Science of the Total Environment 880 (2023) 163151 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Titanium-doped PET nanoplastics of environmental origin as a true-to-life model of nanoplastic Aliro Villacorta a,b, Lourdes Vela a,c, Michelle Morataya-Reyes a, Raquel Llorens-Chiralt d, Laura Rubio a, ⁎ ⁎ Mohamed Alaraby a,e, 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 Faculty of Health Sciences Eugenio Espejo, Universidad UTE, Quito, Ecuador d AIMPLAS, Plastics Technological Centre, Gustave Eiffel, 4, 46980 Paterna, Valencia, Spain e Zoology Department, Faculty of Sciences, Sohag University, 82524 Sohag, Egypt 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 • Nanoplastics from opaque PET milk bot- tles have been obtained. • The hybrid (PET/TiO2NPs) nature of this material has been demonstrated. • TEM/confocal microscopy showed the colocalization of both constituents. • TEM(Ti)NPLs internalize differentially ac- cording to the used cell types. • No toxic effects were observed for this true-to-life NPL. A R T I C L E I N F O A B S T R A C T Editor: Damià Barceló The increased presence of secondary micro/nanoplastics (MNPLs) in the environment requires urgent studies on their potentially hazardous effects on exposed organisms, including humans. In this context, it is essential to obtain repre- Keywords: sentative MNPL samples for such purposes. In our study, we have obtained true-to-life NPLs resulting from the degra- PET(Ti) nanoplastics dation, via sanding, of opaque PET bottles. Since these bottles contain titanium (TiO2NPs), the resulting MNPLs also Obtention contain embedded metal. The obtained PET(Ti)NPLs were extensively characterized from a physicochemical point Characterization Cell internalization of view, confirming their nanosized range and their hybrid composition. This is the first time these types of NPLs are obtained and characterized. The preliminary hazard studies show their easy internalization in different cell lines, without apparent general toxicity. The demonstration by confocal microscopy that the obtained NPLs contain Ti samples offers this material multiple advantages. Thus, they can be used in in vivo approaches to determine the fate of NPLs after exposure, escaping from the existing difficulties to follow up MNPLs in biological samples. 1. Introduction The extended use of plastic goods has results in global environmental pollution by plastic wastes, and their consequent environmental alarms ⁎ Corresponding authors at: Group of Mutagenesis, Department of Genetics and (Chang et al., 2022). Although plastics have a relatively long life, degrada- Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Campus of Bellaterra, 08193 Cerdanyola del Vallès, Barcelona, Spain. tion occurs through different physical-chemical-biological processes gener- E-mail addresses: ricard.marcos@uab.cat (R. Marcos), alba.hernandez@uab.cat ating the so-called micro- and nanoplastics (MNPLs). At these sizes, and (A. Hernández). especially at the nano range, MNPLs can be easily internalized by organisms http://dx.doi.org/10.1016/j.scitotenv.2023.163151 Received 30 January 2023; Received in revised form 13 March 2023; Accepted 25 March 2023 Available online 1 April 2023 0048-9697/© 2023 The Authors. 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. Science of the Total Environment 880 (2023) 163151 (including humans) following different routes, finally resulting in a poten- chemical interaction of the fillers with compounds that possess functional tial health hazard (Rubio et al., 2020a; Gigault et al., 2021; Wu et al., groups, by chemical absorption on the surface of the particles of the filling 2022). Thus, determining the potential hazard risks associated with material of some modifying agents, or by coating the filler particles with a MNPLs exposure is a hot and current topic. suitable coupling agent (Fronza et al., 2019). Although great efforts have been devoted to evaluating the hazards as- Aiming to obtain environmental representative MNPLs, we have used sociated with MNPLs exposure, the lack of representative environmental opaque PET bottles to obtain NPLs, following the protocol recently pub- MNPLs is a big barrier to overcome. Consequently, most of the studies lished (Villacorta et al., 2022). These true-to-life NPLs may help us to un- have been carried out with pristine commercial polystyrene (PS) samples derstand the complexity of the environmental MNPLs, while constituting that, despite their advantages (uniformity, different sizes, surface unrivaled materials to be used to evaluate the potential health hazards of functionalization, and fluorescent staining), are not fully representative of environmental MNPLs. Although different metals and metalloids like arse- the environmental MNPLs (Xu et al., 2022). Although PSMNPLs have nic, cadmium, chromium (VI), and lead are normally used as plastic addi- shown to be very useful for many purposes, the extrapolation of the ob- tives (Smith and Turner, 2020; Turner and Filella, 2021), they are present tained data is doubtful for human health risk assessment associated with in amounts significantly less that TiO2NPs in opaque PET bottles. The pres- MNPLs exposures (Brachner et al., 2020; Coffin et al., 2022). Consequently, ence of such metals can suppose a potential concern from a toxicological the obtention of environmentally relevant (true-to-life) MNPLs is an urgent perspective, not only for their possible leaching in environmental condi- challenge to be achieved. tions but for their behavior in specific physicochemical conditions, such Due to the impossibility of getting enough uniform MNPLs just by pick- the acidic environments encountered in the digestive tract of many animals, ing up environmental samples, different protocols have been proposed by when MNPLs are unintentionally ingested. Beyond their potential toxico- using the physical degradation of plastic goods, followed by the collection logical profile, the obtention of titanium-doped MNPLs can represent a suit- of the different fractions (containing MPLs or NPLs), and their ulterior able true-to-life model to follow up their fate in complex matrices, including characterization. Interestingly, most of the published proposals have used experimental in vivo models. Before determining the potential toxicity of polyethylene terephthalate (PET) goods as the original source. This is MNPLs exposure, their cell uptake must be demonstrated and, if possible, mainly due to the high demand for this plastic type that is mainly used quantified. These aspects have been the subject of recent reviews consider- for packaging, including bottles for multiple purposes, although it is also ing both the exposure route and the model organism used (Paul et al., 2020; a polymer widely used in the textile industry (PlasticsEurope, n.d.). Conse- Huang et al., 2022; Dusza et al., 2023). quently, and because of the deficient recycling mechanisms and the lack of Here we present our approach to obtain PET(Ti)NPLs, as well as their awareness on the part of society, they finally end up in the environment as complete physico-chemical characterization. In addition, preliminary data plastic waste. Among the different experimental approaches, the UV-laser on their behavior in human cells under in vitro exposure conditions is also ablation process was first used to obtain PET-NPLs (Magrì et al., 2018). reported. It is important to point out that this is the first report getting Alternatively, mechanical milling is another fractionation process used to and analyzing NPLs from opaque PET bottles. obtain mainly the MPLs fraction (Astner et al., 2019; Pignattelli et al., 2021; Lionetto et al., 2021). Furthermore, the use of mechanical ground 2. Materials and methods approaches has also been proposed as an easy way to obtain PET-NPLs (Rodríguez-Hernández et al., 2019; Roursgaard et al., 2022; Villacorta 2.1. PET(Ti) nanoplastics obtention et al., 2022). Among the different types of PET bottles, we can distinguish between We have used commercially available opaque-PET bottles to produce transparent and opaque PET ones. Opaque PET bottles are highly used PET nanoplastics containing titanium [PET(Ti)NPLs]. The used procedure many for packing milk because they provide the light protection that was based on the recently published method designed to get PETNPLs UHT milk requires and minimize gas permeability which, together with from water bottles (Villacorta et al., 2022). The process is schematized in their low cost, are some of their advantages in front of other packaging op- Fig. 1. Briefly, pieces of about 12 cm2 were cut from the original opaque tions (Tramis et al., 2021). These characteristics of the opaque PET are due PET bottle and sanded with a diamond rotary burr accessory, taking partic- to the presence of titanium dioxide nanoparticles (TiO2NPs) used as a filler, ular attention not to overheat the surface by passing in only one direction which also allows for reducing the bottle thickness. One of the common and only once on the same area. The obtained opaque PET debris was protocols to increase the compatibility of the fillers introduced in the plas- passed through a 0.20 mm sieve and 4 g of the resulting material was dis- tics intend to modify their surfaces by increasing their hydrophilicity or hy- persed on 40 mL of trifluoroacetic acid pre-heated to 50 °C on a stirring drophobicity. This compatibility critically depends on the particle size of plate at 100 rpm for 2 h and then kept under continuous agitation at the filler because that can increase/decrease viscosity affecting the physical room temperature overnight. The next day, 40 mL of TFA (20 %, v/v) and chemical properties of the final compound. Since surface treatments were added and the mixture was kept under constant stirring for 24 h can decrease the viscosity of the compound or masterbatch, modifications more. The suspension was then passed through the 0.20 mm sieve, to elim- of the surface of fillers are becoming important because of its improvement inate the bigger pieces, and centrifuged at 2500 rcf for 1 h once distributed in adhesion (Mozetič, 2019). Surface modifications can be achieved by the on six 10 mL glass tubes. The obtained pellets were resuspended on 400 mL Fig. 1. Schematic representation of the PET(Ti)NPLs production. Opaque PET fragment from bottles were grinded until a powder is obtained, passed through the sieve (a), stirred, suspended, and heated at different concentrations of TFA (b), distributed on glass tubes and centrifuged (c), resuspended on SDS, sonicated (d), and transferred to a cylinder for sedimentation (e) and then collected, washed, weighted, resuspended and sonicated (f) and aliquots were prepared and stored frozen until needed (g). 2 A. Villacorta et al. Science of the Total Environment 880 (2023) 163151 of a 0.5 % sodium dodecyl sulfate (SDS) solution, vigorously mixed, distrib- were from Bruker (Bruker Corporation, Billerica, Massachusetts, USA). To uted into two 200 mL beakers, and ultrasonicated on an SSE-1 Branson assess the composition, the obtained interferograms were analyzed and sonicator (Branson Ultrasonics Co., Brookfield, CT, USA) for 2 min at contrasted with previously reported data. 25 % amplitude, in 9/9 s sonication/break cycles. The content of each bea- ker was transferred to a graduated cylinder to sediment for 1 h, to eliminate 2.6. PET(Ti)NPLs characterization by mass spectroscopy the bigger fraction. The top 100 mL of each cylinder were collected and cen- trifuged to eliminate SDS. Pellets were washed twice with Milli-Q water To determine the Ti content of the original PET source, 0.10 g of the PET and twice with pure ethanol and dried under sterile air laminar flow. Pellets (Ti) film was weighted on a Mettler Toledo XP205DR analytical balance were then weighted and resuspended on Milli-Q water at concentrations of (Mettler-Toledo S.A.E., Barcelona, Spain). This amount was dispersed in 10 mg/mL. Suspensions were then sonicated for 16 min at 10 % amplitude Milli-Q water to the same concentration than the stock PET(Ti) in a cold-water bath and stock solution aliquots of 1 mL were immediately (10 mg/mL). In an independent way, 0.25 mL of the two dispersions frozen on cryotubes in liquid nitrogen and stored at −80 °C for further use. were vigorously agitated and digested in 5 mL of a mixture (4 mL HNO3 65 % (p/v) and 1 mL of HF 40 % (p/v)) in a microwave oven for 20 min 2.2. PET(Ti)NPLs characterization by transmission electron microscopy (TEM) at 260 °C (ramp 25 min to reach 260 °C). Samples were diluted on HNO3 (1 % v/v) before injection. The Ti content of the samples was determined A carbon-covered cooper grid was dipped into the working solution on an inductively coupled plasma optical emission spectrometer ICP-OES (200 μg/mL) and allowed to evaporate overnight. Particles in the grid Agilent 5900 (Agilent Technologies, Santa Clara, CA, USA). were examined by TEM using a JEOL JEM 1400 instrument (JEOL Ltd., Tokyo, Japan) operated at 120 kV. TEM images were analyzed for particle 2.7. PET(Ti)NPLs labeling and confocal visualization size distribution by measuring the Martin diameter using the ImageJ soft- ware 1.8.0_172 and processed with the GraphPad Prism 7.0 software To visualize/identify PET(Ti)NPLs on confocal microscopy, the reflec- (GraphPad, San Diego, CA). The Savitzky-Golay-like filter was applied to tion property was used for the detection of Ti. To visualize PET, separately smoothen the curve. from Ti, samples were labeled with iDye Poly Pink (iDye), following an ad- aptation of the published protocols to dye microplastics (Karakolis et al., 2.3. PET(Ti)NPLs characterization by scanning electron microscopy (SEM) & 2019; Nguyen and Tufenkji, 2022). Briefly, a working solution of 1 mL of energy dispersive X-ray spectroscopy (EDS) PET(Ti)NPLs at the concentration of 5 mg/mL was prepared from the stock dispersion (10 mg/mL) and transferred into a 1.5 mL tube containing From the stock solution, a working solution of 200 μg/mL was prepared previously weighed 0.01 g of the textile dye (iDye) used for synthetic fibers. by thawing in a warm bath at 37 °C and diluted in Milli-Q water. The work- The mixture was vigorously vortexed and incubated for 2 h at 70 °C on a ing solution was vigorously vortexed and 10 μL of the suspension were de- glass tube. After cooling at room temperature, 9 mL of Milli-Q water was posited on a silica holder. Water was evaporated from the sample and added to the suspension and then centrifuged at 4000 rpm on an Amicon® examined on an SEM Zeiss Merlin (Zeiss, Oberkochen, Germany) coupled Ultra-15 centrifugal Ultracel®-100 K filter 1 × 105 MWCO for 15 min. This with an X-Max 20 mm EDS system (Oxford Instruments, Oxford, UK). In ad- step was performed twice to remove the excess of iDye. The washed parti- dition to collecting SEM images, an area including the nanoparticle surface cles were then collected and suspended in the final volume of 1 mL of was selected for the EDX analysis, the signal was collected and analyzed by Milli-Q water and stored protected from light at 4 °C until needed. Here the INCA Energy software (INCA, Grinnell, IA, USA). on, these stained PET(Ti)NPLs are named iDyePET(Ti)NPLs. For their visu- alization in the confocal microscope, the particle suspension was diluted on 2.4. PET(Ti)NPLs characterization by multi-angle and dynamic light scattering Milli-Q water at a final concentration of 400 μg/mL and two drops of 20 μL (DLS-MADLS), and zeta potential were placed on a slide, covered with a coverslip, and let dry inside the bio- safety cabinet on air laminar flow. PET(Ti)NPLs were prepared similarly to The indicative size of the colloid structures in the suspension of the PET be used as control. Both slides were examined by confocal microscopy using (Ti)NPLs was determined using a Zetasizer® Ultra device from Malvern a Leica TCS SP5 confocal microscope. Excitation wavelength of 561 nm was Panalytical (Cambridge, United Kingdom). To such end, a working solution set on an acousto-optic tunable filter (AOTF 561) and, emission spectra was of 100 μg/mL of PET(Ti)NPLs was used. Additionally, to investigate the in- collected between 580 and 700 nm (lambda scan begin bandwidth fluence of the culture media in the nanoplastic dispersion, the same concen- 575.00–585.00 nm to lambda scan end bandwidth 691.00–701.00 nm). tration was prepared by using the Nanogenotox dispersion protocol Emission was analyzed using the Leica Application Suite X 3.7.5.24914 (Nanogenotox, 2011), and its behavior was evaluated on both Milli-Q (Leica Microsystems CMS GmbH Wetzlar, Germany). Images were collected water and on FBS supplemented RPMI (Roswell Park Memorial Institute) under the same conditions, further explained on Section 2.10. medium supplemented with 10 % fetal bovine serum (FBS), 1 % glutamine (Biowest, France), and 2.5 μg/mL of PlasmocinTM 226 (InvivoGen, 2.8. Cell culture CA, USA). To determine the cell internalization ability of PET(Ti)NPLs, three 2.5. PET(Ti)NPLs characterization by Fourier transform infrared spectroscopy human hematopoietic cell lines were used. Thus, THP-1 monocytes, TK6 (FTIR) lymphoblasts, and Raji-B B-lymphocytes were selected as a widely distrib- uted and accepted lymphoblastic human cell models. All three cell lines To detect functional groups, and to identify samples as PET, materials at were purchased from Sigma Aldrich (MO, USA). Cells were grown in T-25 different stages of the production process were used. Thus, pieces of origi- flasks on RPMI medium (Biowest, France) supplemented with 10 % FBS, nal materials (water- and opaque-bottles), midterm production sanded ma- 1 % glutamine (Biowest, France), and 2.5 μg/mL of Plasmocin (InvivoGen, terial before acid exposure, and final nanoparticle samples were analyzed CA, USA). Cultures, with a density ranging from 5 × 105 to 1 × 106 cells, and compared. For PET(Ti)NPLs from the stock solutions (10 mg/mL), a were maintained at 37 °C in a humidified atmosphere of 5 % CO2. drop was placed on a gold mirror and let dry for one week inside a Petri dish. For the solid samples, 3 × 3 cm pieces for both PET bottle types 2.9. PET(Ti)NPLs cell uptake (internal complexity), as determined by flow were cut directly from bottles. For the above-described materials (suspen- cytometry sion and solids original samples), the analysis was carried out on a Vertex 80 device, while the dust of polymer-metal obtained from the sanded mate- To quantify cell internalization, the internal complexity of the exposed rial was analyzed by using a Tensor 27 device. Both types of equipment cells was used as an indicator of internal structural cell complexity and 3 A. Villacorta et al. Science of the Total Environment 880 (2023) 163151 determined by flow cytometry. To proceed, the three selected cell lines (Villacorta et al., 2022) has been used several times from PET water bottles (TK6, Raji-B, and HTP-1) were grown on U-bottom 96 well plate at a cell to get more material to be spread among the PlasticHeal consortium, show- density of 5 × 105 cells/mL on a final volume of 0.20 mL and treated ing a very high repeatability. with concentrations of 25, 50, and 100 μg/mL for a 24 h period. Once PET(Ti)NPLs uptake takes place, the orthogonal light scattering – com- 3.1. Dry state size distribution and shape monly known as Side Scatter (SSC) – was evaluated using a flow cytometer Cytoflex S (Beckman Coulter CytoFLEX S). A total number of 10,000 events TEM was used to determine the morphology and the size of the obtained (single cells) were scored and evaluated using the CytExpert software and PET(Ti)NPLs. The obtained figures were compared with those previously data was processed using GraphPad Prism Software 7.0. (GraphPad, San obtained from transparent PET bottles (Villacorta et al., 2022). In Fig. 2 it Diego, CA). is indicated the morphology of PETNPLs (a) as compared with PET(Ti) NPLs (b and c). As observed, it is relatively easy to detect differences on 2.10. PET(Ti)NPLs cell uptake determination by confocal microscopy the electrodensity of the sample when Ti is present. The presence of TiO2NPs results in high-density points. Obviously, the degree of agglomer- For the visualization of internalized PET(Ti)NPLs into the cells, confocal ation can also be a factor modulating density. When the Martin diameter microscopy was used using the protocol recently described (Annangi et al., was measured for more than one hundred PET(Ti)NPLs figures, a mean 2023). Nuclei were stained using Hoechst 33342 (excitation of 405 nm and size of 382.07 nm with a polydispersity index of 0.37 was obtained. Inter- emission collected at 415–503), and cell membranes were dyed using estingly, the high electrodensity of TiO2NPs also permitted to determine Cellmask (excitation of 633 nm and emission collected at 645–786). For their size distribution inside the PET(Ti)NPLs complex, as indicated in iDyePET(Ti)NPLs, an excitation wavelength of 561 nm and emission col- Fig. 2d. lected at 570–630 were used. For the localization of Ti, reflection images From the size distribution it is obvious that the average exceeds the were obtained in parallel. Images of each sample were obtained using a range of the nanoparticles (or by extension of a nanoplastic) definition. Leica TCS SP5 confocal microscope and processed using ImageJ processing The last recommendation of the European Commission state that, at least and analysis software, version 265 1.8.0_172. the 50 % of the particles contained in a dispersion must be in the nano- range, and at least one dimension must be in the 1–100 nm range 2.11. Cell viability effects of PET(Ti)NPLs in the used cell lines (European Commission (EC), 2022). Nevertheless, this definition looks ap- propriated for the engineered nanomaterials, and by extension for the pri- To assess the cell viability of the three hematopoietic cell lines after ex- mary nanoplastics, but not seems adequate for secondary nanoplastics posure, cells were seeded on the same conditions as previously described at resulting from the degradation of macroplastic goods, were a large range a cell density of 5 × 105 cells/mL on a final volume of 0.20 mL on U-bottom of sizes and shapes are expected. To describe the new true-to-live obtained 96 well plates and treated with concentrations ranging from 0 to nanoplastics we propose to take into consideration the proposal of 100 μg/mL for a 24 h period at standard conditions. Cells were then Hartmann et al. (2019). These authors propose to categorize MNPLs accord- mixed and diluted 1:100 on ISOFLOW and counted with a ZTM coulter ing to the conventional units of size: nano (1–1000 nm) and micro counter (Beckman Coulter Inc., CA, USA. The average number of cells (1–1000 μm). Although the authors divide the nano range between counted on each treatment was compared with the average number of the nanoplastics (1–100 nm) and submicron-plastics (100–1000 nm), this divi- untreated control cells and data was evaluated using GraphPad Prism Soft- sion add more darkness than light. Thus, from the operational point of view ware 7.0. (GraphPad, San Diego, CA). our sample of PET(Ti)NPLs distribution falls into the nano range. 3. Results and discussion 3.2. Dry state morphology and composition The PET(Ti)NPLs samples obtained according to the protocol outlined To better identify the PET(Ti)NPLs morphology and composition, in Fig. 1 were used for further characterization. It should be remarked the SEM-EDS methodologies were used. Fig. 3a and b show the observed mor- large amount of material obtained in the extraction process above de- phologies. It must be noted that due to the high electrodensity of Ti, the scribed. The resulting 10 vials of 1 mL of the stock dispersion morphological visualization of PET is affected, and the figure definitions (10 mg/mL) were stored at −80 °C. Working solutions were prepared fol- are not optimal. It is important to note the morphology of the TiO2NPs lowing the Nanogenotox protocol and aliquoted in 10 vials containing showing their nanorod shape. Considering the average length of the 0.2 mL of a concentration of 5000 μg/mL. This means that a standard TiO2NPs nanorod present in the PET(Ti)NPLs it is assumable the size distri- PET(Ti)NPLs production generate 100 vials of the working dispersion, bution determined by TEM, since it is difficult to find PET structures not as- which were stored at the same conditions until used. This is a large amount sociated to TiO2NPs. that permit to carry out several rounds of studies aiming to perform a com- As previously indicated, different metals and metalloids like arsenic, plete characterization and they hazard evaluation. The original process cadmium, chromium (VI), and lead are normally used as plastic additives a b c PET d Ti % frequency Fig. 2. TEM figures from in-house secondary PETNPLs (a) compared with PET(Ti)NPLs (b, c). The presence of TiO2NPs is detected as a high density spots. Size distribution for PET(Ti)NPLs was determined, and a bimodal distribution was observed (orange). Due to the good detection of TiO2NPs, their size distribution (Martin diameter) was also determined (blue). 4 A. Villacorta et al. Science of the Total Environment 880 (2023) 163151 a b 2000 c 1500 Intensity (a.u.) 1000 500 0 0 2 4 6 Energy (KeV) Fig. 3. SEM figures of PET(Ti)NPLs show TiO2NPs embedded on nano-PET structures both on secondary electrons view (a), and both secondary (b, left) and backscattered (b, right) electrons. On (a) the white arrows indicate the TiO2NPs, and on (b) the yellow arrow indicates the TiO2NPs that are not easily visible due to the polymer covering. The EDS spectrum observed on (c), show the characteristic peaks of Ti remarked on blue, and the dashed line indicates the Si holder interference. and, consequently, they can be found in their derived MNPLs (Turner and (Ti)NPLs would cover two main aspects, i) are MNPLs containing metal that Filella, 2021). Nevertheless, the levels of titanium present in the MNPLs re- can be used to determine their fate in experimental mammalian models, sulting from opaque bottles, like the obtained in this study, are very much and ii) are true-to-life representative environmental MNPLs. high and impregnating the resulting MNPLs. This could suppose an associ- ated hazard risk for such type of MNPLs. It should be remembered that Ti 3.3. PET(Ti)NPLs size distribution and zeta potential in aqueous solution compound, and by extension TiO2NPs, is a recognized food additive (E171), wide used in many food matrices (Cornu et al., 2022), were the E The hydrodynamic behavior of PET(Ti)NPLs dispersed on Milli-Q water, letter codes for substance that can be used as food additives. In addition or following the Nanogenotox protocol, shows mild differences among to the multiple studies looking for potential hazards associated with E171 them. Independently of these mild differences, they show that there is a use, a recent publication from the panel on Food Additives and Flavorings need of standardized procedures when it comes to nanoplastic dispersions. from the European Food Safety Authority (EFSA) indicates that, “based At this point is relevant to point out that, independently of the dispersion on all the evidence available, E171 can no longer be considered as safe used, all obtained values do not differ importantly from the values obtained when used as a food additive” (EFSA Panel on Food Additives and when TEM approaches were used (382.07 nm). Flavourings (FAF) et al., 2021). As observed on Fig. 4a–d, in terms of size distribution in number per- Nevertheless, PET(Ti)NPLs samples can result very useful for specific centage, the curves whether on water or RPMI supplemented media almost purposes. The need to detect the fate of MNPLs in complex environmental overlap, and the Z-average shows no significant differences except for the matrices has been outlined as a special challenge. In the same way, to deter- behavior of PET(Ti)NPLs on supplemented RPMI, when compared with mine the fate of MNPLs in complex organisms like mammals is another im- water dispersion using the one-way ANOVA with Dunnett multiple compar- portant challenge. To overcome this problem, the use of metal-doped ison test with a significance of 0.05. These data indicate that the surface nanoplastics has been proposed (Mitrano et al., 2019; Clark et al., 2022). coating of the particles is relevant in terms of hydrodynamic behavior Nevertheless, these type of nanoplastics are far from the representative sec- when complex matrices must to be used. This not only apply for in vitro ondary MNPLs present in the environment. From this point of view our PET studies but mainly for in vivo studies, pointing out the need of stablish Fig. 4. Size distribution analysis of PET(Ti)NPLs on MiliQ water (a, c) and on supplemented RPMI media (b, d) are shown by DLS (a, b) and (MADLS) (c, d) on Milli-Q water (light blue line on all charts) and on the Nanogenotox dispersion protocol (green line on all charts). Additionally multiple narrows measurements (purple lines on b and d) are shown. All correlation measurements are shown at the right side of the corresponding line (e, f, respectively). 5 A. Villacorta et al. Science of the Total Environment 880 (2023) 163151 Table 1 how the simultaneous determination of the organic component of the sam- Description of hydrodynamic measurements. ple are consistent and correspond with the spectra of PET previously re- Sample Z-average PDI Z-potential⁎ ported on literature (Chen et al., 2013; Johnson et al., 2021), as well as in PET(Ti)NPLs — Milli-Q 308.00 ± 6.10 0.36 −33.10 (1) our previous work (Villacorta et al., 2022). PET(Ti)NPLs — Nanogenotox 320.00 ± 8.18 0.34 −18.23 (2) As a control, regular commercially available PET samples, as well as the PET(Ti)NPLs – RPMI – Nanogenotox 287.00 ± 16.10 0.40 −9.51 (3) nanoparticles produced from their degradation are also presented (Figs. 5a, PET(Ti)NPLs — RPMI 357.00 ± 36.50× 0.41 −6.96 (4) b). We assume that during the degradation process, a fraction of the original ⁎ Significant differences in Z-potential values were determined by the one-way material is lost. We specifically ask if part of the titanium content could be ANOVA analysis, with a Tukey's multiple comparison test. Comparisons were estab- lost during the process. To such end we determined the percentage of tita- lished between dispersion conditions (1–4) as follows: (1–2**) (1–3***), (1–4***), nium in the original film and in the resulting nanoplastic. Results indicate (2–3*), (2–4*), and (3–4ns). [ns: non-significant; *P < 0.05, ⁎⁎P < 0.01; ⁎⁎⁎P < that the titanium content of the opaque PET films was 3.83 %, which was 0.001]. x significant (P<0.05) in comparison with Milli-Q water dispersion. reduced to 2.60 % in the resulting PET(Ti)NPLs. Results for ICP-OES for the resulting PET(Ti)NPLs were very consistent and the variability among implemented dispersion protocols of nanoplastics as a good practice in replicates was low, never been higher than 1 μg/mL, and the relative stan- terms of keeping the dispersed nanoplastics in the nanoscale range. This dard deviation (RSD) was never higher than 7 %. On the other hand, for is a relevant key factor when different biological endpoints must be further opaque PET film, the differences among replicates were never higher tested. The differences in the Z-average when the BSA is added to the PET than 0.1 mg/g and the RSD was never higher than 0.6 %. We assume that (Ti)NPLs dispersion and tested on supplemented RPMI culture medium during the different ultracentrifugation steps, some titanium (possibly due may result in an electrosteric stabilization and this can be depicted not to its high weight) is lost. Nevertheless, a very important fraction remains only in the already described changes but also in the Z-potential differences associated to the final product of the degradation process. Although no in- (Table 1) where the surface charge of the particle is dramatically shifted. In dications on the potential leakage of titanium from the opaque bottles have terms of significance, and regarding the Z-potential values, only PET(Ti) been found, such plastic can suppose many problems entering in the NPLs suspended on RPMI with or without the Nanogenotox protocol are recycling processes (Tramis et al., 2021). not significantly different. When water dispersion values are compared with the other dispersion protocols, very significant differences for Z- 3.5. PET(Ti)NPLs labeling and confocal visualization potential values are observed. The particles dispersed in complex matrices tend to present a higher value of heterogeneity, which can be confirmed In this study we successfully dye PET(Ti)NPLs with the commercially by observing that the values of this type of dispersion are above 0.40 in available textile dye iDye Poly Pink by heating the nanoplastic/dye mixture terms of the polydispersity index (PDI), while the dispersions in water do allowing the swelling of the polymer at the time that allowing the dye en- not exceed this value. In both cases, the values are not excessively low. tering the polymeric matrix, achieving that within the polymeric matrix we can find both the titanium and the iDye. Particles were then resus- 3.4. Chemical determination of the PET(Ti)NPLs and Ti content quantification pended and washed on Milli-Q water to remove the excess of pigment not attached to the particles, as previously described and depicted on Fig. 6a. As the FTIR methodology is suitable for the analysis of films, suspen- iDyePET(Ti)NPLs shows a significant amount of fluorescence easily sions, and powders, we have used this tool to confirm the chemical identity observed by confocal microscopy where fields with significant particle ag- of our PET(Ti)NPLS. This approach was used not only for the characteriza- glomerates were located and the emission collected (Fig. 6b, c, d). Consis- tion of the final nanomaterial product, but also for the original commercial tent with previous reports (Karakolis et al., 2019) the fluorescence on material (opaque PET films) and for the PET(Ti) powder obtained in the PET was easily observed by this staining approach with the main difference mid-step part of the process of our PET(Ti)NPLS generation. The main rea- that in our study we use nanoplastic instead of microplastics. In a recent son of this sequential analysis was to demonstrate that not changes in the study, authors shows that a commercially available dye (Atto 647N) can original chemical (PET) nature was produced in the nano PET obtaining be used for similar approaches to stain different pristine NPLs (Nguyen process. As observed in the Fig. 5 no significant differences in the obtained and Tufenkji, 2022). It is necessary to remark that in our approach, in spectra are observed, confirming that the different steps carried out in the house made true-to-life nanoplastic have been used showing the suitability process does not modify the PET chemical nature. Thus, we can observe of our method not only for the type of polymer but also for size differences, Fig. 5. FTIR spectra of the principal peaks of the interferogram. On the left chart (a) regular (orange), opaque PET (blue), and opaque PET dust produced from degradation process spectra are shown. On the right chart (b) PETNPLs (orange) from regular PET film and PET(Ti)NPLs (blue) from opaque PET are shown. 6 A. Villacorta et al. Science of the Total Environment 880 (2023) 163151 a b c 200 nm d e 0.25 iDyePET(Ti)NPLs f PET(Ti)NPLs 0.20 Intensity (a.u.) 0.15 0.10 0.05 0.00 600 650 700 O wavelength [nm] Fig. 6. a) Staining procedure: PET(Ti)NPLs were mixed with iDye Poly pink and heated at 70 °C for 2 h, then filtered, and finally resuspended. Labeling was evaluated by confocal microscopy acquiring images of the fluorescence where iDyePET(Ti)NPLs and PET(TI)NPLs were observed agglomerated (b, c, respectively). d) When the signal for both iDye (yellow arrow) and Ti (grey arrow) were used, both signals appear together as indicative of the PET(Ti)NPLs identification. f) The emission spectra for both particles were collected and compared (green signal overlap the X-axis). due to the polydisperse nature of our material. As shown in Fig. 6d, signals hybrid composition of the obtained PET(Ti)NPLs. The orthogonal views corresponding to PET and Ti co-localize, demonstrating the mixed nature of confirm the complex structures previously observed by using electronic mi- PET(Ti)NPLs. Finally, no fluorescence signal for iDye was detectable on croscopy (whether transmission or scanning) but this time inside a cell, as fields were PET(Ti)NPLs were the only component. expected according to the complexity results obtained by flow cytometry. It is necessary to point out that not all the Ti remains attached to the PET 3.6. Cellular uptake evaluation MNPLs, since a fraction of the total is lost during the obtention process, as we describe on the Section 3.4, and quantify by ICP-OES. Nevertheless, The potential harmful effects induced by MNPLs exposure require their the colocalization signals give relevant information supporting the mixed previous internalization. There are different approaches to measure such nature of the obtained true-to-life PET(Ti)NPLs. uptake, and in this study we have used both flow cytometry and confocal microscopy. By using flow cytometry, the intensity of the side-scattered 3.7. PET(Ti)NPLs effects on cell viability light revealed that the particles were taken up in the cells, as initially pro- posed (Suzuki et al., 2007). This method has been used to detect the uptake As a final approach in the exhaustive evaluation of the characteristics of of different nanomaterials including titanium nanoparticles, by measuring the obtained true-to-life PET(Ti)NPLs, the harmful effects in different the complexity of the BEAS-2B cells (Vales et al., 2015). In our study, this human cell lines were determined in terms of cell survival. The obtained re- method has been applied to different hematopoietic cell lines to determine sults are depicted in Fig. 9. potential differences according to the used cell type. If MNPLs can cross the Despite the previous results, showing notorious differences on the up- protective barriers, they move to the general compartment (blood), take among the different selected cell lines, no significant decreases on interacting with their respective components. Thus, blood cells can be con- cell viability were noticeable for any tested concentration nor cell line. sidered as a general target to be used to determine the potential effects of This would confirm that the obtained true-to-life NPLs are not toxic, at any exposure including MNPLs and more specifically PET(Ti)NPLs. The ob- tained results are indicated in Fig. 7, for three well-known leukocytic cell lines: TK6 lymphoblastic, Raji-B lymphocytes, and THP-1 monocytic cells (Rubio et al., 2020b). As observed, the PET(Ti)NPLs uptake differs among the cell lines, leukemic monocytes (THP-1) showing the highest increase on cell complexity after exposures lasting for 24 h, and at all tested concen- trations. Contrary, the lymphoblastic TK6 show practically no uptake. This differential uptake was also reported for polystyrene NPLs using the same leukocytic cells (Rubio et al., 2020b), as well as for three different white pe- ripheral blood cells (lymphocytes, monocytes, and polymorphonuclear cells) (Ballesteros et al., 2020). Accordingly, the complexity of the uptake response must be considered in the selection of the cells to be used in any experiment aiming to evaluated hazardous effects of MNPLs. As indicated, cell uptake can also be determined by using confocal mi- croscopy, as observed in Fig. 8. For this approach we focus our attention Fig. 7. Cellular internalization of PET(Ti)NPLs by Raji-B, TK6, and THP-1 cells after on THP-1 cells since this cell line showed the highest uptake in the flow cy- exposures lasting for 24 h. Graphs show the mean ± SEM of three different tometry approach. Interesting, we could colocalize both the signal coming experiments performed in duplicates. Complexity was compared with untreated from the emission of the iDye on the iDyePET(Ti)NPLs and the signal cells using a One-way ANOVA analysis with Dunnett multiple comparisons post- from Ti. This is observed when Fig. 8b and c are compared, indicating the test. (**P < 0.01; ***P < 0.001). 7 A. Villacorta et al. Science of the Total Environment 880 (2023) 163151 Fig. 8. Confocal microscopy uptake detection of iDyePET(Ti)NPLs in THP-1 cells, following 24 h exposure to 100 μg/mL. a) Control, untreated cells. b) Orthogonal view of THP1 cell with the Ti nanoparticles channel off and showing only iDye signal (yellow arrow). c) Co-localization of the Ti signal (grey arrow) with the PETNPLs emission. Arrows indicate both iDye and Ti signals. d) Imaris image reconstruction. least in the used hematopoietic cell lines and detecting effects on cell viabil- TiO2NP used as food additive concluded that not toxic/harmful were iden- ity. This would agree with previous experiments exposing these cell lines to tify, but a concern on their potential genotoxicity cannot be ruled out (EFSA polystyrene nanoparticles, where not viability effects were observed in that Panel on Food Additives and Flavourings (FAF) et al., 2021). Accordingly, range of exposures lasting for 24 h, and only mild effects when concentra- our data would agree with the assumed non-harmful effects of PET(Ti) tions range to high levels such as 200 μg/mL (Rubio et al., 2020b). The NPLs exposure. It is obvious that cell viability is just the first step in the eval- lack of effects on cell viability of PET(Ti)NPLs is like our previous report uation of the potential toxicity of PET(Ti)NPLs. Consequently, further ex- using true-to-life PETNPLs obtained from sanding water bottles material tensive studies using a wide set of biomarkers are required to ruled out (Villacorta et al., 2022), and from other secondary nano PET particles ob- the existence of any type of harmful effects associated to the secondary tained following other degradation procedures (Magrì et al., 2018; Dhaka MNPLs resulting from the degradation of opaque plastic goods. et al., 2022). Although the PET component is not considered as potentially toxic, what about the other component (TiO2NPs)? It should be remem- 4. Conclusions bered that TiO2NPs were authorized as a food additive (E171) in the EU, according to Annex II of Regulation (EC) No 1333/2008. A recent review True-to-life representative MNPLs, resulting from the degradation of en- conducted by the European Food Safety Authority (EFSA) on the safety of vironmental plastic waste, are urgently required to be used as a model for determining their potential hazards. In this context, the content of this study reports the obtention and characterization of MNPLs resulting from the degradation of opaque milk plastic bottles [PET(Ti)NPLs] which contain TiO2NPs. This is the first study reporting the obtention/characterization of this type of MNPLs. In addition to the multiple physicochemical assays de- termining the hybrid chemical nature of the obtained material, size and shape confirmed their nano range size. The preliminary studies on their bi- ological effects show an important but variable cell uptake, depending on the used cell line. Although no significant cell viability effects were ob- served in any of the used cell lines, further and complex studies are neces- sary to have a clear view of their potential health hazards. It must be pointed out that confocal microscopy images reported that both, in the dis- persion media or internalized inside the cells, PET and Ti signals colocalize. This offers an important advantage to the obtained NPLs since they can be used in in vivo studies to demonstrate their fate inside the organism, thanks to the easy identification/localization/quantification of internalized Ti. Fig. 9. Viability of hematopoietic THP-1, TK6, and Raji-B cells after exposures to PET(Ti)NPLs at concentrations ranging from 0 to 100 μg/mL for 24 h. The graph represents the viability relative to untreated cells. Graphs show the mean ± SEM CRediT authorship contribution statement of three different experiments performed in duplicates. One-way ANOVA analysis with Dunnett multiple comparisons post-test with a confidence level of 95 % was RM and AH planned the experiments. AV, LV, MMR, RLC, LR, and MA performed. carried out the experimental part. AV analyzed the data, carried out the 8 A. Villacorta et al. Science of the Total Environment 880 (2023) 163151 statistical analysis, and prepared tables/figs. AV, RM, and AH wrote the terephthalate plastics. A review. Environ. Chem. Lett. 20 (3), 1777–1800. https://doi. org/10.1007/s10311-021-01384-8. final manuscript. Dusza, H.M., van Boxel, J., van Duursen, M.B.M., Forsberg, M.M., Legler, J., Vähäkangas, K.H., 2023. Experimental human placental models for studying uptake, transport and tox- icity of micro- and nanoplastics. Sci. Total Environ. 860, 160403. https://doi.org/10. Data availability 1016/j.scitotenv.2022.160403. European Commission (EC), 2022. Recommendation on the nanoparticle definition. 10-6 Data will be made available on request. https://ec.europa.eu/environment/chemicals/nanotech/pdf/C_2022_3689_1_EN_ACT_ part1_v6.pd. EFSA Panel on Food Additives and Flavourings (FAF)Younes, M., Aquilina, G., Castle, L., Engel, K.H., Fowler, P., Frutos Fernandez, M.J., Fürst, P., Gundert-Remy, U., Gürtler, R., Declaration of competing interest Husøy, T., Manco, M., Mennes, W., Moldeus, P., Passamonti, S., Shah, R., Waalkens- Berendsen, I., Wölfle, D., Corsini, E., Cubadda, F., De Groot, D., FitzGerald, R., Gunnare, S., Gutleb, A.C., Mast, J., Mortensen, A., Oomen, A., Piersma, A., Plichta, V., Ulbrich, B., The authors declare that they have no known competing financial inter- Van Loveren, H., Benford, D., Bignami, M., Bolognesi, C., Crebelli, R., Dusinska, M., ests or personal relationships that could have appeared to influence the Marcon, F., Nielsen, E., Schlatter, J., Vleminckx, C., Barmaz, S., Carfí, M., Civitella, C., work reported in this paper. Giarola, A., Rincon, A.M., Serafimova, R., Smeraldi, C., Tarazona, J., Tard, A., Wright, M., 2021. Safety assessment of titanium dioxide (E171) as a food additive. EFSA J. 19 (5), e06585. https://doi.org/10.2903/j.efsa.2021.6585. Acknowledgments Fronza, B.M., Lewis, S., Shah, P.K., Barros, M.D., Giannini, M., Stansbury, J.W., 2019. Modifi- cation of filler surface treatment of composite resins using alternative silanes and func- tional nanogels. Dent. Mater. 35 (6), 928–936. https://doi.org/10.1016/j.dental.2019. A. Villacorta was supported by PhD fellowships from the National Agency 03.007. for Research and Development (ANID), CONICYT PFCHA/DOCTORADO Gigault, J., El Hadri, H., Nguyen, B., Grassl, B., Rowenczyk, L., Tufenkji, N., Feng, S., Wiesner, BECAS CHILE/2020-72210237. L. Vela was supported by PhD fellowships M., 2021. Nanoplastics are neither microplastics nor engineered nanoparticles. Nat. from the Fundación Carolina. L. Rubio hold a postdoctoral Juan de la Cierva Nanotechnol. 16 (5), 501–507. https://doi.org/10.1038/s41565-021-00886-4. 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