Biodegradation in Soil of 616 PLA/PBAT Blends Compatibilized with Chain Extender. Journal of Polymers and the Environment 26, 617 330-341. 618 PhysicsOpenLab, 2022. Polymer anaylsis using Raman spectroscopy, 619 https://physicsopenlab.org/2022/05/08/polymer-analysis-using-raman-spectroscopy/. 620 Pires, J.R., Souza, V.G., Fuciños, P., Pastrana, L., Fernando, A.L., 2022. Methodologies to Assess the 621 Biodegradability of Bio-Based Polymers—Current Knowledge and Existing Gaps, Polymers. 622 Pischedda, A., Tosin, M., Degli-Innocenti, F., 2019. Biodegradation of plastics in soil: The effect of 623 temperature. Polymer Degradation and Stability 170, 109017. 624 PlasticsEurope, 2024. Plastics - the fast Facts 2023. 625 Pokhrel, S., Sigdel, A., Lach, R., Slouf, M., Sirc, J., Katiyar, V., Bhattarai, D.R., Adhikari, R., 2021. Starch626 based biodegradable film with poly(butylene adipate-co-terephthalate): preparation, morphology, 627 thermal and biodegradation properties. Journal of Macromolecular Science, Part A 58, 610-621. 628 Pořízka, P., Brunnbauer, L., Porkert, M., Rozman, U., Marolt, G., Holub, D., Kizovský, M., Benešová, M., 629 Samek, O., Limbeck, A., Kaiser, J., Kalčíková, G., 2023. Laser-based techniques: Novel tools for the 630 identification and characterization of aged microplastics with developed biofilm. Chemosphere 313, 631 137373. 632 Ren, S.-Y., Kong, S.-F., Ni, H.-G., 2021. Contribution of mulch film to microplastics in agricultural soil 633 and surface water in China. Environmental Pollution 291, 118227. 634 Rosenboom, J.-G., Langer, R., Traverso, G., 2022. Bioplastics for a circular economy. Nature Reviews 635 Materials 7, 117-137. 636 Rozman, U., Filker, S., Kalčíková, G., 2023a. Monitoring of biofilm development and physico-chemical 637 changes of floating microplastics at the air-water interface. Environmental Pollution 322, 121157. 638 Rozman, U., Jemec Kokalj, A., Dolar, A., Drobne, D., Kalčíková, G., 2022. Long-term interactions 639 between microplastics and floating macrophyte Lemna minor: The potential for phytoremediation of 640 microplastics in the aquatic environment. Science of The Total Environment 831, 154866. 641 Rozman, U., Klun, B., Kuljanin, A., Skalar, T., Kalčíková, G., 2023b. Insights into the shape-dependent 642 effects of polyethylene microplastics on interactions with organisms, environmental aging, and 643 adsorption properties. Scientific Reports 13, 22147. 644 Rozman, U., Turk, T., Skalar, T., Zupančič, M., Čelan Korošin, N., Marinšek, M., Olivero-Verbel, J., 645 Kalčíková, G., 2021. An extensive characterization of various environmentally relevant microplastics – 646 Material properties, leaching and ecotoxicity testing. Science of The Total Environment 773, 145576. 647 Ročnik, T., Oberlintner, A., Novak, U., Likozar, B., Kalčikova, G., 2020. The assessment of 648 biodegradability of chitosan-based films. Book of Abstract, 26th Annual meeting of Slovenian 649 Chemical Society. 16-18. September 2020, Portorož, Slovenia. 650 Simul Bhuyan, M., S, V., S, S., Szabo, S., Maruf Hossain, M., Rashed-Un-Nabi, M., C.R, P., M.P, J., 651 Shafiqul Islam, M., 2021. Plastics in marine ecosystem: A review of their sources and pollution 652 conduits. Regional Studies in Marine Science 41, 101539. 653 van Grinsven, S., Schubert, C., 2023. Soil-biodegradable plastic films do not decompose in a lake 654 sediment over 9 months of incubation. Biogeosciences 20, 4213-4220. 655 Wang, H., Wei, D., Zheng, A., Xiao, H., 2015. Soil burial biodegradation of antimicrobial biodegradable 656 PBAT films. Polymer Degradation and Stability 116, 14-22. 657 Yang, Y., Li, Z., Yan, C., Chadwick, D., Jones, D.L., Liu, E., Liu, Q., Bai, R., He, W., 2022. Kinetics of 658 microplastic generation from different types of mulch films in agricultural soil. Science of The Total 659 Environment 814, 152572. 660 Zidar, P., Kühnel, D., Škapin, A.S., Skalar, T., Drobne, D., Škrlep, L., Mušič, B., Jemec Kokalj, A., 2024. 661 Comparing the effects of pristine and UV–VIS aged microplastics: Behavioural response of model 662 terrestrial and freshwater crustaceans. Ecotoxicology and Environmental Safety 285, 117020. 663 664