Temperature Dominance in Governing Nanoplastic Release and Leachate Composition from Polylactic Acid–Based Disposable Plastics

Temperature Dominance in Governing Nanoplastic Release and Leachate Composition from Polylactic Acid–Based Disposable Plastics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Temperature Dominance in Governing Nanoplastic Release and Leachate Composition from Polylactic Acid–Based Disposable Plastics Mingliang Fang, Ao Guo, Xing Chen, Tong Yang, Ailin Zhao, Jing Yang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8327196/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Nanoplastics released from biodegradable plastics have raised concern, yet their composition and release behavior remain largely unclear. Using polylactic acid (PLA)–based disposable containers, a common alternative to conventional plastics, we developed a quantitative analytical workflow to differentiate and characterize PLA-leached chemicals (PLCs), including PLA nanoparticles (NPs), PLA oligomers (OLAs), and lactic acid (LA). Simulated use of disposable cups (DCs) showed that PLA-DCs released ~6 million particles mL-1 NPs into water, substantially higher than conventional polypropylene (PP) DCs. More interestingly, up to 55% of detected NPs were OLA self-assembled aggregates rather than PLA NPs. Across use scenarios, water temperature was the dominant determinant: PLC concentrations increased nearly two orders of magnitude from 50 to 70 °C, accompanied by a shift from particulate to dissolved OLAs. Integrating national use behaviors with release parameters, global annual PLC exposure from PLA-DCs is projected to increase by 2 folds from 2021 to 2030. Although U.S. coffee cups account for 49% of PLA-DCs, hot coffee consumption contributed >99% of exposure, whereas in China, despite coffee cup consumption being only 20% of the U.S., hot water use in PLA-DCs still resulted in ~80% of the U.S. exposure. These findings highlight the need to establish NP and oligomer release as a new benchmark for evaluating biodegradable disposable plastics and to redefine safe-use temperature thresholds. Physical sciences/Chemistry/Polymer chemistry/Biopolymers Physical sciences/Chemistry/Analytical chemistry Physical sciences/Nanoscience and technology/Nanotoxicology/Regulation and risk management Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Plastics play an indispensable role in modern society 1 . The global production has grown exponentially—reaching 410 million tons in 2023 and projected to rise to 1.2 billion tons annually by 2060 2,3 . To mitigate the long-term accumulation of plastic-related pollutants that ultimately threaten “One Health”, biodegradable plastics have been proposed as a promising alternative to conventional petroleum-based materials 4-6 . By 2024, their global production reached 2.47 million tons, with polylactic acid (PLA) accounting for 67% of total output and dominating the biodegradable plastics market 7,8 . Derived from renewable resources and characterized by excellent mechanical strength and industrial compostability, PLA is widely used in packaging, medical, textile, and agricultural applications, and its adoption in food-contact materials such as disposable cups has grown rapidly 9,10 . Driven by global plastic restriction policies, PLA-based disposable cups (PLA-DCs) are gradually replacing traditional polypropylene (PP) products 11,12 . In China, the proportion of PLA-DCs in the total paper cup market increased to 42% in 2024 13 . Previous studies have demonstrated that plastic-based food containers can release substantial amounts of leachates into beverages, particularly microplastics and nanoplastics 14-16 . Li et al. reported that PP infant feeding bottles released up to 16.2 × 10 6 microplastics per liter during formula preparation 17 . Yang et al. detected 1.8 × 10 5 particles from PLA paper cups under typical hot beverage preparation conditions 18 . Analyses of 36 plastic food packages revealed that most contained compounds disrupting endocrine and metabolic functions 19 . The leachates have raised concerns about potential toxicological risks associated with exposure 20,21 . Chemicals from DCs have shown dose-dependent adverse effects on fetal development and maternal physiology 22 . Recent evidence further indicates that plastics can release oligomers—short-chain degradation fragments derived from polymer backbones 23 . These compounds typically contain 2-40 repeating units, with molecular weights much lower than those of their parent polymers 24 . A field investigation found PLA oligomers (OLAs) in soils under PLA-based mulch films at 16.10 ng g -1 using liquid chromatography–tandem mass spectrometry (LC–MS/MS) 25 . In vivo experiments have shown that OLAs can form from nanoplastics under enzymatic catalysis and induce acute intestinal inflammation 26 . Oligomers can exist in both dissolved and self-assembled particulate forms in water 26 . Yang et al. showed that 34–89% of nanoparticles (NPs) released from polyester textiles during washing were actually water-insoluble oligomers, indicating that NPs from plastics are not solely polymer NPs but also contain oligomeric species—yet this fraction has been largely overlooked 27 . Toxicity studies further suggest that oligomers may be more hazardous than polymer particles: OLA exhibited higher lethality than PLA microplastics in zebrafish, and PCL oligomers were more toxic than PCL particles in Daphnia magna and neurons 23,28 . Thus, resolving NP chemical composition provides a critical foundation for subsequent mechanistic and toxicological evaluation. Current analytical approaches cannot effectively differentiate OLA NPs from PLA NPs due to their similar size and optical signatures, limiting our understanding of their compositional identity and true abundance 29 . Given the rapid growth of PLA product consumption, systematic investigation is urgently needed to elucidate the composition of PLA-leached chemicals (PLCs), use condition–dependent release behaviors, and associated global exposure patterns 30 . In this study, PLA-DCs were selected as a representative biodegradable plastic container. Nanoparticle tracking analysis (NTA) was employed to compare NP release between PLA-DCs and PP-DCs, followed by a selective dissolution strategy to differentiate OLA NPs from PLA NPs. On this basis, we established an integrated analytical workflow combining ultrafiltration, alkaline depolymerization to lactic acid (LA), and LC–MS/MS detection, enabling comprehensive and accurate quantification of PLCs (PLA NPs, OLAs, and LA). Typical daily-use scenarios, including pre-cleaning before use, varying standing times, drinking water temperatures, and beverage types, were further examined to evaluate their impacts on the concentrations and compositions of leached PLCs. Coupling market-scale data of PLA-DCs with beverage consumption statistics, global and regional exposure burdens associated with drinking habits were estimated. Collectively, this work provides the first distinction and quantitative resolution of PLCs released from PLA-DCs, reveals temperature-dominated release processes and exposure differences driven by user behaviors, and offers new insights for evaluating the safety and sustainability of biodegradable plastics through chemical release. Results Release of nanoparticles from PLA -DCs during use Representative DCs used in the experiments were purchased from top-selling brands on major Chinese online retail platforms. NP release from the selected PLA-DCs was evaluated by NTA. The cups were filled with ultrapure water and maintained at 50 °C for 4 h to simulate typical drinking conditions (Fig. 1a), and the measured NP concentrations in PLA-DCs were (5.17–6.83) × 10 6 particles mL -1 (Fig. 1b). In contrast, representative PP-DCs released fewer NPs ((3.77–4.97) × 10 6 particles mL -1 ) under the same conditions, with a significant difference from PLA observed at 2 h (p = 0.005, two-tailed Student’s t test). In addition, the NPs released from PLA-DCs exhibited an average hydrodynamic diameter of 183 nm, slightly smaller than the 224 nm measured for those released from PP-DCs (Supplementary Fig. 1). PLA-DCs released a greater number of small nanoparticles than non-degradable DCs. Moreover, the PLA-derived NPs displayed complex behaviors in cups, with their concentrations first increasing and then declining as the leaching time progressed. The surface morphologies of PLA-DCs and PP-DCs before and after testing were examined using scanning electron microscopy (SEM) (Figs. 1c–f). The inner surface of new PLA-DCs was rough and densely covered with small particles, which almost completely detached after 4 h, concurrently with extensive cracking. The surface of the PP-DCs was relatively smooth and uniform, exhibiting almost no morphological changes. Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC) revealed no detectable changes in functional groups or molecular weight after 4 hours of water standing in the cups (Supplementary Figs. 2 and 3). The elevated PLA NP levels are mainly attributable to the leaching of residual surface particles, rather than the formation of new particles via hydrolysis during use. Nanoparticles released from PLA-DCs comprised PLA and self-assembled OLAs Oligomers were observed to self-assemble into NPs through hydrophobic interactions, revealing that the NPs released from PLA-DCs contain PLA polymer NPs and self-assembled OLA NPs 26 . To distinguish these two components, we developed a selective dissolution method followed by NTA analysis (Fig. 2a). Given the higher polarity and solubility of oligomers than polymers, we dissolved self-assemble OLA NPs into free OLAs using organic solvents, whereas the insoluble PLA NPs were retained 24 . The particle number concentrations of original (C ori ) and residual (C res ) NPs were quantified by NTA. The dissolution efficiencies of NPs in methanol (MeOH), isopropanol (IPA), ethanol (EtOH), and acetonitrile (ACN) were evaluated using mixed synthetic OLA standard and PLA NP reference standard (100 nm in diameter). Compared with water, 40% (v/v) MeOH, IPA, and ACN all induced pronounced dissolution of PLA NPs, resulting in low recoveries (22.71%–26.81%) (Fig. 2b). In contrast, 40% EtOH showed minimal dissolution of PLA NPs and incomplete dissolution of OLA NPs, whereas 60% EtOH completely dissolved OLA NPs and yielded a PLA NP recovery of 101.11 ± 4.94 % (mean ± s.d., n = 3) (Supplementary Fig. 4). Meanwhile, replacing the solvent did not affect the quantification linearity, accuracy, or the particle size distribution (Fig. 2c and Supplementary Fig. 5). Typical drinking water conditions were further simulated using PLA-DCs including PLA-coated paper DCs (PLA-CP-DCs) and PLA injection-molded DCs (PLA-IM-DCs), which were filled with water and maintained at 30, 50, and 70 °C for 1 h, respectively. Two types of NPs were detected in all samples, with OLA NPs at (1.73-3.87) × 10 6 particles mL -1 and PLA NPs at (2.37-5.80) × 10 6 particles mL -1 (Fig. 2d), revealing that the occurrence of OLA NPs is a general phenomenon in PLA-DCs. Temperature differentially affected the two NP types in water, with PLA NP concentrations increasing steadily and OLA NP concentrations first rising and then decreasing. In PLA-CP-DCs, OLA NPs accounted for 40.62% of the total NPs at 30 °C, comparable to PLA NPs. However, the proportion decreased to 32.03% at 70 °C, significantly lower than that of PLA NPs (p = 0.003, two-tailed Student’s t-test). These differences result from the distinct nature of the particles: PLA NPs are polymeric fragments with temperature-independent solubility, while OLA NPs are reversible aggregates that dissociate into free molecules in water as temperature increases. After dissolving OLA NPs, the measured particle size distribution also shifted: the peak diameter at 50 °C increased from 105 to 145 nm in PLA-CP-DCs and from 165 to 185 nm in PLA-IM-DCs (Figs. 2e, f). In addition, residual NPs with larger sizes were also observed after dissolution with 60% EtOH at 30 and 70 °C, indicating that this phenomenon is robust across temperatures (Supplementary Fig. 6). Therefore, OLA NPs are generally smaller than PLA NPs, which also explains the slightly smaller overall NP size observed in PLA-DCs compared with PP-DCs, partly due to the contribution of OLA NPs. Comprehensive quantitative analytical framework for PLA nanoparticles , oligomers, and lactic acid The number concentration of NPs already reflects the non-negligible amount of substances leached from PLA-DCs. During use, three types of PLCs are generated in water: free LA, OLAs in both dissolved and particulate forms, and PLA NPs (Fig. 3a). For the quantification of free LA concentration (C LA ), samples were directly freeze-dried, re-dissolved in ACN, and subjected to instrumental analysis with 13 C 3 -LA as an internal standard (Supplementary Fig. 7). Method validation showed good linearity between 20-20,000 ng mL -1 , with instrumental detection limit (IDL) of 2.03 ng mL -1 , method detection limit (MDL) of 3.54 ng mL -1 , and method quantification limit (MQL) of 11.26 ng mL -1 (Supplementary Table 1). No detectable background above MQL was observed in procedural blanks. The spike recoveries at the three concentration levels were 97.03–108.90%. The insolubility of both OLA and PLA NPs in water and their high molecular weights rendered direct MS quantification impractical 32 . To enable accurate and unified quantification, an integrated analytical workflow was developed based on alkaline depolymerization to fully hydrolyze each component into LA, whose yield was quantified by LC-MS/MS. PLA and OLAs share the same LA repeating unit but differ in their degree of polymerization and molecular weight. Quantification of OLAs and PLA NPs required efficient separation of the two fractions. To accurately quantify OLAs without interference from PLA NPs, a selective separation strategy was developed. As OLAs generally have molecular weights below 10 kDa, 60% EtOH was applied to selectively dissolve OLA NPs, followed by ultrafiltration to remove insoluble PLA NPs 33 . Spiking tests with a mixed OLA standard solution showed partial adsorption of oligomers on the membrane, yielding OLA recoveries of 30.44–86.30% after a single filtration. The following two EtOH washes increased recoveries to 60.96–86.30%, with no further improvement upon additional washing (Fig. 3b). In addition, depolymerization tests of OLAs and PLA were conducted in an autoclave at 95 °C (0.1 MPa), 121 °C (0.12 MPa), and 134 °C (0.21 MPa) using 0, 0.5, 1, and 2% (w/v) NaOH solutions, respectively (Fig. 3c and Supplementary Fig. 8). Near-maximal LA yields were achieved under 1% NaOH at 121 °C for 4 h, which was selected as the treatment condition in this study. Under this condition, the depolymerization efficiency of OLAs reached 92.56%. The depolymerized LA concentration (C LA+OLA ), representing the total amount of OLAs and free LA, was then determined. The concentration of particulate OLAs was estimated using particle size distributions and number concentrations obtained by NTA, together with the density of PLA material, assuming spherical geometry. Details of the calculation are provided in Supplementary Note 1. Water samples were freeze-dried and redissolved in dichloromethane (DCM) to dissolve all PLCs while minimizing residual PLA NPs. During depolymerization, the efficiency for PLA particles was 64.02%, likely due to their high degree of polymerization and large physical size, which limited hydrolysis (Fig. 3d and Supplementary Fig. 9). To correct this, a linear calibration curve (0.1-100 μg mL -1 , R 2 = 0.9999) was established using the PLA NP standard (Supplementary Fig. 10). The depolymerized LA concentration (C LA+OLA+PLA ) obtained by LC–MS/MS, representing the total amount of PLA NPs, OLAs, and free LA, was then determined. The PLA particle fraction was calculated as the difference between the C LA+OLA+PLA and C LA+OLA . Quality assurance and quality control (QA/QC) validation yielded MDL and MQL values of 2.69 and 8.56 ng mL -1 for OLAs, and 12.45 and 39.60 ng mL -1 for PLA NPs. The corresponding recoveries were 70.47–77.06% for OLAs and 98.20–98.34% for PLA NPs. These results demonstrate that the developed method enables effective separation and precise quantification of individual PLC components. Temperature-dependent release behavior of P LA-leached chemicals The developed analytical method was applied to examine all PLCs from PLA-DCs at different temperatures. Ultrapure water at 50 °C and 70 °C was maintained in the cups for 6 h, and samples were collected at 0.5, 1, 2, 4, and 6 h to quantify the LA, OLAs, and PLA NPs. The amounts and temporal variation trends of PLCs differed across temperatures. At 50 °C, LA concentrations remained low (<100 ng mL -1 ) throughout the 6 h, showing only minor fluctuations (Fig. 4a). OLAs exhibited a modest upward trend over time, while PLA NPs increased steadily and reached 1.09 × 10 3 ng mL -1 at 6 h. The continuous increase in total PLC concentrations indicates that release during this stage was dominated by leaching. At 1 h, the total concentration of PLCs was 7.79 × 10 2 ng mL -1 , comprising 45.7% PLA particles, 46.9% OLAs, and 7.4% LA (Fig. 4b). Among OLAs, the dissolved (25.1%) and particulate (21.8%) fractions were comparable, with the dissolved form being slightly higher. At 70 °C, LA concentration increased steadily over time, reaching a level threefold higher than the 0.5 h value after 6 h (Fig. 4c). In contrast, OLAs exhibited a rapid initial release within the first 0.5 h followed by a gradual decline, and these complementary kinetic trends suggest the potential hydrolytic conversion of OLAs to LA. PLA NPs peaked at 2.85 × 10 4 ng mL -1 at 1 h, earlier than at 50 °C, and then slowly decreased. The elevated temperature accelerates diffusion and release, shifting the overall peak of the release process to earlier time points. The amount of PLCs increased markedly at 70 °C, with concentrations nearly one to two orders of magnitude higher than those at 50 °C, reaching a total leachate concentration of 4.66 × 10 4 ng mL -1 at 1 h, comprising 61.1% PLA NPs, 37.4% OLAs, and 1.5% LA (Fig. 4d). Among the OLAs, the particulate fraction accounted for only 4.0%, while the dissolved fraction reached 33.4%, indicating that elevated temperature strongly promotes the dissolution and dispersion of OLAs. The temperature of water in the cups affected not only the total release of PLCs but also their kinetic profiles, compositional ratios, and the equilibrium among different leachate components. Compared with 50 °C, the fraction of PLA NPs at 70 °C was higher, whereas the proportion of particulate OLAs decreased markedly, consistent with the non-monotonic variation in OLA NP concentration observed above. Elevated temperature accelerated polymer chain hydrolysis, promoting ester bond cleavage within PLA NPs and OLAs, and facilitating the transformation of OLAs from particulate to dissolved forms. Short-chain oligomers released under various simulated use conditions As revealed above, oligomers represent a major fraction of PLCs. Short-chain PLA oligomers with lower molecular weights may pose higher oral exposure and health risks, highlighting the need to elucidate their release characteristics under more realistic use conditions 34 . Seven representative short-chain OLAs with degrees of polymerization of 2, 4, 6, 7, 8, 10, and 12 (denoted as OLA 2 , OLA 4 , OLA 6 , OLA 7 , OLA 8 , OLA 10, and OLA 12 , respectively) were selected. Their release behavior was systematically investigated under four practical drinking-use scenarios: pre-cleaning before use, varying standing times before drinking, different water temperatures, and different types of beverages. The short-chain OLAs were quantified by LC–MS/MS using synthesized standards and their deuterated analogs as internal standards. Method validation yielded MDLs and MQLs of 0.11–1.99 and 0.35–6.33 ng mL -1 , respectively, with recoveries of 83.35–99.76%, demonstrating high sensitivity and accuracy (Supplementary Table 2). The effect of beverage type on the release of OLAs was examined according to the EU Commission Regulation on food simulants (Figs. 5a, b) 35 . A 3% (w/v) acetic acid solution was used to simulate acidic beverages such as juice and yoghurt, a 20% (v/v) EtOH solution represented low-alcohol and organic beverages such as beer and coffee, and ultrapure water served as the control. At room temperature, the total concentration of released short-chain OLAs in 3% acetic acid and 20% EtOH was 1.51 and 2.26 times that in water, respectively, indicating that the promoting effects of different beverage media followed the order: beer > juice > water. Acidic media may accelerate ester bond cleavage at chain ends, whereas EtOH increases the solubility and mobility of oligomers 36,37 . The leaching of PLCs is highly dependent on temperature. The concentrations of short-chain OLAs were monitored in cups at 30 °C and 70 °C over time intervals of 0.5, 1, and 2 h, respectively (Figs. 5c, d). The total concentration of short-chain OLAs increased from 2.80-3.87 ng mL -1 at 30 °C to 416.36-473.43 ng mL -1 at 70 °C, approximately a 150-fold enhancement. Among the seven OLAs, those with lower degrees of polymerization (DP) exhibited higher release levels than higher-DP oligomers, with OLA 2 being the predominant species at both 30 °C and 70 °C, while the relative abundance of OLA 8 increased at 70 °C, indicating that shorter-chain OLAs diffuse and release more readily and that elevated temperature exerts differential effects on individual OLA species. The effect of standing time before drinking on the release of short-chain OLAs was examined by extending the monitoring period to 4, 8, and 12 h, respectively (Supplementary Fig. 11). At 30 °C, the total concentration showed a fluctuating increase, reaching up to 3.9 times the initial level after 12 h, whereas at 70 °C, OLA 2 , OLA 4 , and OLA 12 remained relatively stable, while the other four OLAs increased continuously. This kinetics contrasts with the overall decline in total OLAs at 70 °C and reflects dynamic transformations among oligomeric fractions in water, driven by the stepwise hydrolysis of longer-chain oligomers into shorter, more soluble species 23 . Pre-cleaning, a common practice before drinking, was further evaluated for PLA-DCs to assess its impact on short-chain OLAs release. At 30 °C, pre-cleaning reduced the total concentration of OLAs to 47.19–59.13% of the level without pre-cleaning, whereas at 70 °C, it slightly increased to 117.76–120.62%. These results suggest that pre-cleaning effectively reduces OLAs release at lower temperature but cannot mitigate the drastic leaching driven by high temperature. Nevertheless, it remains a practical usage strategy to lower exposure during drinking. The release levels of short-chain OLAs under different practical usage scenarios correspond to variations in the oral exposure of drinkers. Temperature exerted the strongest influence, followed by beverage type and standing time, while pre-cleaning had a comparatively minor effect (Fig. 5e). High temperature and alcoholic or acidic beverages markedly promoted OLA migration, whereas pre-cleaning suppressed release when applied before use. To reduce potential exposure, several practical measures are proposed: (1) rinse with cold water before use, (2) avoid hot beverages, (3) shorten drinking duration, and (4) limit acidic or alcoholic drinks (Supplementary Fig. 12). These recommendations provide practical guidance for safer use of PLA-DCs. Global and Behavioral Drivers of Chemical Exposure from PLA-DCs Driven by the promotion of biodegradable materials and eco-friendly policies, PLA has become the most widely used biodegradable plastic, increasingly replacing petroleum-based plastics in DC market (Supplementary Fig. 13). With PLA-DCs accounting for 42% of total demand in China in 2024 and projected to reach 55% by 2030, evaluating PLC exposure during daily use is essential 13 . To estimate PLC exposure from the use of PLA-DCs, we assumed a cup volume of 250 mL and a drinking duration of 0.5 h. Global market forecasts project that the DC market will reach a total value of 12.8–17.9 billion USD during 2021–2030, corresponding to an estimated annual consumption of 92.16–197.44 billion PLA-DCs worldwide (Supplementary Fig. 14) 38 . On this basis, global PLC exposure was estimated to have increased continuously from 2021 to 2024, reaching 776.25 t in 2024 and comprising 353.13 t of OLAs and 413.11 t of PLA NPs (Fig. 6a). By 2030, the total exposure is projected to rise by 67% to 1,293.97 t. Based on market shares in 2024 for the Asia-Pacific, North America, Europe, Latin America, and the Middle East and Africa, the annual regional exposure to PLCs was estimated, revealing the highest exposure in the Asia-Pacific region (284.88 t), followed by North America (218.12 t) and Europe (184.74 t), while the Middle East and Africa showed the lowest (35.71 t) (Fig. 6b and Supplementary Fig. 15). Furthermore, PLC exposure under different beverage scenarios was extrapolated from the measured concentrations of short-chain OLAs in food simulants (Supplementary Table 3). It was reported that the U.S. had the highest consumption of DCs for coffee, followed by China (Supplementary Fig. 16) 39 . The two countries differ in their drinking habits: Americans typically drink cold water except for coffee, while Chinese consumers usually drink hot water 40 . In 2018, the U.S. consumed 36.72 million PLA-DCs for coffee drinking per day, corresponding to an exposure of 842.95 kg PLCs, whereas 39.23 million used for cold water contributed only 0.54 kg (Fig. 6c). In contrast, 7.40 million PLA-DCs per day for coffee drinking were consumed across China, resulting in an exposure of 169.83 kg of PLCs, while 47.83 million cups used for hot water contributed an additional 677.52 kg. These findings further highlight that hot drinking habits related to PLA-DC use are key factors influencing PLC exposure. Considering beverage intake data for adults from 13 countries across Asia, Europe, and the Americas, per capita PLC exposure was estimated under the assumption that all beverages were consumed from PLA-DCs (Supplementary Fig. 17) 41 . Across all countries, hot beverages represented the dominant exposure source (11.02-94.58 mg day -1 ), followed by room-temperature water (0.01-0.10 mg day -1 ) (Fig. 6d). Alcoholic beverages contributed substantially in Europe, while fruit juices and dairy products were more influential in the Americas. With the increasing adoption of PLA-DCs, PLC exposure of consumers is jointly governed by product consumption volume, beverage type, and drinking habits. Discussion With the extensive promotion of biodegradable plastics, the nanoparticles and other leachates produced during their routine use have raised concern. Nanoparticle analysis showed that PLA-based disposable cups released more NPs with smaller particle sizes than PP cups, indicating that substituting petroleum-based plastics with biodegradable alternatives in food-contact applications may not represent a safer option from an NPs exposure perspective. The NPs observed in water are attributable to the leaching of residual particles left from manufacturing. OLAs not only exist in dissolved form but can also self-assemble into NPs in water. By exploiting the solubility of OLA NPs in 60% (v/v) EtOH to separate them from PLA NPs, we found that 32–55% of NPs released from PLA-DCs were oligomer-derived, suggesting that previous studies may have overestimated nanoplastic release. Since certain oligomers exhibit higher bioavailability and toxicity than polymer NPs, elucidating both the composition and physical states of leachates is critical for reliable health risk assessment 24,42 . Here, we established a comprehensive analytical workflow that integrates selective dissolution followed by ultrafiltration to separate oligomers from polymer NPs, alkaline depolymerization to monomers, and LC–MS/MS quantification. For the first time, this enables the distinction and precise characterization of leachates released from PLA-DCs (LA, OLAs, and PLA NPs), providing a more holistic and broadly applicable evaluation framework. The developed workflow was applied to simulated daily-use scenarios and demonstrated that water temperature is a major determinant of PLC release. When temperature increased from 50°C to 70°C, PLC concentrations rose by nearly two orders of magnitude, along with a higher proportion of PLA NPs and a greater conversion of particulate OLAs to dissolved forms. Notably, the OLA concentration at 70 °C reached 1.74 × 10 4 ng mL -1 , exceeding the reported exposure level of 1 × 10 4 ng mL -1 for the OLA mixture that induced mortality in zebrafish embryos, suggesting that consuming hot water in PLA-DCs may pose a non-negligible health risk. PLA-DCs are increasingly used for hot beverages such as milk tea and coffee, where temperatures often exceed 70°C, suggesting that temperature-driven exponential release may result in substantial underestimation of exposure and overlooked safety risks. The safe-use temperature limits for biodegradable disposable plastics need to be redefined, and regulatory guidelines for food-contact materials should incorporate NP and oligomer release as temperature-related benchmarks, rather than relying solely on physical performance criteria such as melting or leakage 43 . In addition to temperature, acidic or alcoholic beverages and prolonged standing further increased leachate release, whereas low-temperature pre-rinsing prior to use effectively suppressed it. A typical 150 mL PLA-DC can release up to 10.61 mg PLCs in hot water, and with the rapid expansion of PLA-DC consumption, global human exposure is estimated to rise by 2 folds over ten years. Differences in drinking habits across countries substantially influence exposure—for example, China’s preference for hot water increases exposure by nearly 4 folds, while in the U.S., hot coffee consumption contributes over 99% of total exposure—indicating that consumer use behavior should be included in exposure assessments. This study elucidated key aspects of PLC release from PLA-DCs but mainly focused on PLA NPs, OLAs, and LA. Real leachates are more complex, containing additives and other uncharacterized species, leading to incomplete chemical coverage 44 . In addition, the simulated-use experiments were performed under controlled aqueous conditions with standardized food simulants, whereas real beverages contain sugars, proteins, caffeine, and other components that can alter PLC solubility, leaching kinetics, and stability 45 . Thus, extrapolation to realistic consumption scenarios remains subject to uncertainty, and the global exposure estimation was based on simplified assumptions. Overall, this work addressed the critical need to distinguish oligomers from polymer NPs and accurately characterize PLC composition and their release behaviors. The workflow can be extended to other burgeoning biodegradable plastics such as PBAT, PCL, and PBS, facilitating evaluation of their leachate profiles and degradation dynamics, and supporting manufacturers in balancing biodegradability with consumer health protection. Based on the dominant influence of temperature and drinking habits on global exposure differences, we propose a new perspective for guiding the use specifications of biodegradable disposable plastic containers using oligomer and NP release levels, especially with respect to temperature control. We also emphasize the need to evaluate product quality under various real-life use scenarios. These insights are essential for developing regulatory policies and promoting the safe and sustainable application of environmentally friendly products. Methods Materials and chemicals Three types of 500 mL cups (PLA-CP-DC, PLA-IM-DC, and PP-DC) were purchased from the top-selling brands on Chinese online retail platforms. The polymer composition of all cups was further confirmed by FTIR, GPC, and differential scanning calorimetry (DSC) (Supplementary Fig. 18). Ultrapure water was produced using a Milli-Q Reference system (Merck, Germany; resistivity = 18.2 MΩ·cm, total organic carbon < 5 µg L -1 ). All solvents used in this study were of HPLC grade. NanoStandard Series particle size standards (100 nm diameter) used for NTA calibration were obtained from Applied Microspheres (Wiesbaden, Germany). A mixed OLA standard and seven individual short-chain OLA standards were synthesized at Fudan University, while the deuterated OLA analogs were synthesized at Nanjing University. Detailed synthesis procedures are provided in Supplementary Note 2. NP release and material characterization under simulated use To simulate NP release under typical use, 500 mL of ultrapure water preheated to 50 °C was poured to each PLA-DC and PP-DC, which were kept on a thermostatic magnetic stirrer at 50 rpm for 4 hours to mimic gentle agitation. Aliquots of 5 mL were collected at 0.5, 1, 2, and 4 h for NTA to determine NPs concentration and size distribution. A procedural blank was included to monitor background contamination. Cup walls before and after exposure were sectioned and analyzed in situ by SEM and FTIR, or dissolved in N,N-dimethylformamide (DMF) for GPC to assess physicochemical changes, with detailed characterization procedures provided in Supplementary Note 3. NP quantification and component separation NTA was used to quantify NPs based on Brownian motion to obtain particle number concentrations and hydrodynamic size distributions, but this technique cannot resolve chemical composition 46 . Measurements were performed using a ZetaView instrument (Particle Metrix, Germany). To minimize background contamination, NP counts in ultrapure water and solvents were confirmed to be <1 × 10 6 particles mL -1 , all glassware was pretreated with MeOH and ultrapure water followed by 450 °C heating for 4 h, and no plastic materials were used during handling except ultrafiltration tubes and pipette tips. Detailed analytical procedures of NTA are provided in Supplementary Note 4. A selective dissolution method was then developed to separate PLA NPs and OLA NPs released from PLA-DCs. Five organic solvents (40% MeOH, IPA, ACN, EtOH, and 60% EtOH) were evaluated, and 60% EtOH was identified as the optimal solvent, selectively dissolving OLA NPs while leaving PLA NPs intact. PLA-CP-DCs and PLA-IM-DCs were filled with 500 mL of ultrapure water at 30, 50, and 70 °C and stirred at 50 rpm for 1 h. Aliquots of 5 mL were collected to examine NP composition, levels, and temperature effects. For each sample, 3 mL was used to determine the original NP concentration (C ori ), and 2 mL was diluted 2.5-fold with 3 mL EtOH and vortexed before analyzing the residual NP concentration (C res ). The concentrations of PLA NPs and OLA NPs were calculated according to Eqs. (1) and (2): Comprehensive quantitative analysis of PLCs A comprehensive analytical workflow was developed to quantify PLCs, including free LA, OLAs, and PLA NPs. PLA-DCs were filled with 500 mL of ultrapure water at 50 °C and 70 °C and maintained at the respective temperatures for 6 h. Aliquots of 3 mL were collected at 0.5, 1, 2, 4, and 6 h from the same cup to examine the release behavior of the three PLCs under the two temperatures. For free LA quantification, 1 mL of sample was spiked with 2 µg of 13 C 3 -LA as an internal standard, freeze-dried, reconstituted in 1 mL acetonitrile, centrifuged, and analyzed by LC–MS/MS to obtain C LA . For OLAs quantification, 1 mL of sample was spiked with 2 µg of 13 C 3 -LA, freeze-dried, and redissolved in 1 mL of 60% (v/v) EtOH. The solution was ultrafiltered through a 10 kDa membrane, and the filter was rinsed twice with 1 mL EtOH. The filtrate was dried and mixed with 1 mL of 1% (w/v) NaOH solution, followed by depolymerization in an autoclave at 121 °C for 240 min. The product pH was adjusted to 7, NaCl was precipitated by refrigerated centrifugation, and monomeric LA was extracted with MeOH and analyzed by LC–MS/MS to yield C LA+OLA . For PLA NP quantification, 1 mL of sample was spiked with 2 µg of 13 C 3 -LA, freeze-dried, and dissolved in 1 mL DCM to extract all leachates. After drying, 1 mL of 1% (w/v) NaOH was added for depolymerization, followed by neutralization and desalting as described above, and the resulting LA was analyzed by LC–MS/MS to obtain C LA+OLA+PLA .The concentration of OLAs and PLA NPs was calculated according to Eq. (3) and (4), where MW [OLA] (72 g·mol -1 ) and MW LA (90 g·mol -1 ) represent the molecular weights of the [-O-CH(CH₃)-CO-] repeating unit and LA, respectively 47 . More detailed procedures for PLC quantification are provided in Supplementary Note 5. Detection of short-chain OLAs release under realistic use conditions The release behavior of short-chain OLAs from PLA-DCs was investigated under four simulated drinking scenarios: varying water temperature, standing time, beverage type, and pre-cleaning before use. To examine the effect of pre-cleaning, six PLA-DCs were evenly assigned to two groups. One group was rinsed with ultrapure water at the corresponding temperature for ~30 s, while the other was used directly. The cups were filled with ultrapure water at 30 °C or 70 °C and kept isothermal for 12 h under stirring at 50 rpm. Aliquots of 1 mL were collected at 0.5, 1, 2, 4, 8, and 12 h, spiked with 2 µg of d-OLAs, concentrated tenfold by refrigerated centrifugation, reconstituted in 100 µL ACN, centrifuged (15,000 rpm, 30 min), and analyzed by LC–MS/MS (Supplementary Fig. 19). Details of the selection of the concentration procedure are provided in Supplementary Note 6. For beverage type experiments, food simulants were prepared following European Commission Regulation (2011): 3% (w/v) acetic acid for acidic beverages, 20% (v/v) ethanol for alcoholic or organic-containing beverages, and ultrapure water as a control. At 30 °C, 500 mL of each simulant was added to the PLA-DCs and stirred at 50 rpm. Aliquots of 1 mL were collected at 0.5, 1, and 2 h, concentrated, centrifuged, and analyzed by LC–MS/MS as described above. LC–MS/MS analysis Quantification of LA and OLAs (OLA 2 , OLA 4 , OLA 6 , OLA 7 , OLA 8 , OLA 10 , OLA 12 ) was performed on an Agilent 6495 triple quadrupole MS system (Agilent Technologies, USA) operating in multiple reaction monitoring (MRM) mode. Chromatographic separation was achieved using a HILIC column (150 × 2.1 mm, 5 μm; HILICON, Sweden) for LA and a C18 column (50 × 2.1 mm, 1.8 μm; Agilent Technologies, USA) for seven short-chain OLAs (Supplementary Tables 4–7). A 200 ng mL -1 calibration standard was injected after every ten samples to monitor potential sensitivity drift, and blank solvent runs were inserted between samples to verify carryover. Quality assurance and quality control (QA/QC) were assessed based on linearity, sensitivity, and recovery. All samples pretreatment using 13 C 3 -LA or d-OLAs as internal standards. Procedural blanks and duplicate samples were included throughout sample preparation and analysis to ensure accuracy. Exposure assessment of PLCs The potential exposure to PLCs from PLA-DCs was estimated on global and regional scales, taking into account market value, cup usage, and beverage consumption data, as well as differences in drinking habits. A cup volume of 250 mL and a drinking duration of 0.5 h were assumed, consistent with the dominant global market size (151–350 mL) and typical consumer behavior 38,48 . Based on the experimentally measured PLC concentrations, the per-cup exposure was calculated. Global and regional annual exposures were estimated by multiplying the per-cup exposure with the annual PLA-DC consumption derived from the corresponding market values (Supplementary Note 7). Country-specific exposure from coffee cups in the U.S. and China was further estimated using national disposable cup consumption data (Supplementary Note 8). To evaluate the contribution from different beverage types, it was assumed that all beverages were consumed from PLA-DCs. Daily beverage consumption data for 13 countries were combined with the experimentally determined PLC concentrations in beverage simulants to calculate beverage-specific exposure (Supplementary Note 9). Statistical analysis All statistical data are presented as means ± s.d., based on at least three independent experiments. Differences between groups were evaluated using two-tailed Student’s t-tests or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. All statistical analyses were performed using Origin software (version 2025b), and differences were considered statistically significant at p < 0.05. Declarations Data availability All data are included in the manuscript and/or the Supplementary Information. These data are also available via Zenodo at https://zenodo.org/records/17633664. Detailed experimental methods, exposure-estimation procedures, and validation data for the quantification of PLCs are provided in the Supplementary Information and Supplementary Data. Source data are provided with this paper. Acknowledgments M.F. was sponsored by the National Key R&D Program (grant no. 2024YFA0918900), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB0750300), National Natural Science Foundation of China (grant no. 22376032), Agilent University Relations (ACT-UR Program, grant no. 4863) and the Xiaomi Young Investigator Award. M.S. was sponsored by the National Natural Science Foundation of China (grant nos. 22125606 and 22241604). The corresponding author Changzhi Shi is funded by the Shanghai Tongji Gao Tingyao Environmental Science & Technology Development Foundation. The authors would like to thank C. Xu from Fudan University for providing assistance with polymer material characterization. Contributions A.G., C.S., and M.F. conceived and designed the research. C.S. and M.F. jointly supervised the study. A.G. purchased the cups, performed material characterization, and prepared water samples under different simulated use conditions. A.G. and C.S. conducted the NP experiments and NTA analyses and completed data processing and exposure estimation. A.G., X.C., and J.Y. performed the mass spectrometry analyses. T.Y. and A.Z. provided technical advice on experimental design and contributed to manuscript revision. C.S., A.G., and M.F. wrote the manuscript with input from all authors, all of whom participated in the discussion and interpretation of the results. Corresponding author Correspondence to Changzhi Shi or Mingliang Fang. Ethics declarations Competing interests The authors declare no competing interests. References Cowger, W. et al. Global producer responsibility for plastic pollution. Science advances 10 , eadj8275 (2024). OECD. Global Plastics Outlook Database . OECD Publishing, Paris. https://doi.org/10.1787/c0821f81-en (2022). Houssini, K., Li, J. & Tan, Q. Complexities of the global plastics supply chain revealed in a trade-linked material flow analysis. Communications Earth & Environment 6 , 257 (2025). Rosenboom, J.-G., Langer, R. & Traverso, G. Bioplastics for a circular economy. Nature Reviews Materials 7 , 117–137 (2022). Deng, Y. et al. Potential health risks associated with biodegradable plastics and future research prospects: a focus on biodegradable microplastics. Progress in Chemistry 37 , 59–75 (2025). Monclús, L. et al. Mapping the chemical complexity of plastics. Nature 643 , 349–355 (2025). 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Physical Chemistry Chemical Physics 20 , 17839–17846 (2018) Wang, L. et al. An in situ depolymerization and liquid chromatography–tandem mass spectrometry method for quantifying polylactic acid microplastics in environmental samples. Environmental Science & Technology 56 , 13029–13035 (2022). Ranjan, V. P., Joseph, A. & Goel, S. Microplastics and other harmful substances released from disposable paper cups into hot water. Journal of Hazardous Materials 404 , 124118 (2021). Additional Declarations There is NO Competing Interest. Supplementary Files GASupplementary.docx Supplementary Information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8327196","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":569375095,"identity":"76c1175e-6203-4d2d-84f1-8bd118edc14c","order_by":0,"name":"Mingliang Fang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDACdsYHQNKGn4EhAcRlJkILM7MBkEyTbCBVy2EStOg2MzN+Lvh1XsLgePKzBwwV1okN7GcP4NVidpiZWXpm320JgzPPzA0YzqQnNvDkJRDQwn9Amrfndp3BjQQzCca2w4kNEjwGBG35zdtzTsLgRvo3CcZ/xGlhk+b5cQCoJQdoSwORWqx5G5IlJM+8KZNIOJZu3MaTQ0DL8Wbm2zx/7CT4jqdvk/hQYy3bz34GvxYwYGyDMhKAmI2wehD4Q5yyUTAKRsEoGKEAAJEVQpFbUwk/AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2204-9783","institution":"Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Mingliang","middleName":"","lastName":"Fang","suffix":""},{"id":569375096,"identity":"79789c72-bdce-4843-9d2d-57fa4cb2c246","order_by":1,"name":"Ao Guo","email":"","orcid":"","institution":"Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, China","correspondingAuthor":false,"prefix":"","firstName":"Ao","middleName":"","lastName":"Guo","suffix":""},{"id":569375097,"identity":"cf6a55d6-4579-43f9-8bc0-b44c4e609e0f","order_by":2,"name":"Xing Chen","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xing","middleName":"","lastName":"Chen","suffix":""},{"id":569375098,"identity":"a0e8191c-8d88-4ac3-95ad-18483b70a894","order_by":3,"name":"Tong Yang","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Yang","suffix":""},{"id":569375099,"identity":"bbb073a4-49de-444f-a5d6-8df2ea971b12","order_by":4,"name":"Ailin Zhao","email":"","orcid":"","institution":"Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, China","correspondingAuthor":false,"prefix":"","firstName":"Ailin","middleName":"","lastName":"Zhao","suffix":""},{"id":569375100,"identity":"9e5ea57b-e4d6-4a29-b1bf-5cc52ec48e72","order_by":5,"name":"Jing Yang","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Yang","suffix":""},{"id":569375101,"identity":"b716f84e-892b-448a-9d77-0b2f2c311fa9","order_by":6,"name":"Changzhi Shi","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Changzhi","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-12-10 11:57:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8327196/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8327196/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99798143,"identity":"d6ad962d-b88e-4492-b8c9-8e795218adbe","added_by":"auto","created_at":"2026-01-08 13:47:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13262290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNPrelease from PLA-DCs under simulated drinking water conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Schematic illustration of the experimental design for NP release from PLA-DCs and PP-DCs. Each cup was filled with 500 mL of ultrapure water at 50 °C to simulate typical drinking-water conditions, and samples were collected at 0.5, 1, 2, and 4 h. The number of NPs released during use was measured using nanoparticle tracking analysis (NTA). \u003cstrong\u003eb\u003c/strong\u003e NP concentrations in PLA-DCs and PP-DCs at 50 °C across the four sampling time points (mean ± s.d., n = 3). Statistical differences between the two cup types were evaluated using a two-tailed Student’s t-test.\u003cstrong\u003e c\u003c/strong\u003e SEM image of the inner surface of new PLA-DC. \u003cstrong\u003ed\u003c/strong\u003eSEM image of the PLA-DC surface after 4 hours of water standing. \u003cstrong\u003ee \u003c/strong\u003eSEM image of the inner surface of new PP-DC. \u003cstrong\u003ef\u003c/strong\u003e SEM image of the PP-DC surface after 4 hours of water standing. Scale bars: main images, 1 μm; insets, 200 nm.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8327196/v1/b03691dc9d4e692c44d258d6.png"},{"id":99765658,"identity":"10e9c918-62f2-4a8d-bdb1-3bc77ed8d9c3","added_by":"auto","created_at":"2026-01-08 08:03:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3700140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComposition and differentiation of NPs released from PLA-DCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Schematic of selective dissolution method for separating OLA NPs and PLA NPs. OLAs self-assemble into NPs in water but dissolve as free OLAs in organic solvents. The relative abundance of OLA NPs and PLA NPs was determined by comparing the concentrations of original NPs (C\u003csub\u003eori\u003c/sub\u003e) and residual NPs (C\u003csub\u003eres\u003c/sub\u003e). \u003cstrong\u003eb\u003c/strong\u003e Dissolution efficiency of 40% MeOH, IPA, ACN, EtOH, and 60% EtOH on PLA NP reference standard (100 nm in diameter) and mixed synthetic OLA standard (mean ± s.d., n = 3).\u003cstrong\u003ec\u003c/strong\u003e Normalized size distribution of PLA NP reference standard in ultrapure water and 60% EtOH. \u003cstrong\u003ed\u003c/strong\u003e Concentrations of OLA NPs and PLA NPs released from PLA-CP-DCs and PLA-IM-DCs at 30, 50, and 70 °C, respectively (mean ± s.d., n = 3). At 70 °C, which exceeds the tolerance temperature of PLA-IM-DCs, the concentrations of NPs were not measurable\u003csup\u003e31\u003c/sup\u003e. Statistical significance was assessed using a two-tailed Student’s t-test. \u003cstrong\u003ee\u003c/strong\u003e Normalized size distributions of NPs in PLA-IM-DCs before and after dissolution with 60% EtOH at 50 °C. \u003cstrong\u003ef \u003c/strong\u003eNormalized size distributions of NPs in PLA-CP-DCs before and after dissolution with 60% EtOH at 50 °C.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8327196/v1/25dee394e0ad5d80308b9a83.png"},{"id":99798567,"identity":"2bcad89e-7cd0-4b7b-998d-c34cedb5aa27","added_by":"auto","created_at":"2026-01-08 13:48:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3856133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComprehensive quantitative analytical workflow and performance evaluation for PLA-leached chemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eSchematic workflow for the quantitative analysis of PLCs (LA, OLAs, and PLA NPs) integrating dissolution, ultrafiltration, alkaline depolymerization, and LC–MS/MS detection.\u003cstrong\u003e b \u003c/strong\u003eRecovery rates of the mixed OLA standard with increasing numbers of EtOH washes after ultrafiltration (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test.\u003cstrong\u003ec \u003c/strong\u003eDepolymerization efficiency of the mixed OLA standard (OLA std.) at 95, 121, and 134 °C after 0, 2, 4, and 6 h of reaction, respectively (mean ± s.d., n = 3).\u003cstrong\u003ed \u003c/strong\u003eDepolymerization efficiency of the PLA NP reference standard (PLA std.) at 95, 121, and 134 °C after 0, 2, 4, and 6 h of reaction (mean ± s.d., n = 3) , respectively.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8327196/v1/3cdb2f1e6361c28d8b76a174.png"},{"id":99797763,"identity":"50b6c641-221c-479e-a692-43d0a496d7c6","added_by":"auto","created_at":"2026-01-08 13:46:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1782799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelease behavior of PLA-leached chemicals at different temperatures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Concentrations of PLCs including LA, OLAs, and PLA NPs released from PLA-DCs filled with 50 °C water for 0.5, 1, 2, 4, and 6 h, respectively. \u003cstrong\u003eb \u003c/strong\u003eCompositional distribution of PLCs from PLA-DCs filled with 50 °C water for 1 h, with the inner ring showing the proportions of dissolved and particulate OLAs.\u003cstrong\u003e c \u003c/strong\u003eConcentrations of PLCs released from PLA-DCs filled with 50 °C water for 0.5, 1, 2, 4, and 6 h, respectively. \u003cstrong\u003ed\u003c/strong\u003e Compositional distribution of PLCs from PLA-DCs filled with 70 °C water for 1 h, with the inner ring showing the proportions of dissolved and particulate OLAs. Data represent means ± s.d. of three independent replicates.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8327196/v1/8c61effadfe3241f2d422046.png"},{"id":99797798,"identity":"dd3f251f-4291-4bd6-9066-a9afc9b74f9a","added_by":"auto","created_at":"2026-01-08 13:46:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3131664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelease behavior of short-chain oligomers under various use conditions of PLA-DCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Food simulants used to represent common beverages: 3% (w/v) acetic acid for acidic drinks (juice and yoghurt), and 20% (v/v) EtOH for low-alcohol or organic beverages (beer and coffee). \u003cstrong\u003eb\u003c/strong\u003e Concentrations of short-chain OLAs released in water, simulated juice and beer at 30 °C. \u003cstrong\u003ec\u003c/strong\u003e Concentrations of short-chain OLAs released from PLA-DCs at 30 °C for 0.5, 1, and 2 h, with and without pre-cleaning. \u003cstrong\u003ed\u003c/strong\u003e Concentrations of short-chain OLAs released from PLA-DCs at 70 °C for 0.5, 1, and 2 h, with and without pre-cleaning. \u003cstrong\u003ee\u003c/strong\u003e Comparative influence of four conditions (beverage type, water temperature, standing time, and pre-cleaning before use) on the total concentration of short-chain OLAs, expressed as ratios relative to the baseline condition of 30 °C water after 1 hour. \u0026nbsp;Data represent means ± s.d. of three independent replicates.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8327196/v1/c19ed5f5098a15409c14460b.png"},{"id":99798268,"identity":"045169ad-2838-460d-b397-b1c5a50d1926","added_by":"auto","created_at":"2026-01-08 13:47:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":695809,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExposure assessment of PLCs\u003c/strong\u003e \u003cstrong\u003eduring the large-scale application of PLA-DCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Estimated global annual exposures of OLAs, PLA NPs, and total PLCs from 2021 to 2024, with projections to 2030. \u003cstrong\u003eb\u003c/strong\u003e Geographic distribution of PLC exposure in 2024, covering the Asia-Pacific, Europe, North America, Latin America, and the Middle East and Africa regions. \u003cstrong\u003ec\u003c/strong\u003e Daily consumption of coffee and non-coffee PLA-DCs in the U.S. and China, together with the corresponding daily PLC exposures. Coffee PLA-DCs were used for hot coffee, while non-coffee PLA-DCs were used for cold water in the U.S. and hot water in China. \u003cstrong\u003ed\u003c/strong\u003e Per-capita daily PLC exposure from different beverage types in 13 representative countries across Asia, Europe, and the Americas, assuming all beverages were consumed in PLA-DCs.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8327196/v1/7cc718bf77b7b952fbb0b4a6.png"},{"id":99805560,"identity":"2c6a60b9-deb5-4d26-8efa-bd050f67e988","added_by":"auto","created_at":"2026-01-08 14:16:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27296758,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8327196/v1/378144fd-9dfc-46e2-aae3-5ab8c15f9344.pdf"},{"id":99798577,"identity":"9ef91ab3-e2c2-43c7-bed0-387e478672b2","added_by":"auto","created_at":"2026-01-08 13:48:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3004233,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"GASupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8327196/v1/2fe3f1954e1bebffea3ed156.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Temperature Dominance in Governing Nanoplastic Release and Leachate Composition from Polylactic Acid–Based Disposable Plastics","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlastics play an indispensable role in modern society\u003csup\u003e1\u003c/sup\u003e. The global production has grown exponentially\u0026mdash;reaching 410 million tons in 2023 and projected to rise to 1.2 billion tons annually by 2060\u003csup\u003e2,3\u003c/sup\u003e. To mitigate the long-term accumulation of plastic-related pollutants that ultimately threaten \u0026ldquo;One Health\u0026rdquo;, biodegradable plastics have been proposed as a promising alternative to conventional petroleum-based materials\u003csup\u003e4-6\u003c/sup\u003e. By 2024, their global production reached 2.47 million tons, with polylactic acid (PLA) accounting for 67% of total output and dominating the biodegradable plastics market\u003csup\u003e7,8\u003c/sup\u003e. Derived from renewable resources and characterized by excellent mechanical strength and industrial compostability, PLA is widely used in packaging, medical, textile, and agricultural applications, and its adoption in food-contact materials such as disposable cups has grown rapidly\u003csup\u003e9,10\u003c/sup\u003e. Driven by global plastic restriction policies, PLA-based disposable cups (PLA-DCs) are gradually replacing traditional polypropylene (PP) products\u003csup\u003e11,12\u003c/sup\u003e. In China, the proportion of PLA-DCs in the total paper cup market increased to 42% in 2024\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePrevious studies have demonstrated that plastic-based food containers can release substantial amounts of leachates into beverages, particularly microplastics and nanoplastics\u003csup\u003e14-16\u003c/sup\u003e. Li et al. reported that PP infant feeding bottles released up to 16.2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e microplastics per liter during formula preparation\u003csup\u003e17\u003c/sup\u003e. Yang et al. detected 1.8 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e particles from PLA paper cups under typical hot beverage preparation conditions\u003csup\u003e18\u003c/sup\u003e. Analyses of 36 plastic food packages revealed that most contained compounds disrupting endocrine and metabolic functions\u003csup\u003e19\u003c/sup\u003e. The leachates have raised concerns about potential toxicological risks associated with exposure\u003csup\u003e20,21\u003c/sup\u003e. Chemicals from DCs have shown dose-dependent adverse effects on fetal development and maternal physiology\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eRecent evidence further indicates that plastics can release oligomers\u0026mdash;short-chain degradation fragments derived from polymer backbones\u003csup\u003e23\u003c/sup\u003e. These compounds typically contain 2-40 repeating units, with molecular weights much lower than those of their parent polymers\u003csup\u003e24\u003c/sup\u003e. A field investigation found PLA oligomers (OLAs) in soils under PLA-based mulch films at 16.10 ng g\u003csup\u003e-1\u003c/sup\u003e using liquid chromatography\u0026ndash;tandem mass spectrometry (LC\u0026ndash;MS/MS)\u003csup\u003e25\u003c/sup\u003e.\u0026nbsp;In vivo experiments have shown that OLAs can form from nanoplastics under enzymatic catalysis and induce acute intestinal inflammation\u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOligomers can exist in both dissolved and self-assembled particulate forms in water\u003csup\u003e26\u003c/sup\u003e. Yang et al. showed that 34\u0026ndash;89% of nanoparticles (NPs) \u0026nbsp; released from polyester textiles during washing were actually water-insoluble oligomers, indicating that NPs from plastics are not solely polymer NPs but also contain oligomeric species\u0026mdash;yet this fraction has been largely overlooked\u003csup\u003e27\u003c/sup\u003e. Toxicity studies further suggest that oligomers may be more hazardous than polymer particles: OLA exhibited higher lethality than PLA microplastics in zebrafish, and PCL oligomers were more toxic than PCL particles in \u003cem\u003eDaphnia magna\u003c/em\u003e and neurons\u003csup\u003e23,28\u003c/sup\u003e. Thus, resolving NP chemical composition provides a critical foundation for subsequent mechanistic and toxicological evaluation.\u0026nbsp;Current analytical approaches cannot effectively differentiate OLA NPs from PLA NPs due to their similar size and optical signatures, limiting our understanding of their compositional identity and true abundance\u003csup\u003e29\u003c/sup\u003e. Given the rapid growth of PLA product consumption, systematic investigation is urgently needed to elucidate the composition of PLA-leached chemicals (PLCs), use condition\u0026ndash;dependent release behaviors, and associated global exposure patterns\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, PLA-DCs were selected as a representative biodegradable plastic container. Nanoparticle tracking analysis (NTA) was employed to compare NP release between PLA-DCs and PP-DCs, followed by a selective dissolution strategy to differentiate OLA NPs from PLA NPs. On this basis, we established an integrated analytical workflow combining ultrafiltration, alkaline depolymerization to lactic acid (LA), and LC\u0026ndash;MS/MS detection, enabling comprehensive and accurate quantification of PLCs (PLA NPs, OLAs, and LA). Typical daily-use scenarios, including pre-cleaning before use, varying standing times, drinking water temperatures, and beverage types, were further examined to evaluate their impacts on the concentrations and compositions of leached PLCs. Coupling market-scale data of PLA-DCs with beverage consumption statistics, global and regional exposure burdens associated with drinking habits were estimated. Collectively, this work provides the first distinction and quantitative resolution of PLCs released from PLA-DCs, reveals temperature-dominated release processes and exposure differences driven by user behaviors, and offers new insights for evaluating the safety and sustainability of biodegradable plastics through chemical release.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eRelease of nanoparticles from PLA\u003c/strong\u003e\u003cstrong\u003e-DCs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;during use\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative DCs used in the experiments were purchased from top-selling brands on major Chinese online retail platforms. NP release from the selected PLA-DCs was evaluated by NTA. The cups were filled with ultrapure water and maintained at 50 \u0026deg;C for 4 h to simulate typical drinking conditions (Fig. 1a), and the measured NP concentrations in PLA-DCs were (5.17\u0026ndash;6.83) \u0026times; 10\u003csup\u003e6\u003c/sup\u003e particles mL\u003csup\u003e-1\u003c/sup\u003e (Fig. 1b).\u0026nbsp;In contrast, representative PP-DCs released fewer NPs ((3.77\u0026ndash;4.97) \u0026times; 10\u003csup\u003e6\u003c/sup\u003e particles mL\u003csup\u003e-1\u003c/sup\u003e) under the same conditions, with a significant difference from PLA observed at 2 h (p = 0.005, two-tailed Student\u0026rsquo;s t test). In addition, the NPs released from PLA-DCs exhibited an average hydrodynamic diameter of 183 nm, slightly smaller than the 224 nm measured for those released from PP-DCs (Supplementary Fig. 1).\u0026nbsp;PLA-DCs released a greater number of small nanoparticles than non-degradable DCs. Moreover, the PLA-derived NPs displayed complex behaviors in cups, with their concentrations first increasing and then declining as the leaching time progressed.\u003c/p\u003e\n\u003cp\u003eThe surface morphologies of PLA-DCs and PP-DCs before and after testing were examined using scanning electron microscopy (SEM) (Figs. 1c\u0026ndash;f). The inner surface of new PLA-DCs was rough and densely covered with small particles, which almost completely detached after 4 h, concurrently with extensive cracking. The surface of the PP-DCs was relatively smooth and uniform, exhibiting almost no morphological changes. Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC) revealed no detectable changes in functional groups or molecular weight after 4 hours of water standing in the cups (Supplementary Figs. 2 and 3). The elevated PLA NP levels are mainly attributable to the leaching of residual surface particles, rather than the formation of new particles via hydrolysis during use.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eNanoparticles released from PLA-DCs comprised PLA and self-assembled OLAs\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eOligomers were observed to self-assemble into NPs through hydrophobic interactions, revealing that the NPs released from PLA-DCs contain PLA polymer NPs and self-assembled OLA NPs\u003csup\u003e26\u003c/sup\u003e. To distinguish these two components, we developed a selective dissolution method followed by NTA analysis (Fig. 2a). Given the higher polarity and solubility of oligomers than polymers, we dissolved self-assemble OLA NPs into free OLAs using organic solvents, whereas the insoluble PLA NPs were retained\u003csup\u003e24\u003c/sup\u003e. The\u0026nbsp;particle number\u0026nbsp;concentrations of original (C\u003csub\u003eori\u003c/sub\u003e) and residual (C\u003csub\u003eres\u003c/sub\u003e) NPs were quantified by NTA.\u003c/p\u003e\n\u003cp\u003eThe dissolution efficiencies of NPs in methanol (MeOH), isopropanol (IPA), ethanol (EtOH), and acetonitrile (ACN) were evaluated using mixed synthetic OLA standard and PLA NP reference standard (100 nm in diameter). Compared with water, 40% (v/v) MeOH, IPA, and ACN all induced pronounced dissolution of PLA NPs, resulting in low recoveries (22.71%\u0026ndash;26.81%) (Fig. 2b). In contrast, 40% EtOH showed minimal dissolution of PLA NPs\u0026nbsp;and incomplete dissolution of OLA NPs, whereas 60% EtOH completely dissolved OLA NPs\u0026nbsp;and yielded a PLA NP recovery of 101.11 \u0026plusmn; 4.94 % (mean \u0026plusmn; s.d., n = 3) (Supplementary Fig. 4).\u0026nbsp;Meanwhile, replacing the solvent did not affect the quantification linearity, accuracy, or the particle size distribution (Fig. 2c and Supplementary Fig. 5).\u003c/p\u003e\n\u003cp\u003eTypical drinking water conditions were further simulated using PLA-DCs\u0026nbsp;including\u0026nbsp;PLA-coated paper DCs (PLA-CP-DCs) and PLA injection-molded DCs (PLA-IM-DCs), which were filled with water and maintained at 30, 50, and 70 \u0026deg;C for 1 h, respectively. Two types of NPs were detected in all samples, with OLA NPs at (1.73-3.87) \u0026times; 10\u003csup\u003e6\u003c/sup\u003e particles mL\u003csup\u003e-1\u003c/sup\u003e and PLA NPs at (2.37-5.80) \u0026times; 10\u003csup\u003e6\u003c/sup\u003e particles mL\u003csup\u003e-1\u003c/sup\u003e (Fig. 2d), revealing that the occurrence of OLA NPs is a general phenomenon in PLA-DCs. Temperature differentially affected the two NP types in water, with PLA NP concentrations increasing steadily and OLA NP concentrations first rising and then decreasing. In PLA-CP-DCs, OLA NPs accounted for 40.62% of the total NPs at 30 \u0026deg;C, comparable to PLA NPs. However, the proportion decreased to 32.03% at 70 \u0026deg;C, significantly lower than that of PLA NPs (p = 0.003, two-tailed Student\u0026rsquo;s t-test). These differences result from the distinct nature of the particles: PLA NPs are polymeric fragments with temperature-independent solubility, while OLA NPs are reversible aggregates that dissociate into free molecules in water as temperature increases. After dissolving OLA NPs, the measured particle size distribution also shifted: the peak diameter at 50 \u0026deg;C increased from 105 to 145 nm in PLA-CP-DCs and from 165 to 185 nm in PLA-IM-DCs (Figs. 2e, f). In addition, residual NPs with larger sizes were also observed after dissolution with 60% EtOH at 30 and 70 \u0026deg;C, indicating that this phenomenon is robust across temperatures (Supplementary Fig. 6). Therefore, OLA NPs are generally smaller than PLA NPs, which also explains the slightly smaller overall NP size observed in PLA-DCs compared with PP-DCs, partly due to the contribution of OLA NPs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComprehensive\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003equantitative\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanalytical framework for\u003c/strong\u003e \u003cstrong\u003ePLA nanoparticles\u003c/strong\u003e\u003cstrong\u003e, oligomers, and lactic acid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe number concentration of NPs already reflects the non-negligible amount of\u0026nbsp;substances leached from PLA-DCs. During use, three types of PLCs are generated in water: free LA, OLAs in both dissolved and particulate forms, and PLA NPs (Fig. 3a).\u003c/p\u003e\n\u003cp\u003eFor the quantification of free LA concentration (C\u003csub\u003eLA\u003c/sub\u003e), samples were directly freeze-dried, re-dissolved in ACN, and subjected to instrumental analysis with \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e3\u003c/sub\u003e-LA as an internal standard (Supplementary Fig. 7). Method validation showed good linearity between 20-20,000 ng mL\u003csup\u003e-1\u003c/sup\u003e, with instrumental detection limit (IDL) of 2.03 ng mL\u003csup\u003e-1\u003c/sup\u003e, method detection limit (MDL) of 3.54 ng mL\u003csup\u003e-1\u003c/sup\u003e, and method quantification limit (MQL) of 11.26 ng mL\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003e(Supplementary Table 1). No detectable background above MQL was observed in procedural blanks. The spike recoveries at the three concentration levels were 97.03\u0026ndash;108.90%.\u003c/p\u003e\n\u003cp\u003eThe insolubility of both OLA and PLA NPs in water and their high molecular weights rendered direct MS quantification impractical\u003csup\u003e32\u003c/sup\u003e. To enable accurate and unified quantification, an integrated analytical workflow was developed based on alkaline depolymerization to fully hydrolyze each component into LA, whose yield was quantified by LC-MS/MS.\u003c/p\u003e\n\u003cp\u003ePLA and OLAs share the same LA repeating unit but differ in their degree of polymerization and molecular weight. Quantification of OLAs and PLA NPs required efficient separation of the two fractions. To accurately quantify OLAs without interference from PLA NPs, a selective separation strategy was developed. As OLAs generally have molecular weights below 10 kDa, 60% EtOH was applied to selectively dissolve OLA NPs, followed by ultrafiltration to remove insoluble PLA NPs\u003csup\u003e33\u003c/sup\u003e. Spiking tests with a mixed OLA standard solution showed partial adsorption of oligomers on the membrane, yielding OLA recoveries of 30.44\u0026ndash;86.30% after a single filtration. The following two EtOH washes increased recoveries to 60.96\u0026ndash;86.30%, with no further improvement upon additional washing (Fig. 3b). In addition, depolymerization tests of OLAs and PLA were conducted in an autoclave at 95 \u0026deg;C (0.1 MPa), 121 \u0026deg;C (0.12 MPa), and 134 \u0026deg;C (0.21 MPa) using 0, 0.5, 1, and 2% (w/v) NaOH solutions, respectively (Fig. 3c and Supplementary Fig. 8). Near-maximal LA yields were achieved under 1% NaOH at 121 \u0026deg;C for 4 h, which was selected as the treatment condition in this study. Under this condition, the depolymerization efficiency of OLAs reached 92.56%. The depolymerized LA concentration (C\u003csub\u003eLA+OLA\u003c/sub\u003e), representing the total amount of OLAs and free LA, was then determined. The concentration of particulate OLAs was estimated using particle size distributions and number concentrations obtained by NTA, together with the density of PLA material, assuming spherical geometry. Details of the calculation are provided in Supplementary Note 1.\u003c/p\u003e\n\u003cp\u003eWater samples were freeze-dried and redissolved in dichloromethane (DCM) to dissolve all PLCs while minimizing residual PLA NPs. During depolymerization, the efficiency for PLA particles was 64.02%, likely due to their high degree of polymerization and large physical size, which limited hydrolysis (Fig. 3d and Supplementary Fig. 9). To correct this, a linear calibration curve (0.1-100 \u0026mu;g mL\u003csup\u003e-1\u003c/sup\u003e, R\u003csup\u003e2\u003c/sup\u003e = 0.9999) was established using the PLA NP standard (Supplementary Fig. 10). The depolymerized LA concentration (C\u003csub\u003eLA+OLA+PLA\u003c/sub\u003e) obtained by LC\u0026ndash;MS/MS, representing the total amount of PLA NPs, OLAs, and free LA, was then determined. The PLA particle fraction was calculated as the difference between the C\u003csub\u003eLA+OLA+PLA\u003c/sub\u003e and C\u003csub\u003eLA+OLA\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eQuality assurance and quality control (QA/QC) validation yielded MDL and MQL values of 2.69 and 8.56 ng mL\u003csup\u003e-1\u003c/sup\u003e for OLAs, and 12.45 and 39.60 ng mL\u003csup\u003e-1\u003c/sup\u003e for PLA NPs. The corresponding recoveries were 70.47\u0026ndash;77.06% for OLAs and 98.20\u0026ndash;98.34% for PLA NPs. These results demonstrate that the developed method enables effective separation and precise quantification of individual PLC components.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemperature-dependent release behavior of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003eLA-leached chemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe developed analytical method was applied to examine all PLCs from PLA-DCs at different temperatures. Ultrapure water at 50 \u0026deg;C and 70 \u0026deg;C was maintained in the cups for 6 h, and samples were collected at 0.5, 1, 2, 4, and 6 h to quantify the LA, OLAs, and PLA NPs.\u003c/p\u003e\n\u003cp\u003eThe amounts and temporal variation trends of PLCs differed across temperatures. At 50 \u0026deg;C, LA concentrations remained low (\u0026lt;100 ng mL\u003csup\u003e-1\u003c/sup\u003e) throughout the 6 h, showing only minor fluctuations (Fig. 4a). OLAs exhibited a modest upward trend over time, while PLA NPs increased steadily and reached 1.09 \u0026times; 10\u003csup\u003e3\u0026nbsp;\u003c/sup\u003eng mL\u003csup\u003e-1\u003c/sup\u003e at 6 h.\u0026nbsp;The continuous increase in total PLC concentrations indicates that release during this stage was dominated by leaching.\u0026nbsp;At 1 h, the total concentration of PLCs was 7.79\u0026nbsp;\u0026times; 10\u003csup\u003e2\u003c/sup\u003e ng mL\u003csup\u003e-1\u003c/sup\u003e, comprising 45.7% PLA\u0026nbsp;particles, 46.9% OLAs, and 7.4% LA (Fig. 4b). Among OLAs, the dissolved (25.1%) and particulate (21.8%) fractions were comparable, with the dissolved form being slightly higher. At 70 \u0026deg;C, LA concentration increased steadily over time, reaching a level\u0026nbsp;threefold higher than the 0.5 h value after 6 h\u0026nbsp;(Fig. 4c).\u0026nbsp;In contrast, OLAs exhibited a rapid initial release within the first 0.5 h followed by a gradual decline, and these complementary kinetic trends suggest the potential hydrolytic conversion of OLAs to LA.\u0026nbsp;PLA NPs peaked at 2.85 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e ng mL\u003csup\u003e-1\u003c/sup\u003e at 1 h, earlier than at 50 \u0026deg;C, and then slowly decreased.\u0026nbsp;The elevated temperature accelerates diffusion and release, shifting the overall peak of the release process to earlier time points.\u0026nbsp;The amount of PLCs increased markedly at 70 \u0026deg;C, with concentrations nearly one to two orders of magnitude higher than those at 50 \u0026deg;C, reaching a total leachate concentration of 4.66 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e ng mL\u003csup\u003e-1\u003c/sup\u003e at 1 h,\u0026nbsp;comprising 61.1% PLA NPs, 37.4% OLAs, and 1.5% LA (Fig. 4d). Among the OLAs, the particulate fraction accounted for only 4.0%, while the dissolved fraction reached 33.4%, indicating that elevated temperature strongly promotes the dissolution and dispersion of OLAs.\u003c/p\u003e\n\u003cp\u003eThe temperature of water in the cups affected not only the total release of PLCs but also their kinetic profiles, compositional ratios, and the equilibrium among different leachate components. Compared with 50 \u0026deg;C, the fraction of PLA NPs at 70 \u0026deg;C was higher, whereas the proportion of particulate OLAs decreased markedly, consistent with the non-monotonic variation in OLA NP concentration observed above. Elevated temperature accelerated polymer chain hydrolysis, promoting ester bond cleavage within PLA NPs and OLAs, and facilitating the transformation of OLAs from particulate to dissolved forms.\u003c/p\u003e\n\u003ch2\u003eShort-chain oligomers released under various simulated use conditions\u003c/h2\u003e\n\u003cp\u003eAs revealed above, oligomers represent a major fraction of PLCs. Short-chain PLA oligomers with lower molecular weights may pose higher oral exposure and health risks, highlighting the need to elucidate their release characteristics under more realistic use conditions\u003csup\u003e34\u003c/sup\u003e. Seven representative short-chain OLAs with degrees of polymerization of 2, 4, 6, 7, 8, 10, and 12 (denoted as OLA\u003csub\u003e2\u003c/sub\u003e, OLA\u003csub\u003e4\u003c/sub\u003e, OLA\u003csub\u003e6\u003c/sub\u003e, OLA\u003csub\u003e7\u003c/sub\u003e, OLA\u003csub\u003e8\u003c/sub\u003e, OLA\u003csub\u003e10,\u003c/sub\u003e and OLA\u003csub\u003e12\u003c/sub\u003e, respectively) were selected. Their release behavior was systematically investigated under four practical drinking-use scenarios: pre-cleaning before use, varying standing times before drinking, different water temperatures, and different types of beverages. The short-chain OLAs were quantified by LC\u0026ndash;MS/MS using synthesized standards and their deuterated analogs as internal standards. Method validation yielded MDLs and MQLs of 0.11\u0026ndash;1.99 and 0.35\u0026ndash;6.33 ng mL\u003csup\u003e-1\u003c/sup\u003e, respectively, with recoveries of 83.35\u0026ndash;99.76%, demonstrating high sensitivity and accuracy (Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003eThe effect of beverage type on the release of OLAs was examined according to the EU Commission Regulation on food simulants (Figs. 5a, b)\u003csup\u003e35\u003c/sup\u003e. A 3% (w/v) acetic acid solution was used to simulate acidic beverages such as juice and yoghurt, a 20% (v/v) EtOH solution represented low-alcohol and organic beverages such as beer and coffee, and ultrapure water served as the control. At room temperature, the total concentration of released short-chain OLAs in 3% acetic acid and 20% EtOH was 1.51 and 2.26 times that in water, respectively, indicating that the promoting effects of different beverage media followed the order: beer \u0026gt; juice \u0026gt; water. Acidic media may accelerate ester bond cleavage at chain ends, whereas EtOH increases the solubility and mobility of oligomers\u003csup\u003e36,37\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe leaching of PLCs is highly dependent on temperature. The concentrations of short-chain OLAs were monitored in cups at 30 \u0026deg;C and 70 \u0026deg;C over time intervals of 0.5, 1, and 2 h, respectively (Figs. 5c, d). The total concentration of short-chain OLAs increased from 2.80-3.87 ng mL\u003csup\u003e-1\u003c/sup\u003e at 30 \u0026deg;C to 416.36-473.43\u0026nbsp;ng mL\u003csup\u003e-1\u003c/sup\u003e at 70 \u0026deg;C, approximately a 150-fold enhancement.\u0026nbsp;Among the seven OLAs, those with lower degrees of polymerization\u0026nbsp;(DP)\u0026nbsp;exhibited higher release levels\u0026nbsp;than\u0026nbsp;higher-DP oligomers, with OLA\u003csub\u003e2\u003c/sub\u003e being the predominant species at both 30 \u0026deg;C and 70 \u0026deg;C, while the relative abundance of OLA\u003csub\u003e8\u003c/sub\u003e increased at 70 \u0026deg;C, indicating that shorter-chain OLAs diffuse and release more readily and that elevated temperature exerts differential effects on individual OLA species. The effect of standing time before drinking on the release of short-chain OLAs was examined by extending the monitoring period to 4, 8, and 12 h, respectively (Supplementary Fig. 11). At 30 \u0026deg;C, the total concentration showed a fluctuating increase, reaching\u0026nbsp;up to\u0026nbsp;3.9 times the initial level after 12 h, whereas at 70 \u0026deg;C, OLA\u003csub\u003e2\u003c/sub\u003e, OLA\u003csub\u003e4\u003c/sub\u003e, and OLA\u003csub\u003e12\u003c/sub\u003e remained relatively stable, while the other four OLAs increased continuously. This kinetics contrasts with the overall decline in total OLAs at 70 \u0026deg;C and reflects dynamic transformations among oligomeric fractions\u0026nbsp;in\u0026nbsp;water, driven by the stepwise hydrolysis of longer-chain\u0026nbsp;oligomers into shorter, more soluble species\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePre-cleaning, a common practice before drinking, was further evaluated for PLA-DCs to assess its impact on short-chain OLAs release. At 30 \u0026deg;C, pre-cleaning reduced the total concentration of OLAs to 47.19\u0026ndash;59.13% of the level without pre-cleaning, whereas at 70 \u0026deg;C, it slightly increased to 117.76\u0026ndash;120.62%. These results suggest that pre-cleaning effectively reduces OLAs release at lower temperature but cannot mitigate the drastic leaching driven by high temperature. Nevertheless, it remains a practical usage strategy to lower exposure during drinking.\u003c/p\u003e\n\u003cp\u003eThe release levels of short-chain OLAs under different practical usage scenarios correspond to variations in the oral exposure of drinkers. Temperature exerted the strongest influence, followed by beverage type and standing time, while pre-cleaning had a comparatively minor effect (Fig. 5e). High temperature and alcoholic or acidic beverages markedly promoted OLA migration, whereas pre-cleaning suppressed release when applied before use. To reduce potential exposure, several practical measures are proposed: (1) rinse with cold water before use, (2) avoid hot beverages, (3) shorten drinking duration, and (4) limit acidic or alcoholic drinks (Supplementary Fig. 12). These recommendations provide practical guidance for safer use of PLA-DCs.\u003c/p\u003e\n\u003ch2\u003eGlobal and Behavioral Drivers of Chemical Exposure from PLA-DCs\u003c/h2\u003e\n\u003cp\u003eDriven by the promotion of biodegradable materials and eco-friendly policies, PLA has become the most widely used biodegradable plastic, increasingly replacing petroleum-based plastics in DC market (Supplementary Fig. 13). With PLA-DCs accounting for 42% of total demand in China in 2024 and projected to reach 55% by 2030, evaluating PLC exposure during daily use is essential\u003csup\u003e13\u003c/sup\u003e. To estimate PLC exposure from the use of PLA-DCs, we assumed a cup volume of 250 mL and a drinking duration of 0.5 h. Global market forecasts project that the DC market will reach a total value of 12.8\u0026ndash;17.9 billion USD during 2021\u0026ndash;2030, corresponding to an estimated annual consumption of 92.16\u0026ndash;197.44 billion PLA-DCs worldwide (Supplementary Fig. 14)\u003csup\u003e38\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOn this basis, global PLC exposure was estimated to have increased continuously from 2021 to 2024, reaching 776.25 t in 2024 and comprising 353.13 t of OLAs and 413.11 t of PLA NPs (Fig. 6a). By 2030, the total exposure is projected to rise by 67% to 1,293.97 t. Based on market shares in 2024 for the Asia-Pacific, North America, Europe, Latin America, and the Middle East and Africa, the annual regional exposure to PLCs was estimated, revealing the highest exposure in the Asia-Pacific region (284.88 t), followed by North America (218.12 t) and Europe (184.74 t), while the Middle East and Africa showed the lowest (35.71 t) (Fig. 6b and Supplementary Fig. 15).\u003c/p\u003e\n\u003cp\u003eFurthermore, PLC exposure under different beverage scenarios was extrapolated from the measured concentrations of short-chain OLAs in food simulants (Supplementary Table 3).\u0026nbsp;It was reported that the U.S. had the highest consumption of DCs for coffee, followed by China (Supplementary Fig. 16)\u003csup\u003e39\u003c/sup\u003e. The two countries differ in their drinking habits: Americans typically drink cold water except for coffee, while Chinese consumers usually drink hot water\u003csup\u003e40\u003c/sup\u003e. In 2018, the U.S. consumed 36.72 million PLA-DCs for coffee drinking per day, corresponding to an exposure of 842.95 kg PLCs, whereas 39.23 million used for cold water contributed only 0.54 kg (Fig. 6c). In contrast, 7.40 million PLA-DCs per day for coffee drinking\u0026nbsp;were\u0026nbsp;consumed\u0026nbsp;across\u0026nbsp;China, resulting in an exposure of 169.83 kg of PLCs, while 47.83 million cups used for hot water contributed an additional 677.52 kg. These findings further highlight that hot drinking habits related to PLA-DC use are key factors influencing PLC exposure. Considering beverage intake data for adults from 13 countries across Asia, Europe, and the Americas, per capita PLC exposure was estimated under the assumption that all beverages were consumed from PLA-DCs\u0026nbsp;(Supplementary Fig. 17)\u003csup\u003e41\u003c/sup\u003e.\u0026nbsp;Across all countries, hot beverages represented the dominant exposure source (11.02-94.58 mg day\u003csup\u003e-1\u003c/sup\u003e), followed by room-temperature water (0.01-0.10\u0026nbsp;mg day\u003csup\u003e-1\u003c/sup\u003e) (Fig. 6d). Alcoholic beverages contributed substantially in Europe, while fruit juices and dairy products were more influential in the Americas. With the increasing adoption of PLA-DCs, PLC exposure of consumers is jointly governed by product consumption volume, beverage type, and drinking habits.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWith the extensive promotion of biodegradable plastics, the nanoparticles and other leachates produced during their routine use have raised concern. Nanoparticle analysis showed that PLA-based disposable cups released more NPs with smaller particle sizes than PP cups, indicating that substituting petroleum-based plastics with biodegradable alternatives in food-contact applications may not represent a safer option from an NPs exposure perspective.\u0026nbsp;The NPs observed in water are attributable to the leaching of residual particles left from manufacturing. OLAs not only exist in dissolved form but can also self-assemble into NPs in water. By exploiting the solubility of OLA NPs in 60% (v/v) EtOH to separate them from PLA NPs, we found that 32\u0026ndash;55% of NPs released from PLA-DCs were oligomer-derived, suggesting that previous studies may have overestimated nanoplastic release. Since certain oligomers exhibit higher bioavailability and toxicity than polymer NPs, elucidating both the composition and physical states of leachates is critical for reliable health risk assessment\u003csup\u003e24,42\u003c/sup\u003e.\u0026nbsp;Here, we established a comprehensive analytical workflow that integrates selective dissolution followed by ultrafiltration to separate oligomers from polymer NPs, alkaline depolymerization to monomers, and LC\u0026ndash;MS/MS quantification. For the first time, this enables the distinction and precise characterization of leachates released from PLA-DCs (LA, OLAs, and PLA NPs), providing a more holistic and broadly applicable evaluation framework.\u003c/p\u003e\n\u003cp\u003eThe developed workflow was applied to simulated daily-use scenarios and demonstrated that water temperature is a major determinant of PLC release. When temperature increased from 50\u0026deg;C to 70\u0026deg;C, PLC concentrations rose by nearly two orders of magnitude, along with a higher proportion of PLA NPs and a greater conversion of particulate OLAs to dissolved forms.\u0026nbsp;Notably, the OLA concentration at 70 \u0026deg;C reached 1.74 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e ng mL\u003csup\u003e-1\u003c/sup\u003e, exceeding\u0026nbsp;the reported exposure level of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e ng mL\u003csup\u003e-1\u003c/sup\u003e for the OLA mixture that induced mortality in zebrafish embryos, suggesting that consuming hot water in PLA-DCs may pose a non-negligible health risk.\u0026nbsp;PLA-DCs are increasingly used for hot beverages such as milk tea and coffee, where temperatures often exceed 70\u0026deg;C, suggesting that temperature-driven exponential release may result in substantial underestimation of exposure and overlooked safety risks. The safe-use temperature limits for biodegradable disposable plastics need to be redefined, and regulatory guidelines for food-contact materials should incorporate NP and oligomer release as temperature-related benchmarks, rather than relying solely on physical performance criteria such as melting or leakage\u003csup\u003e43\u003c/sup\u003e. In addition to temperature, acidic or alcoholic beverages and prolonged standing further increased leachate release, whereas low-temperature pre-rinsing prior to use effectively suppressed it. A typical 150 mL PLA-DC can release up to 10.61 mg PLCs in hot water, and with the rapid expansion of PLA-DC consumption, global human exposure is estimated to rise by 2 folds over ten years. Differences in drinking habits across countries substantially influence exposure\u0026mdash;for example, China\u0026rsquo;s preference for hot water increases exposure by nearly 4 folds, while in the\u0026nbsp;U.S., hot coffee consumption contributes over 99% of total exposure\u0026mdash;indicating that consumer use behavior should be included in exposure assessments.\u003c/p\u003e\n\u003cp\u003eThis study elucidated key aspects of PLC release from PLA-DCs but mainly focused on PLA NPs, OLAs, and LA. Real leachates are more complex, containing additives and other uncharacterized species, leading to incomplete chemical coverage\u003csup\u003e44\u003c/sup\u003e. In addition, the simulated-use experiments were performed under controlled aqueous conditions with standardized food simulants, whereas real beverages contain sugars, proteins, caffeine, and other components that can alter PLC solubility, leaching kinetics, and stability\u003csup\u003e45\u003c/sup\u003e. Thus, extrapolation to realistic consumption scenarios remains subject to uncertainty, and the global exposure estimation was based on simplified assumptions.\u003c/p\u003e\n\u003cp\u003eOverall, this work addressed the critical need to distinguish oligomers from polymer NPs and accurately characterize PLC composition and their release behaviors. The workflow can be extended to other burgeoning biodegradable plastics such as PBAT, PCL, and PBS, facilitating evaluation of their leachate profiles and degradation dynamics, and supporting manufacturers in balancing biodegradability with consumer health protection.\u003c/p\u003e\n\u003cp\u003eBased on the dominant influence of temperature and drinking habits on global exposure differences, we propose a new perspective for guiding the use specifications of biodegradable disposable plastic containers using oligomer and NP release levels, especially with respect to temperature control. We also emphasize the need to evaluate product quality under various real-life use scenarios. These insights are essential for developing regulatory policies and promoting the safe and sustainable application of environmentally friendly products.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials and chemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree types of 500 mL cups (PLA-CP-DC, PLA-IM-DC, and PP-DC) were purchased from the top-selling brands on Chinese online retail platforms. The polymer composition of all cups was further confirmed by FTIR, GPC, and differential scanning calorimetry (DSC) (Supplementary Fig. 18). Ultrapure water was produced using a Milli-Q Reference system (Merck, Germany; resistivity = 18.2 M\u0026Omega;\u0026middot;cm, total organic carbon \u0026lt; 5 \u0026micro;g L\u003csup\u003e-1\u003c/sup\u003e). All solvents\u0026nbsp;used\u0026nbsp;in\u0026nbsp;this\u0026nbsp;study\u0026nbsp;were of HPLC grade. NanoStandard Series particle size standards (100 nm diameter) used for NTA calibration were obtained from Applied Microspheres (Wiesbaden, Germany). A mixed OLA standard and seven individual short-chain OLA standards were synthesized at Fudan University, while the deuterated OLA analogs were synthesized at Nanjing University. Detailed synthesis procedures are provided in Supplementary Note\u0026nbsp;2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNP release and material characterization under simulated use\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo simulate NP release under typical use, 500 mL of ultrapure water preheated to 50 \u0026deg;C was poured to each PLA-DC and PP-DC, which were kept on a thermostatic magnetic stirrer at 50 rpm for 4 hours to mimic gentle agitation. Aliquots of 5 mL were collected at 0.5, 1, 2, and 4 h for NTA to determine NPs concentration and size distribution. A procedural blank was included to monitor background contamination. Cup walls before and after exposure were sectioned and analyzed in situ by SEM and FTIR, or dissolved in N,N-dimethylformamide (DMF) for GPC to assess physicochemical changes, with detailed characterization procedures provided in Supplementary Note 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNP quantification and component separation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNTA was used to quantify NPs based on Brownian motion to obtain particle number concentrations and hydrodynamic size distributions, but this technique cannot resolve chemical composition\u003csup\u003e46\u003c/sup\u003e. Measurements were performed using a ZetaView instrument (Particle Metrix, Germany). To minimize background contamination, NP counts in ultrapure water and solvents were confirmed to be \u0026lt;1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e particles mL\u003csup\u003e-1\u003c/sup\u003e, all glassware was pretreated with MeOH and ultrapure water followed by 450 \u0026deg;C heating for 4 h, and no plastic materials were used during handling except ultrafiltration tubes and pipette tips.\u0026nbsp;Detailed analytical procedures of NTA are provided in Supplementary Note 4.\u003c/p\u003e\n\u003cp\u003eA selective dissolution method was then developed to separate PLA NPs and OLA NPs released from PLA-DCs. Five organic solvents (40% MeOH, IPA, ACN, EtOH, and 60% EtOH) were evaluated, and 60% EtOH was identified as the optimal solvent, selectively dissolving OLA NPs while leaving PLA NPs intact. PLA-CP-DCs and PLA-IM-DCs were filled with 500 mL of ultrapure water at 30, 50, and 70 \u0026deg;C and stirred at 50 rpm for 1 h. Aliquots of 5 mL were collected to examine NP composition, levels, and temperature effects. For each sample, 3 mL was used to determine the original NP concentration (C\u003csub\u003eori\u003c/sub\u003e), and 2 mL was diluted 2.5-fold with 3 mL EtOH and vortexed before analyzing the residual NP concentration (C\u003csub\u003eres\u003c/sub\u003e). The concentrations of PLA NPs and OLA NPs were calculated according to Eqs. (1) and (2):\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1767859143.png\" width=\"839\" height=\"103\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComprehensive quantitative analysis of PLCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comprehensive analytical workflow was developed to quantify PLCs, including free LA, OLAs, and PLA NPs. PLA-DCs were filled with 500 mL of ultrapure water at 50 \u0026deg;C and 70 \u0026deg;C and maintained at the respective temperatures for 6 h. Aliquots of 3 mL were collected at 0.5, 1, 2, 4, and 6 h from the same cup to examine the release behavior of the three PLCs under the two temperatures.\u003c/p\u003e\n\u003cp\u003eFor free LA quantification, 1 mL of sample was spiked with 2 \u0026micro;g of \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e3\u003c/sub\u003e-LA as an internal standard, freeze-dried, reconstituted in 1 mL acetonitrile, centrifuged, and analyzed by LC\u0026ndash;MS/MS to obtain C\u003csub\u003eLA\u003c/sub\u003e. For OLAs quantification, 1 mL of sample was spiked with 2 \u0026micro;g of \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e3\u003c/sub\u003e-LA, freeze-dried, and redissolved in 1 mL of 60% (v/v) EtOH. The solution was ultrafiltered through a 10 kDa membrane, and the filter was rinsed twice with 1 mL EtOH. The filtrate was dried and mixed with 1 mL of 1% (w/v) NaOH solution, followed by depolymerization in an autoclave at 121 \u0026deg;C for 240 min. The product pH was adjusted to 7, NaCl was precipitated by refrigerated centrifugation, and monomeric LA was extracted with MeOH and analyzed by LC\u0026ndash;MS/MS to yield C\u003csub\u003eLA+OLA\u003c/sub\u003e. For PLA NP quantification, 1 mL of sample was spiked with 2 \u0026micro;g of \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e3\u003c/sub\u003e-LA, freeze-dried, and dissolved in 1 mL DCM to extract all leachates. After drying, 1 mL of 1% (w/v) NaOH was added for depolymerization, followed by neutralization and desalting as described above, and the resulting LA was analyzed by LC\u0026ndash;MS/MS to obtain C\u003csub\u003eLA+OLA+PLA\u003c/sub\u003e.The concentration of OLAs and PLA NPs was calculated according to Eq. (3) and (4), where MW\u003csub\u003e[OLA]\u003c/sub\u003e (72 g\u0026middot;mol\u003csup\u003e-1\u003c/sup\u003e) and MW\u003csub\u003eLA\u003c/sub\u003e (90 g\u0026middot;mol\u003csup\u003e-1\u003c/sup\u003e) represent the molecular weights of the [-O-CH(CH₃)-CO-] repeating unit and LA, respectively\u003csup\u003e47\u003c/sup\u003e. More detailed procedures for PLC quantification are provided in Supplementary Note 5.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1767859180.png\" width=\"839\" height=\"124\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of short-chain OLAs release under realistic use conditions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe release behavior of short-chain OLAs from PLA-DCs was investigated under four simulated drinking scenarios: varying water temperature, standing time, beverage type, and pre-cleaning before use. To examine the effect of pre-cleaning, six PLA-DCs were evenly assigned to two groups. One group was rinsed with ultrapure water at the corresponding temperature for ~30 s, while the other was used directly. The cups were filled with ultrapure water at 30 \u0026deg;C or 70 \u0026deg;C and kept isothermal for 12 h under stirring at 50 rpm. Aliquots of 1 mL were collected at 0.5, 1, 2, 4, 8, and 12 h, spiked with 2 \u0026micro;g of d-OLAs, concentrated tenfold by refrigerated centrifugation, reconstituted in 100 \u0026micro;L ACN, centrifuged (15,000 rpm, 30 min), and analyzed by LC\u0026ndash;MS/MS (Supplementary Fig. 19).\u0026nbsp;Details of the selection of the concentration procedure are provided in Supplementary Note 6.\u003c/p\u003e\n\u003cp\u003eFor beverage type experiments, food simulants were prepared following European Commission Regulation (2011): 3% (w/v) acetic acid for acidic beverages, 20% (v/v) ethanol for alcoholic or organic-containing beverages, and ultrapure water as a control. At 30 \u0026deg;C, 500 mL of each simulant was added to the PLA-DCs and stirred at 50 rpm. Aliquots of 1 mL were collected at 0.5, 1, and 2 h, concentrated, centrifuged, and analyzed by LC\u0026ndash;MS/MS as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC\u0026ndash;MS/MS analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantification of LA and OLAs (OLA\u003csub\u003e2\u003c/sub\u003e, OLA\u003csub\u003e4\u003c/sub\u003e, OLA\u003csub\u003e6\u003c/sub\u003e, OLA\u003csub\u003e7\u003c/sub\u003e, OLA\u003csub\u003e8\u003c/sub\u003e, OLA\u003csub\u003e10\u003c/sub\u003e, OLA\u003csub\u003e12\u003c/sub\u003e) was performed on an Agilent 6495 triple quadrupole MS system (Agilent Technologies, USA) operating in multiple reaction monitoring (MRM) mode. Chromatographic separation was achieved using a HILIC column (150 \u0026times; 2.1 mm, 5 \u0026mu;m; HILICON, Sweden) for LA and a C18 column (50 \u0026times; 2.1 mm, 1.8 \u0026mu;m; Agilent Technologies, USA) for seven short-chain OLAs (Supplementary Tables 4\u0026ndash;7). A 200 ng mL\u003csup\u003e-1\u003c/sup\u003e calibration standard was injected after every ten samples to monitor potential sensitivity drift, and blank solvent runs were inserted between samples to verify carryover. Quality assurance and quality control (QA/QC) were assessed based on linearity, sensitivity, and recovery. All samples pretreatment using \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e3\u003c/sub\u003e-LA or d-OLAs as internal standards. Procedural blanks and duplicate samples were included throughout sample preparation and analysis to ensure accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExposure assessment of PLCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe potential exposure to PLCs from PLA-DCs was estimated on global and regional scales, taking into account market value, cup usage, and beverage consumption data, as well as differences in drinking habits. A cup volume of 250 mL and a drinking duration of 0.5 h were assumed, consistent with the dominant global market size (151\u0026ndash;350 mL) and typical consumer behavior\u003csup\u003e38,48\u003c/sup\u003e. Based on the experimentally measured PLC concentrations, the per-cup exposure was calculated. Global and regional annual exposures were estimated by multiplying the per-cup exposure with the annual PLA-DC consumption derived from the corresponding market values (Supplementary Note 7). Country-specific exposure from coffee cups in the\u0026nbsp;U.S.\u0026nbsp;and China was further estimated using national disposable cup consumption data (Supplementary Note 8). To evaluate the contribution from different beverage types, it was assumed that all beverages were consumed from PLA-DCs. Daily beverage consumption data for 13 countries were combined with the experimentally determined PLC concentrations in beverage simulants to calculate beverage-specific exposure (Supplementary Note 9).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp; s.d., based on at least three independent experiments. Differences between groups were evaluated using two-tailed Student\u0026rsquo;s t-tests or one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons. All statistical analyses were performed using Origin software (version 2025b), and differences were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are included in the manuscript and/or the Supplementary Information. These data are also available via Zenodo at https://zenodo.org/records/17633664. Detailed experimental methods, exposure-estimation procedures, and validation data for the quantification of PLCs are provided in the Supplementary Information and Supplementary Data. Source data are provided with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.F. was sponsored by the National Key R\u0026amp;D Program (grant no. 2024YFA0918900), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB0750300), National Natural Science Foundation of China (grant no. 22376032), Agilent University Relations (ACT-UR Program, grant no. 4863) and the Xiaomi Young Investigator Award. M.S. was sponsored by the National Natural Science Foundation of China (grant nos. 22125606 and 22241604). The corresponding author Changzhi Shi is funded by the Shanghai Tongji Gao Tingyao Environmental Science \u0026amp; Technology Development Foundation.\u0026nbsp;The authors would like to thank C. Xu from Fudan University for providing assistance with polymer material characterization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.G., C.S., and M.F. conceived and designed the research. C.S. and M.F. jointly supervised the study. A.G. purchased the cups, performed material characterization, and prepared water samples under different simulated use conditions. A.G. and C.S. conducted the NP experiments and NTA analyses and completed data processing and exposure estimation. A.G., X.C., and J.Y. performed the mass spectrometry analyses. T.Y. and A.Z. provided technical advice on experimental design and contributed to manuscript revision. C.S., A.G., and M.F. wrote the manuscript with input from all authors, all of whom participated in the discussion and interpretation of the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Changzhi Shi or Mingliang Fang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCowger, W. et al. 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Microplastics and other harmful substances released from disposable paper cups into hot water. \u003cem\u003eJournal of Hazardous Materials\u003c/em\u003e \u003cstrong\u003e404\u003c/strong\u003e, 124118 (2021). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8327196/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8327196/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Nanoplastics released from biodegradable plastics have raised concern, yet their composition and release behavior remain largely unclear. Using polylactic acid (PLA)–based disposable containers, a common alternative to conventional plastics, we developed a quantitative analytical workflow to differentiate and characterize PLA-leached chemicals (PLCs), including PLA nanoparticles (NPs), PLA oligomers (OLAs), and lactic acid (LA). Simulated use of disposable cups (DCs) showed that PLA-DCs released ~6 million particles mL-1 NPs into water, substantially higher than conventional polypropylene (PP) DCs. More interestingly, up to 55% of detected NPs were OLA self-assembled aggregates rather than PLA NPs. Across use scenarios, water temperature was the dominant determinant: PLC concentrations increased nearly two orders of magnitude from 50 to 70 °C, accompanied by a shift from particulate to dissolved OLAs. Integrating national use behaviors with release parameters, global annual PLC exposure from PLA-DCs is projected to increase by 2 folds from 2021 to 2030. Although U.S. coffee cups account for 49% of PLA-DCs, hot coffee consumption contributed \u003e99% of exposure, whereas in China, despite coffee cup consumption being only 20% of the U.S., hot water use in PLA-DCs still resulted in ~80% of the U.S. exposure. These findings highlight the need to establish NP and oligomer release as a new benchmark for evaluating biodegradable disposable plastics and to redefine safe-use temperature thresholds.","manuscriptTitle":"Temperature Dominance in Governing Nanoplastic Release and Leachate Composition from Polylactic Acid–Based Disposable Plastics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-08 08:03:52","doi":"10.21203/rs.3.rs-8327196/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-sustainability","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"natsustain","sideBox":"Learn more about [Nature Sustainability](http://www.nature.com/natsustain/)","snPcode":"","submissionUrl":"","title":"Nature Sustainability","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"db2c9adf-1c75-4bc0-a39e-2eb056586847","owner":[],"postedDate":"January 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60580720,"name":"Physical sciences/Chemistry/Polymer chemistry/Biopolymers"},{"id":60580721,"name":"Physical sciences/Chemistry/Analytical chemistry"},{"id":60580722,"name":"Physical sciences/Nanoscience and technology/Nanotoxicology/Regulation and risk management"}],"tags":[],"updatedAt":"2026-01-08T08:03:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-08 08:03:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8327196","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8327196","identity":"rs-8327196","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}