Journal of Hazardous Materials 439 (2022) 129593 Contents lists available at ScienceDirect Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat A new source of representative secondary PET nanoplastics. Obtention, characterization, and hazard evaluation Aliro Villacorta a, b, Laura Rubio a, c, Mohamed Alaraby a, d, Montserrat López-Mesas e, Victor Fuentes-Cebrian e, Oscar H. Moriones f, g, Ricard Marcos a, *, Alba Hernández a, * a Group of Mutagenesis, Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain b Facultad de Recursos Naturales Renovables, Universidad Arturo Prat, Iquique, Chile c Nanobiology Laboratory, Department of Natural and Exact Sciences, Pontificia Universidad Católica Madre y Maestra, PUCMM, Santiago de los Caballeros, Dominican Republic d Zoology Department, Faculty of Sciences, Sohag University, 82524 Sohag, Egypt e GTS-UAB Research Group, Department of Chemistry, Faculty of Science, Universitat Autònoma de Barcelona, 08193 Bellaterra, Cerdanyola del Vallès, Spain f Institut Català de Nanociència i Nanotecnologia (ICN2-UAB-CSIC-BIST), Campus UAB, Bellaterra, 08193 Barcelona, Spain g Universitat Autonòma de Barcelona (UAB), Campus UAB, Bellaterra, 08193 Barcelona, Spain H I G H L I G H T S G R A P H I C A L A B S T R A C T • A proposed method to get PET nano­ plastic samples from plastic bottles is presented. • Completed physicochemical character­ ization is provided. • Cell uptake was evaluated following different methods. • Different hazard effects were determined. • The method is extrapolated to other plastic and avoids metal contamination. A R T I C L E I N F O A B S T R A C T Editor: Dr. T Meiping Micro and nanoplastics (MNPLs) are emergent environmental pollutants requiring urgent information on their potential risks to human health. One of the problems associated with the evaluation of their undesirable effects is Keywords: the lack of representative samples, matching those resulting from the environmental degradation of plastic wastes. Nanoplastics To such end, we propose an easy method to obtain polyethylene terephthalate nanoplastics from water plastic Polyethylene terephthalate bottles (PET-NPLs) but, in principle, applicable to any other plastic goods sources. An extensive characterization Physicochemical characterization indicates that the proposed process produces uniform samples of PET-NPLs of around 100 nm, as determined by Cell uptake Cytotoxicity using AF4 and multi-angle and dynamic light scattering methodologies. An important point to be highlighted is that to avoid the metal contamination resulting from methods using metal blades/burrs for milling, trituration, * Correspondence to: Group of Mutagenesis, Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Campus of Bellaterra, 08193 Cerdanyola del Vallès, Barcelona, Spain. E-mail addresses: ricard.marcos@uab.cat (R. Marcos), alba.hernandez@uab.cat (A. Hernández). https://doi.org/10.1016/j.jhazmat.2022.129593 Received 10 March 2022; Received in revised form 4 July 2022; Accepted 11 July 2022 Available online 14 July 2022 0304-3894/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A. Villacorta et al. Journal of Hazardous Materials 439 (2022) 129593 or sanding, we propose to use diamond burrs to produce metal-free samples. To visualize the toxicological profile of the produced PET-NPLs we have evaluated their ability to be internalized by cells, their cytotoxicity, their ability to induce oxidative stress, and induce DNA damage. In this preliminary approach, we have detected their cellular uptake, but without the induction of significant biological effects. Thus, no relevant increases in toxicity, reactive oxygen species (ROS) induction, or DNA damage -as detected with the comet assay- have been observed. The use of representative samples, as produced in this study, will generate relevant data in the discussion about the potential health risks associated with MNPLs exposures. 1. Introduction ablation process to obtain PET-NPLs (Magrì et al., 2018). Others have used mechanical milling followed by sieve fractionation into MPLs Despite the multiple advantages of plastic materials, a very dramatic fractions applied to agricultural plastics (a mulch film prepared biode­ side-effect resulting from this big success is the increased presence of gradable polymer polybutyrate adipate-co-terephthalate and plastic wastes in the environment. Unfortunately, and considering the low-density polyethylene (Astner et al., 2019). As well as the trituration present capacities of recycling such wastes, this environmental catas­ of a few millimeters’ of industrial PET pellets (Pignattelli et al., 2021). trophe will still be increasing and remain for a long (Borrelle et al., The use of mechanical ground approaches was also proposed to obtain 2020). PET-NPLs (Rodríguez-Hernández et al., 2019). Once into the environment, plastic wastes degrade due to different Two important aspects need to be pointed out regarding the so-far abiotic (i.e., photodegradation, physical abrasion, or hydrolysis) and proposed alternative protocols to obtain MNPLs. First, polyethylene biotic mechanisms (Ali et al., 2021a), generating a new source of con­ terephthalate (PET) is used in most of these approaches. This is because, taminants: the micro/nanoplastics (MNPLs). With a size between less as a thermoplastic polymer, PET do not undergo chemical reactions than 10 nm and a few mm, MNPLs can be highly mobilized by water and when heated, and can re-ground several times, what it is very useful for air and, consequently, they can be found in all the environmental niches recycling purposes. This, among other reasons, is why PET is one of the on a global scale (Evangeliou et al., 2020; Batool et al., 2021). Another major contributors to worldwide plastic wastes (Gwada et al., 2019). characteristic linked to the MNPLs’ size is their potential to enter the Second, many of the proposed methods to obtain degraded MNPLs, such food web as they can be easily uptaken by living organisms, including as mechanical milling, allows only to obtain MPLs, but not NPLs. This is humans. In this way, they have become a new group of emergent con­ very relevant from the toxicological point of view, since it is considered taminants with a potential human health risk. In fact, it has been that NPLs have the ability to cross biological primary barriers and, demonstrated that at the nanoscale level MNPLs could exert important consequently, reach the different organs and tissues after an exposure biological effects such as reactive oxygen species (ROS) generation, the (Zitouni et al., 2021). activation of the immune system by inflammatory responses, and even According to the above indicated, it is clear that there is an urgent more, genotoxic DNA damage (Rubio et al., 2020; Ballesteros et al., need to generate new representative MNPLs samples for further use in 2020; Rai et al., 2021). hazard evaluations (Balakrishnan et al., 2019). In this study, we thor­ It should be noted that environmental MNPLs have two different oughly describe our in-house method to obtain PET-NPLs, provide origins. One is associated with the abiotic/biotic degradation of big exhaustive characterization of the obtained materials, and assess its plastic sizes (macroplastics), as above indicated. In this case, these usefulness in hazard assessment approaches by analyzing the cellular MNPLs are denominated secondary MNPLs. On the contrary, some uptake, cytotoxicity, reactive oxygen (ROS) production, and DNA MNPLs are synthesized at these small size ranges for specific purposes damage induction in human lymphoblastic cell line models after an and are denominated primary MNPLs. One typical example of primary acute exposure. MNPLs is the micro/nanobeads used in exfoliating hand cleansers and facial scrubs. In fact, they are also intentionally added to other many 2. Materials and methods products such as paints, coatings, and inks, or in construction, agricul­ ture/horticulture, and medical products, among others. For these 2.1. PET-NPLs obtention MNPLs, the estimated used amount in Europe is around 145,000 tons per year, and to minimize their impact on the environment and potentially The PET raw material used to produce PET-NPLs was obtained from on human health, a restriction on the intentionally-added-microplastic commercially available water plastic bottles. The developed procedure use has been proposed (ECHA, 2018). Regarding the environmental was adapted from the previously reported by Rodríguez-Hernández levels of secondary MNPLs they are very difficult to estimate. Never­ et al. (2019). A schematic overview of the used protocol is outlined in theless, it must be remembered that the world production of plastics on Fig. 1. Briefly, pieces of about 12 cm2 from the bottom part of the water 2019 was 368 million tons, that its production is exponentially growing bottles were initially sanded with aluminum oxide/silicon carbide ro­ over time, that about 40 % was used for packaging (or single-use), and tary burrs. Due to the observed metal-contamination (as explained in the about 80 % is not properly recycled ending into the environment or Results section) a diamond rotary burr sander accessory, attached to a landfills (PlasticsEurope 2019; Ali et al., 2021b). flexible shaft powered by a multitool Dremel 3000, was choose for the Studies aiming to determine the potential hazard of MNPLs enface sanding process. The obtained PET debris was passed through a 0.20 mm big challenges, one being the selection of environmentally representa­ sieve (CISA R-92), and 4 g of the resulting material were dispersed in 40 tive MNPLs to be used as reference materials. Due to the impossibility of mL of pre-heated (50 ºC) trifluoroacetic acid (TFA, 90 % v/v), in a collecting real MNPLs from the environment, especially at the nano proportion of 10 mL TFA per gram of sieved PET, on a stirring plate at range, practically all available studies have used the only MNPLs type 100 rpm for 2 h. After a complete dispersion, the mixture was kept at that is commercially available in a wide range of sizes, surface modifi­ room temperature overnight in continuous agitation. Next day, 40 mL of cations, and labeling, which is polystyrene (PS) MNPLs. Hence, all our TFA 20 % (v/v) was added to the sample and the mixture was kept under present knowledge on the potential risk of MNPLs for human health is constant and vigorous stirring for 24 h. After that, the obtained disper­ basically limited to one polymer type. To avoid this bias, some recent sion was distributed on six 10 mL glass tubes and centrifuged at 2500 alternatives have been proposed aiming at artificially degrade plastic RCF for 1 h. Once the supernatants were discarded, the obtained pellets items to obtain new sources of secondary MNPLs to be included in the were resuspended on 400 mL of 0.50 % sodium dodecyl sulfate (SDS) hazard assessment studies. The pioneering study used the UV-laser solution, vigorously mixed, and distributed into two 200 mL beakers, for 2 A. Villacorta et al. Journal of Hazardous Materials 439 (2022) 129593 ultrasonication on an SSE-1 Branson sonicator. Sonication lasted for 2 as briefly indicated. A drop of particle suspension at a concentration of min at 25% amplitude, in 9/9s sonication/break cycles. The volume of 1 mg/mL was placed on a gold mirror and let dry for one week inside a each beaker was transferred to a graduated cylinder, where sedimen­ petri dish. Analysis was carried out on a Hyperion 2000 micro- tation of larger particles took place for 1 h. The top 100 mL of the sus­ spectrometer using the gold mirror as reference. This analysis was car­ pension (from each graduated cylinder) was collected and centrifuged as ried out at the Molecular Spectroscopy and Optical Microscopy facility before for SDS elimination, and further washed twice with Mili-Q water at the Institut Català de Nanociència i Nanotecnologia (ICN2). To assess the and twice with pure ethanol, and let to dry. Pellets were then resus­ composition, interferograms were analyzed and contrasted with previ­ pended on Mili-Q at the desired final concentration, and sonicated at 10 ous reports. % amplitude for 16 min in a cold-water bath, aliquoted, and immedi­ ately frozen on cryotubes in liquid nitrogen and stored at − 80 ºC for 2.2.4. Multi angle and dynamic light scattering (DLS-MADLS) and zeta further use. Each one of the cryotubes contains 1 mL of a concentration potential of 12.50 mg/mL. The indicative size of the colloid structures in suspension of the hy­ pothetical hard sphere that behave similarly to the particle suspension of 2.2. Characterization of particle suspension the PET-NPLs was analyzed using a Zetasizer® Ultra from Malvern Panalytical. A working solution of 1 mg/mL of PET-NPLs suspension was 2.2.1. Scanning electron microscopy/Energy dispersive X-ray microscopy thawed and then diluted on Milli-Q water for further analysis at a con­ (SEM-EDS) centration of 200 μg/mL. To investigate better the fraction on the The stock solution was thawed on a warm bath at 37 ◦ C, and a nanoscale, avoiding the masking generated by, agglomerates or particles working solution of 200 μg/mL was prepared on Milli-Q water. A holey bigger in size, a sonication for 16 min at 10 % amplitude (as described carbon grid was dipped into the working solution and water was allowed above), and a filtration step with a 450 µm PES membrane Millex®-HP to evaporate overnight; additionally, a drop was placed on the Si holder. from Millipore express® was included. To visualize the influence of Then, particles in the grid were examined on a SEM Zeiss Merlin (Zeiss, anionic surfactant as a dispersant of the sample the same procedure Oberkochen, Germany) coupled with an X-Max 20 mm EDS system described was carried out maintaining the SDS 0.5 % v/v original con­ (Oxford Instruments, Oxford, UK). In addition to collecting SEM images, centration. Once prepared, the samples were placed on DTS1070 cuvette for the EDX analysis an area including the nanoparticle surface was for surface charge determination by zeta potential measurements, for selected, and the sample signal was collected and analyzed by the INCA both water and SDS 0.5 % dispersant. DLS, MADLS and particle con­ Energy software. centration measurement were carried out by placing 1.5 mL of the prepared solutions on DTS0012 cuvettes. 2.2.2. Transmission electron microscopy (TEM) A working solution of 200 μg/mL was prepared on Milli-Q water and 2.2.5. Asymmetric flow field flow fractionation (AF4) mounted on a holey carbon grid as described above. Particles in the grid The PET nanoparticles separation was performed using the AF2000 were examined by TEM on a JEOL JEM 1400 instrument (JEOL LTD, Asymmetric Flow FFF system from Postnova Analytics GmbH (Lands­ Tokyo, Japan) operated at 120 kV. TEM imaging was analyzed for berg, Germany) with two PN1130 Isocratic Pumps, a Kloehn V6 Pump, a particle size distribution using ImageJ software 1.8.0_172 processing PN5300 Autosampler, a PEEK AF4 Channel with a 10 kDa regenerated and analysis software. cellulose membrane and a 350 µm height spacer. The mobile phase and the PET suspension were prepared in NovaChem Surfactant 100 (IES­ 2.2.3. Fourier transform infrared spectroscopy (FTIR) MAT, Madrid, Spain) at 0.20 % (v/v). The size data (gyration radius, Rg) To detect the functional groups, and to identify the samples as PET, was obtained using a PN3609 MALS Detector (9 angles), calibrated with these were analyzed by Fourier-transform infrared spectroscopy (FTIR), a 125 nm NIST Polystyrene Latex standard (Postnova Analytics GmbH), Fig. 1. Schematic representation of the frag­ mentation and grind process with the mixed burrs or the diamond covered burr (a). Repre­ sentation of the fractioned pieces (b), PET powder obtained (c) that will be passed through the sieve (d), stirred, suspended, and heated at different concentrations of TFA (e), distributed on glass tubes (f) and centrifuged (g), washed and then resuspended on SDS, sonicated, and transferred to a cylinder for sedimentation (h) and then collected, washed (i), weighted (j), resuspended, sonicated and stored frozen until needed (k). 3 A. Villacorta et al. Journal of Hazardous Materials 439 (2022) 129593 using the software AF2000 Software and used to calculate the geometric were dyed using Cellmask (excitation of 633 nm and emission collected diameter (Dgeo) by dividing 2*Rg by the typical form factor of a sphere at 645–786). For PET-NPLs labeled with Nile red, an excitation wave­ (0.78) (Lohrke et al., 2008). The method consisted in three steps, used a length of 514 nm and emission collected at 546–628 was used. Images of constant detector flow of 0.50 mL/min and injections of 20 µL of sam­ each sample were obtained using a Leica TCS SP5 confocal microscope ples of 1 mg/mL of PET-NPLs suspended in NovaChem 0.20 % were and processed using ImageJ processing and analysis software. version performed. The first step was a focusing step of 3 min with an injection 1.8.0_172. flow of 0.20 mL/min, a cross-flow of 2.00 mL/min and a focus flow of 2.30 mL/min, the second was a separation step, with focus-flow of 2.5. Cell viability assay 0 mL/min, cross-flow from 2.00 mL/min exponential to 0.10 mL/min in 40 min and a final step at a constant flow of 0.10 mL/min of cross-flow Cell viability after exposure to PET-NPLs, was determined using the during 20 min Beckman counter method. To proceed, cells were seeded at a cell density of 5 × 105 cells/mL on 96 well plates and exposed to a range of con­ 2.3. Cell culture centrations from 0 up to 200 μg/mL of PET-NPLs (200 µg/mL per well). After the exposure time, cells were mixed and diluted 1:100 in ISOFLOW Two human lymphoblastic cell lines were used to detect the potential and counted with a ZTM series coulter-counter (Beckman Coulter Inc., biological effects of the obtained PET-NPLs, the THP-1 (monocytes) and CA, USA). The average number of cells counted in each treatment was the TK6 (lymphoblasts). Both cell lines were obtained from Sigma compared with the average number of the untreated control cells. The Aldrich (MO, USA) and grown in T-25 flasks containing Roswell Park results from the viability assay permit to determine the non-cytotoxic Memorial Institute (RPMI) medium (Biowest, France) supplemented concentrations to be used in further experiments. with 10 % fetal bovine serum (FBS), 1% glutamine (Biowest, France), and 2.5 μg/mL of Plasmocin™ (InvivoGen, CA, USA). Cultures, with a 2.6. Reactive oxygen species (ROS) quantification density ranging from 5 × 105 and 1 × 106 cells were maintained at 37 ºC in a humidified atmosphere of 5 % CO2. Due to the combined capacity of passively diffuse into the cell and being highly reactive, the reduced form of ethidium bromide (dihy­ 2.4. Cell uptake determination droethidium, DHE) was used to detect cytosolic superoxide. Cellular suspensions were seeded on 96 U-type well plates at a cell density of To confirm the cell uptake of the obtained PET-NPLs, three different 5 × 105 and treated with concentrations of 5, 10, 25, and 50 μg/mL of approaches have been used: flow cytometry (internal complexity), TEM PET-NPLs for 3 and 24 h. After the exposure, cells were centrifuged at (internalization), and confocal microscopy (Nile red staining). 1000 rpm for 5 min, resuspended on 10 μM DHE diluted on PBS 1X, to a final cell density of 1 × 106, and incubated for 30 min at 37 ºC. Cells 2.4.1. Internal complexity were kept on ice for immediate analysis by using a flow cytometer As an indicator of structural complexity of THP1 cells, once the PET- (Beckman Coulter CytoFLEX S). A total number of 20000 events (single NPLs uptake takes place the orthogonal light scattering -commonly cells) were scored and evaluated using the CytExpert software. known as Side Scatter (SSC)- was evaluated at concentrations ranging from 0 to 50 μg/mL after exposures lasting for 3 h. The highest con­ 2.7. DNA damage detection: The comet assay centration (50 μg/mL) was tested in different scenarios in which PET- NPLs were added directly after being rapidly thawed from − 80 ºC on The levels of DNA damage (DNA breaks) in both selected cell lines a 37 ºC water bath, then vortexed and resuspended on RPMI media with were evaluated using the published protocol (García-Rodríguez et al., or without FBS, vortexed again and applied directly to the culture and 2019). Both, the overall DNA damage, and the oxidized DNA base levels compared with regular culture conditions with and without FBS. Cells were determined with the absence/presence of formamidopyrimidine were analyzed by flow cytometry on a CytoFlex s and CytExpert software DNA glycosylase (FPG) enzyme. This enzyme detects oxidized DNA was used to properly collect means from 10,000 events on the live cell bases, excise them, and generate a transient single-strand DNA break population. Data were analyzed using GraphPad Prism 7.0. detected by the comet assay and denominated as specific oxidative DNA damage (ODD). THP1 cells were exposed to concentrations of 5, 10, 25, 2.4.2. TEM analysis and 50 μg/mL of PET-NPLs for 3 h. Treated cells were centrifuged at Exposed cells were fixed in 2 % (w/v) paraformaldehyde and 2.5 % 0.30 RCF for 8 min at 4 ºC. The obtained pellet was washed and resus­ (v/v) glutaraldehyde (Merck, Darmstadt, Germany) in 0.10 M cacody­ pended on cold PBS 1x to get 106 cell density per mL. Cells were mixed late buffer (Sigma-Aldrich, Steinheim, Germany) at pH 7.4. After that, 1:10 with 0.75 % low melting point agarose at 37 ºC and dropped on cells were processed as already published (Rubio et al., 2016). Briefly, triplicates onto GelBond® films (GBFs) (Life Sciences, Lithuania). GBFs samples were post-fixed with osmium, dehydrated in ethanol, embedded containing the samples were treated overnight on cold lysis buffer at in Epon, polymerized at 60 ºC, and cut with an ultramicrotome. Finally, 4 ºC and washed on successive 5 and 50 min of enzyme buffer immer­ ultrathin sections placed in copper grids, were contrasted with con­ sion, immediately followed by 30 min incubation at 37 ºC with FPG for ventional uranyl acetate and Reynolds’s lead citrate solutions and the detection of ODD, or without FPG for the detection of general gen­ observed using a Jeol 1400 (Jeol LTD) transmission electron microscope otoxic damage. GBFs were subjected to electrophoresis (20 V, 300 mA, equipped with a CCD GATAN ES1000 W Erlangshen camera. 4;ºC), washed twice with cold PBS 1X, fixed in absolute ethanol, and Nile Red stained particles and confocal visualization. Nile red staining of air-dried overnight at room temperature. After staining with SYBR Gold, PET-NPLs was carried out following the procedure described in Rodrí­ GBFs were visualized in an epifluorescent microscope (Olympus BX50, guez-Hernández et al. (2019) with some important modifications. Hamburg, Germany). The levels of DNA damage, as percentage of DNA Briefly, an aliquot (1 mg/mL) was centrifuged for 25 min at 16100 RCF. in the tail, was quantified with the Komet 5.5 Image analysis system The supernatant was removed, and 1 mL of Nile red solution (0.50 % in (Kinetic Imaging Ltd, Liverpool, UK). A total of 100 comet images DMSO) was added to the PET-NPLs pellet. The mixture was stirred at randomly selected were analyzed per sample. room temperature for 24 h (protected from light) at 200 rpm. Successive washes, at least twelve times, with ethanol in 0.10 M PBS, pH 7.40 and resuspended on RPMI supplemented media, prior to cell exposure. For confocal visualization, nuclei were stained using Hoechst 33342 (exci­ tation of 405 nm and emission collected at 415–503), cell membranes 4 A. Villacorta et al. Journal of Hazardous Materials 439 (2022) 129593 3. Results and discussion 3.1.2. Dry state size distribution and shape The TEM images of PET-NPLs reveal a polydisperse anisodiametric 3.1. Characterization of the obtained PET-NPL samples particles population with notorious lack of shape regularity. The Martin diameter was measured, and the relative percentage of particle size is 3.1.1. Morphology and composition represented in Fig. 3a using GraphPad Prism 7.0. The obtained average To identify size, shape, and composition of the obtained PET-NPLs, size of 40.08 nm presents a standard deviation of 55.68 nm and a SEM-EDS was used. In Figs. 2a and 2b (as well as in Fig. S1) SEM polydispersity index of 0.52 evidencing the heterogeneity of the PET morphology of the obtained PET-NPLs is visualized. Surface irregular­ particles on the suspension produced by this method. Interestingly, the ities of the produced particles can be easily observed on the cooper and graphic shows a bimodal distribution with a big peak around 50 mm and on the carbon sections of the holder. Not only the differences in size are a second small peak around 500 nm. As an overall, this means that our noticeable, but also the holey structures that can be appreciated. This protocol produces PET particles inside the nano range. means that not only the particles are on the nanoscale, but also their Although the term MNPLs is increasingly used, no general agreement surface/volume ratios are different than in a solid body. Interestingly, exists on the borders between microplastic and nanoplastic sizes. Engi­ when energy dispersive X-ray microscopy was used, metal contamina­ neered nanomaterials are classically considered to range between 1 and tion resulting from the sanded process was detected. Thus, in the sam­ 100 nm, but this strict definition leaves out other sizes placed in the ples obtained using aluminum oxide/silicon carbide rotary burrs such nano range. Although engineered materials are built at the desired size, elements were detected. This metal contamination was not present when secondary MNPLs are constituted by a wide range of sizes and shapes a diamond rotary burr was used. This can be observed on Figs. 2c and 2d, and some agreement is necessary to define the borders between MPLs and it is summarized on the included table (Fig. 2e). These findings raise and NPLs. At present, many authors consider that MNPLs range from few an important alarm regarding the abrasive methods to be used in the nm to several mm. To clarify the boundaries criteria, Hartmann et al. case of mechanically produced MNPLs, mostly when the potential haz­ (2019) proposed to categorize MNPLs according to the conventional ard of the obtained MNPLs need to be evaluated. Surprisingly, this po­ units of size: nano (1–1000 nm) and micro (1–1000 µm). Nevertheless, tential metal contamination of the obtained samples is not taken into to maintain the classical definition of nanomaterial/nanoplastic, they consideration in neither of the recent published papers using mechanical proposed to divide the nano range (1–1000 nm) between nanoplastics milling or the trituration of few millimeters’ industrial pellets (Astner (1–100 nm) and submicron-plastics (100–1000 nm). Such classification et al., 2019; Parolini et al., 2020; Lionetto et al., 2021; Pignattelli et al., needs to have the general agreement, mostly from those coming from the 2021). The contamination by metals, as consequence of the mechanical nanomaterials field. A close inspection of our TEM figures confirms the milling of other polymers pellets, has also been detected by us, as heterogenicity of the obtained material, which can be considered a indicated in Fig. S2, where the results of milling polylactic acid plastic’ realistic characteristic, thereby making it more representative from the pellets are indicated. Therefore, from this point forward all the results environmental point of view. Thus, in addition of the small PET particles presented in this study corresponds to the PET-NPLs produced with the of around 50 nm (Fig. 3b), bigger sizes are also present, including rigid diamond burr rotating tool. fibers (Figs. 3c, 3d), different large shapes (Fig. 3e), and agglomerates From our data, a warning signal must be put to all the hazard data (Fig. 3f). Interestingly in Fig. 3f, it is evident that a high amount of PET generated using MNPLs obtained using milling procedures, due to the in the nano range is contained in the agglomerates; nevertheless, the potential metal associated contamination. It is well-know the toxic ef­ potential biological effects discussed further in this section do not seem fects of metals, including those included during the manufacture of to be highly affected by the presence of such agglomerates. plastics (Turner and Filella, 2021). 3.1.3. Chemical composition To confirm the chemical identity of the obtained PET-NPL samples, Fig. 2. Scanning electron microscopy results. Amorphous particle structures from a diverse range of geometry can be observed as aggregates metallic section of the holder (a) as in the polymer, where differences in sizes and shapes as well as holey structures can also be detected (b). Notorious differences on the EDS spectra are noticed according to the type of rotating burr utilized where only the diamond covered (c) was able to produce not contaminated samples (d). Aluminum contamination is marked on blue and silica on violet dashed lines, while copper signal marked with green solid lines corresponds to background signal produced by the cooper grid holder. Summary of EDS for both conditions on the table at the bottom (e). 5 A. Villacorta et al. Journal of Hazardous Materials 439 (2022) 129593 Fig. 3. PET-NPLs characterized by TEM. Size distribution of sizes was obtained from measures of more than 100 images showing a bimodal distribution, with the main peak with sizes around 50 nm, and a second peak with sizes around 400–500 nm (a). Fields with many small sized materials are observed (b), as well as some irregular shapes and fibers (c-e). Agglomerates permit to visualize the small sizes of their components (f). FTIR analysis was carried out at the premises of the ICN2. To assess the on PET spectrum (Andanson and Kazarian, 2008; Chen et al., 2013; composition of our samples the obtained interferograms were analyzed Johnson et al., 2021), and then assigned as reported on Fig. 4. As and contrasted with previous reports of the representative bands present observed, the obtained peaks match well with the expected, confirming Fig. 4. FTIR spectra with the identity of principal peaks of the interferogram (a) and its assignment (b). Vertical lines indicate coincident peaks. 6 A. Villacorta et al. Journal of Hazardous Materials 439 (2022) 129593 the PET nature of our samples. approximately 60 nm (155 nm of Dgeo), obtaining a weighted Dgeo of 145 ± 7 nm. 3.1.4. Aqueous solution size distribution and zeta potential From the bimodal distribution observable by TEM, we tried to reduce 3.2. Cell uptake of PET-NPLs the content of the higher-size peak (including agglomerates) by using sonication and/or filtration. This approach is schematized on the Sup­ To demonstrate the potential applicability of the obtained PET-NPLs, plementary Fig. S3. According to such approaches, we have observed as a representative MNPLs material, it is important to find out whether it that the best way to reduce agglomerates includes filtration and soni­ can be uptake by the cells after having applied a PET-NPLs dispersion cation. Thus, the characterization of such filtered/sonicated samples by and treatment protocol. Herein we have used three different approaches a Zetasizer device is indicated in this section. Other combinations of to confirm the cellular uptake in TK6 and THP1 cell models. filtration/sonication and dispersion procedures (Nanogenotox, 2011) are included in Fig. S3. Hydrodynamic diameter measurements by using 3.2.1. Internalization of PET-NPLs stained by Nile red by confocal dynamic light scattering shows mild differences in the average size (of microscopy around 30 nm) between the suspension of nanoparticles in water, or Nanoplastics, in opposition to metallic nanoparticles, are difficult to with the addition of SDS. The obtained diameter (161.01 ± 3.94 nm) be visualized by confocal microscopy due to their lack of reflection. and polydispersity index (PI, 0.22) for aqueous dispersions, slightly Hence, a staining is needed to explore its toxicokinetic behavior by differ from those observed using SDS, 129.01 ± 0.94 nm and 0.21, imaging techniques. Among the different staining dyes available that respectively. The corresponding signals, expressed as the mean of three can be applied to plastic particles after its synthesis/obtention, stand up independent measurements, are depicted in Fig. 5a. The correlograms the Nile red. It produced the best results when applied to virgin and for each condition by triplicates are observed in Fig. 5b. It is interesting weathered synthetic polymers and textile fibers, in comparison with to notice that for the mentioned measurements a fixed scattered other seven dyes (Prata et al., 2019). In fact, a recent review points out collection angle of 174.7◦ was employed, but when the combination of the main pros and cons of the use of the staining dye in the MNPLs three angles (174.7◦ , 90◦ , and 12.78◦ ) are used, the differences or in­ context (Shruti et al., 2022) In our case, Nile red staining allowed us to fluence of the dispersion media is less noticeable. This can be observed visualize the internalized PET-NPLs inside cells. Due to its affinity for on Fig. 5c, where the curves for both conditions, are almost overlapping. lipidic structures, Nile red signal could induce errors. Nevertheless, we Thus, DLS and even better MADLS are excellent tools for the initial have detected fluorescent signal into the nucleus (Fig. 7a-c) where not characterization of the obtained PET-NPLs. Another important approach structures exist, confirming the usefulness of this staining. Furthers to describe the relative behavior of the particles in suspension is the zeta intracellular signals are indicated in Fig. 7g-j, confirming the cell uptake. potential (ZP), which indicates the charge on the surface of the In fact, the use of Nile red staining was previously found to be useful to PET-NPLs and the stability of the colloid dispersion. In both cases, detect the cell uptake of other sources of in-house produced PET-NPLs summarized on the table (Fig. 5d), the obtained ZP values can be (Rodríguez-Hernández et al., 2019). considered as indicative of moderately stable to highly stable suspen­ sions. It is important to remark the important influence of the sample 3.2.2. Cell complexity as detected by flow cytometry sonication, prior to the characterization, as can be observed in the The first approach was an indirect one checking for structural cell Supplementary material (Figs. S3 and S4). It is also remarkable that it is changes due to PET-NPLs exposure. It is expected that cells internalizing possible to calculate the number of particles from the intensity values PET-NPLs experience changes on their internal structures increasing (Fig. 5d). The obtained values indicate that the used protocol permits to their granularity, which can be assessed using flow cytometry. As cell obtain highly concentrated samples. granularity increase, an increment of side scattered light is expected, From the results obtained by the analysis of PET by AF4-MALS, after and this effect commonly known as cell-complexity can be observed on the void peak at 5 min, a large peak appeared, from min 10–35 with an Fig. 7e,f. This methodology has shown to be useful in detection cell increase of gyration radius from 30 nm up to 600 nm (Fig. 6a). The 90 % uptake of different nanomaterials (Vila et al., 2017). From our results, of PET size ranged from 40 nm up to 75 nm of gyration radius (103 up to we show that cell uptake is modulated by two factors, such as the 194 nm of Dgeo), being 5 % from 75 nm to 100 nm and the rest of the exposure time and the presence/absence of FBS. As observed on Fig. 7b, sample above 100 nm (Dgeo above 258 nm), as it can be seen in the cell uptake increases with the exposure time showing higher uptake Cumulative tendency in Fig. 6b. Moreover, the differential distribution after exposures lasting for 3 h regarding the observed after 1 h exposure. of the PET size, in Fig. 6b, indicated the highest number of particles at In addition, PET-NPLs dispersed in culture media with or without FBS Fig. 5. Size distribution analysis by DLS. (a) Filtered and sonicated PET-NPLs samples were used with both water and SDS (0.5%) as dispersants, and 174.7 as the angle of scattering collection. (b) The correlation coefficients of the measurements. (c) Particle size distribution by using MADLS with three angles of scattering collection (174.7, 90, and 12.78) with water or SDS (0.5 %) as dispersants. 7 A. Villacorta et al. Journal of Hazardous Materials 439 (2022) 129593 Fig. 6. AF4-MALS plots obtained for a 20 µL injection of 1 mg/mL of PET, (a) Fractogram (left axis: MALS 90º detector signal (V); right axis: Gyration Radius (Rg, nm)) and (b) Differential and Cumulative graphics. Fig. 7. Confocal orthogonal view of internalized Nile red stained PET-NPLs (green signal) on THP1 cells after 24 h exposure to 50 μg/mL is presented as XY, YZ and XZ (a, b and c respectively) three dimensional views and d is the control. Small dot like structures is observed at the cytoplasm of THP1 cells (g) and fluorescence at 546–628 on h is collected on the same points. Merged images are visualized on i where also the big fluorescent structure corresponds to fluorescent agglomerates. Relative increment on complexity at early exposition to 50 μg/mL of PET-NP on THP1 cells is represented on e (**p < 0.018, ***p < 0.0009 and ****p < 0.0004) and influence FBS on the media composition (f) (*p < 0.0162 and **p < 0.0087). For both cases One-way ANOVA with Dunnett’s post-test was used for the statistical analysis. TEM images show an increment on vacuoles at 5 (l) or 50 (m) μg/mL of PET-NPs, relative to control (k), PET-NPs like structures are show by the red arrows and mitochondrial abnormalities or holey mitochondrion are present only on PET-NPs treated cells (yellow and brown arrows). 8 A. Villacorta et al. Journal of Hazardous Materials 439 (2022) 129593 are significantly different in terms of complexity, with a higher uptake in the absence of serum. 3.2.3. Cell uptake detection by TEM TEM has successfully been applied to detect nanomaterials uptake by many cell types, including MNPLs (Vila et al., 2017; Cortés et al., 2020). Our TEM figures show substantial amounts of PET-NPLs incorporated into cytoplasmic vesicles (Fig. 7l). Although no specific studies have been addressed to understand the cell uptake of PET-NPLs, their pres­ ence in the lysosomes of Caco-2 cells (Magrì et al., 2021) can suggest that their uptake is by endocytosis, as previously demonstrated for PS-NPLs where that was considered as the main cellular absorption pathway (Magrì et al., 2018; Cortés et al., 2020). Interestingly, we detect PETNPLs into mitochondria inducing some alterations. These effects Fig. 9. Reactive oxygen species (ROS) induction in THP-1 cells after PET-NPLs have been also reported for PSNPLs (Cortés et al., 2020; Trevisan et al., exposures lasting for 3 h at concentrations ranging from 0 to 50 μg/mL at 3 h exposition. Data is expressed as percentage of ROS production relative to the 2020). untreated control ± SEM. One-way ANOVA with Dunnett’s post-test was per­ Taking all three approaches together, it seems clear that cells can formed. Statistical significance indicated on the graph as ****p < 0.0001. uptake our lab-produced PET-NPLs. compared to untreated controls. Nevertheless, the positive control used 3.3. Cytotoxicity (antimycin) induced important levels of ROS, confirming the goodness of the used protocol. Although one of the most recognized mechanisms Cell viability curves were studied to determine the cytotoxic effects of action of nanomaterials is the induction of ROS (Karkossa et al., of PET-NPLs exposure on THP1 and TK6 cell-lines. As shown in Fig. 8 no 2021), MNPLs seem to have a different behavior. Recent results with real significant cytotoxicity was observed up to the concentration of 100 μg/ PET samples did not show ROS induction in human intestinal Caco-2 mL. Only mild effects were observed at the high concentration of cells (Magrì et al., 2021). This inability of PET-NPLs to induce oxida­ 200 μg/mL of PET-NPLs on TK6, where the viability was compromised tive stress in vitro, agree with many studies using PSNPLs as a model of close to 80 % for exposures lasting for 48 h. Since our aim is to work with MNPLs where exposure did not increase ROS levels in Caco-2 cells after non-cytotoxic doses, the following experiments were carried out at acute exposures (Domenech et al., 2020) or after long-term exposures concentrations up to 50 μg/mL. (Domenech et al., 2021). Nevertheless, the potential induction of This lack of toxicity of the obtained PET-NPLs agree with the re­ oxidative stress seems to be dependent on the cell type as demonstrated ported for other secondary PET nanomaterials obtained using different with different hematopoietic cell lines where ROS was clearly observ­ methodological approaches (Magrì et al., 2018; Dhaka et al., 2022). able (Rubio et al., 2020), as well as dependent on the particle size (Kik Although the toxic effects of MNPLs can depend on distinctive charac­ et al., 2021). teristics, i.e., the size, most of the studies agree with their lack of acute toxic effects. This has been proved when the effects of different MPLs 3.5. Genotoxic evaluation of PET-NPLs by using the comet assay (polyethylene, polypropylene, polyvinylchloride, and PET) were eval­ uated in three different cell lines (Stock et al., 2021). This means that Among the different biomarkers evaluating health risk, those other milder, sub-toxic cellular effects, and not cell death, must be detecting genotoxicity stand out. This is because, due to the main role of selected as biomarker when evaluating the harmful effects associated DNA in cell functionality, DNA damage severely compromises human with MNPLs exposure. Further efforts looking for different exposure health (Carbone et al., 2020). Although the genotoxicity assays can scenarios, considering acute exposure to higher concentrations, evaluate both primary or fixed DNA damage, those detecting primary repeated doses, or long-term exposures, are necessary to decipher the damage are much more sensitive. Thus, the comet assay is used to toxic effects of PET-NPLs obtained from environmental plastic samples. measures the induction of single/double DNA breaks as well as the in­ duction of oxidative damage on the DNA bases. Its simplicity and 3.4. ROS induction by PET-NPLs sensitivity have spread its use in many fields, including the hazard assessment of nanomaterials (García-Rodríguez et al., 2019). The ability of our PET-NPLs to increase the intracellular levels of ROS In our study, we have not been able to demonstrate the ability of our was evaluated using the DHE detection assay, in THP1 cells. The results PET-NPLs to damage DNA, as indicated in Fig. 10. In addition to the lack indicated in Fig. 9 show that exposures lasting for 3 h were unable to of direct strand breaks (Fig. 10a) PET-NPLs exposure also did not induce induce statistically significant differences in the ROS levels when oxidative damage on the DNA bases (Fig. 10b). These results agree with Fig. 8. Relative cell viability of THP1 (green) and TK6 (red) cell lines at concentrations ranging from 0 to 200 μg/mL, after PET-NPLs exposures lasting for 24 h (a) and 48 h (b). Data represented as percentage of living cells relative to the untreated control treatment at 24 (green) and 48 (blue) h of exposure. Data is expressed as percentage of cells relative to the untreated control ± SEM. One-way ANOVA with Dunnett’s post-test was used for the sta­ tistical analysis. Statistical significance indi­ cated on the graph as * p < 0.01. 9 A. Villacorta et al. Journal of Hazardous Materials 439 (2022) 129593 Fig. 10. THP1 genotoxic (a) and oxidative DNA damage (b) at concentrations ranging from 0 to 50 μg/mL and exposure lasting for 3 h. Damage is expressed as the percentage DNA in tail ± SEM. One-way ANOVA with Dunnett’s post-test was performed. Statistical significance indicated on the graph as **P < 0.01 ****p < 0.0001. respectively. Methyl methanesulfonate (MMS) was used as a positive control. our previous data showing that PET-NPLs were unable to induce approaches detecting other types of milder cell effects, out of cell death, oxidative stress. No other studies are using PET where the genotoxic should be used when evaluating the harmful effects associated with potential was evaluated. Nevertheless, a few studies on the genotoxic MNPLs exposure (Stock et al., 2021). In such a context, having the fa­ potential of polystyrene MNPLs have been reported showing contra­ cility of easily obtaining different types of MNPLs can result very helpful. dictory results. In in vitro studies, negative findings using the micronu­ It is obvious that the original sources of the environmental commercial cleus assay were observed in the intestinal/placenta barrier cells PET plastics can differ in factors such as crystallinity and plasticizer exposed to 50/500 nm polystyrene NPLs (Hesler et al., 2020). Negative content. This can suppose differences in the obtained samples, hence results were also reported in human intestinal Caco-2 cells exposed to differences in their induced biological effects. However, this should not 50 nm polystyrene NPLs both as a component of an in vitro barrier necessarily be considered a problem but rather a challenge, since the (Domenech et al., 2020) or after long-term exposures (Domenech et al., obtained/evaluated samples are more representative of the environ­ 2021). Nevertheless, positive effects were observed in sh27 fibroblasts mentally MNPLs pollutants. using the micronucleus assay (Poma et al., 2019). This would indicate differences between cell types in both MNPLs uptake and genotoxic ef­ CRediT authorship contribution statement fects as reported when different types of human hematopoietic cell lines were used (Rubio et al., 2020). RM and AH planned the experiments. AV, LR, MA, MLM, VFC, and As a summing up, we consider that the proposed method to obtain OHM carried out the experimental part. AV analyzed the data, carried uniform and reproducible secondary NPLs from representative envi­ out the statistical analysis, and prepared tables/figures. AV, RM, and AH ronmental plastic goods is adequate, since the nano sized fraction of the wrote the final manuscript. suspension is very high (about 95%). In addition, we consider that the obtained samples mimic the naturally occurring degradation phenom­ Declaration of Competing Interest enon The characterization of the working material by TEM showed a bimodal distribution in the nano range with most of the particles ranging The authors declare that they have no known competing financial around 50 nm. Simple filtering and sonication steps produce a uniform interests or personal relationships that could have appeared to influence distribution around 100 nm when DLS and AF4 measurements are the work reported in this paper. applied. We are confident that this methodology can be easily used to obtain NPLs from other plastics materials, scaping from the constrictions Data availability posed using pristine forms of commercial polystyrene NPLs. Materials resulting from environmentally representative plastics permit to answer Data will be made available on request. the main open questions asking for the risks of environmental MNPLs resulting from degradation processes. As reported, our protocol using Acknowledgments non-metallic burrs during the sanding process has a great advantage over those using metallic blades which contaminate the resulting A. Villacorta was supported by Ph.D. fellowships from the National MNPLs. Such metallic contamination would invalidate any approach Agency for Research and Development (ANID), CONICYT PFCHA / aiming to evaluate the potential health risk of such types of MNPLs. It DOCTORADO BECAS CHILE / 2020 − 72210237. L. Rubio was sup­ must be recalled that most of the studies evaluating the hazardous ef­ ported by the Fondo Nacional de Innovación y Desarrollo Científico y fects of MNPLs use sizes in the micro range. Although they can provide Tecnológico (FONDOCYT) República Dominicana (Project 2018–2019- useful information, it must be remembered that plastic particles sized in 2B2–093). This project (PlasticHeal) has received funding from the the nano-range are more prone to cross epithelial barriers and, conse­ European Union’s Horizon 2020 research and innovation program quently, spread over the different tissues and organs. Accordingly, to be under grant agreement No 965196. L. Rubio was supported by a contract able to get nanoplastics -instead of microplastics- resulting from the Juan de la Cierva (IJC2020-2686I/AEI/10.13039/501100011033). degradation of representative environmental plastic pollutants can represent an important step to get more sound data answering about their potential effects on human health. Environmental Implication Although our hazard approach can be considered just as a pre­ liminary one, it is important to highlight that the obtained PET-NPLs are In spite of the intrinsic interest in understanding the potential haz­ easily internalized by the used cell models without important toxic or ards associated with the environmental micro-/nanoplastics exposure, genotoxic effects. Due to the lack of gross biological effects, reported by most of the studies use pristine commercial samples. Thus, a challenge is many authors using different sized MNPLs, the use of methodological to get/evaluate environmental representative samples. 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