Progress 03/01/24 to 02/28/25
Outputs
Target Audience:The target audience includes : 1) environmental heath risk assessors; 2) the lay public; 3) lawmakers; 4) the chemical and plastics industries; and 5) the agricultural community Changes/Problems:AIM-1: No problems were faced and no changes to report AIM2: We had two main material constrains, one in terms of the amount of MNPs to conduct the plant experiments, and another in terms of the consumable materials interfering with the analysis of organic contaminants. Thus: - We re-designed the plant exposure studies, still complying with our proposal objectives. - We agreed on a specific material type (glass for boscalid, plastic for PFOS and heavy metals) to handle the EPs without interference in the analyses. AIM-3: No problems were faced and no changes to report. What opportunities for training and professional development has the project provided?Aim 1 Aim 1 of this project facilitated the training of a number of postdoctoral fellows and students, who have acquired proficiency in cryo-milling and UV weathering system method development, and thermal degradation of plastics while also expanding their skill set in particle characterization techniques. Aim 2 A new CAES postdoctoral fellow was brought on staff in January of 2024. This post-doc has been building analytical skills and applying data analysis tools to interpret measured results. The post-doc also attended a workshop on Identifying and Prioritizing Research and Programmatic Needs in the Detection, Mitigating, and Remediating PFAS in Agriculture and Food Systems - A collaboration between USDA ARS - Center of Excellence for Environmental Monitoring and Mitigation and the University of Maine at Washington DC - Arlington Westin, USA (10-12th September 2024). The NJIT Py-GCMS project allowed the continued training of one postdoctoral and one PhD student, who have gained further expertise in Py-GCMS methods development and sample preparation for plants and biological matrices. Aim 3 The in vitro triculture Aim 3 project allowed the training of two postdoctoral fellows and a number of undergraduate students, who have gained expertise in performing simulated digestion, preparation and use of the in vitro triculture small intestinal epithelial model, and physicochemical characterization of MNPs. The CAES postdoctoral researcher has been building specific analytical skills, including method development strategies for the analysis of organic and inorganic chemicals, as well as optimizing operating conditions for various instruments (inductively coupled plasma optical by emission or mass spectroscopy, ICP-OES and ICP-MS; and high resolution mass spectroscopy, LC-MS/MS). How have the results been disseminated to communities of interest?Dr. Jason C. White (Co-PI) and Dr. Nubia Zuverza-Mena (senior key person), attended the 2024 Sustainable Nanotechnology Organization meeting in Providence, RI (Nov. 8-10, 2024); Dr. Zuverza-Mena presented: "Uptake of Micro-Nanoplastics and Environmental Pollutants in Lettuce (Lactuca sativa) is Size-Dependent" in the session Translating Nano EHS to Nanoplastics where Dr. White served in the planning committee. Dr. Mandeep Kaur, CAES postdoctoral scientist, presented poster on different effects of PVC on the bioaccumulation of environmental pollutants by Lactuca sativa L. at the 114th Plant Science Day organized by The Connecticut Agricultural Experiment Station, Lockwood Farm,CT, USA (August 7, 2024); presented at the 2024 ACS Fall Meeting in Denver (August 17-22, 2024) and at the International Phytotechnology Conference- IPC18 on "Phytotechnologies for sustainable environment and food safety" in India (October 22nd- 24th 2024), and she also presented at the series of UConn-CAES symposium organized by Institute of Material Science, University of Connecticut, USA (June 4th, 2024). Sarah Alotaibi from NJIT Prenseted a Poster at Pittcon 2024 entitled"Comparative Sample preparation and Quantitation of Microplastics in Plants using Pyrolysis GC-MS" Dr. Demokritou presented a number of invited lectures on micronanoplastics and potential health effects at national meetings including the 2025 SOT annual meeting, and lectures at various Universities (NYU, FIU, U. Utrecht, Stevens Institute of Technology, ETH Zurich, NJIT). We have also published 3 scientific papers including a review paper of current knowledge about the toxicology of MNPs in Nanoimpact, a manuscript describing our MNP genotoxicity studies in Nanomaterials, as well as a manuscript describing our mechanistic uptake studies of MNP both in using the triculture system and the organoid based duodenum on a chip platform (both papers published in the Journal of Hazardous Materials) Several additonal manuscripts are in preparation and under review Dr Demokritou and NAMC team also participated and delivered lecture to the Communities in NJ on plastic pollution and health during an event organized by the Environmental Occupational Health Sciences Institute in the fall of 2025 increasing awareness on this emerging contaminant. Our published research was showcased extensively in the media including articles in major newspapers and media outlets. What do you plan to do during the next reporting period to accomplish the goals?Aim 1 The goals for AIM 2 were completed in Year 2. Sufficient quantities of PVC-C, PVC-CW, PVC-I, and PET-CW have been generated and characterized for the needs of the AIM 2 and AIM 3 studies. Aim 2 1. Studies of MNP and EP impacts and accumulation in plants: In year 3 of this project, a significant amount of work is planned under Aim 2. We will finalize the analysis and data interpretation from the experiments carried on in year 2 (studies on aged-PVC co-exposed with EPs in hydroponics/lettuce and soil/wheat). In addition, we will start conducting investigations in hydroponically grown lettuce on the uptake/translocation of EPs in upon exposure to two relevant microplastics (MNPs). The MNPs will be either PET (aged, 10 um) or PVC (incinerated, 0.1 um); the EP cocktail will include As (V), Cr (VI), Pb (II) and boscalid at 0.5 mg/L each, as well as PFOS at 5 µg/L. Equivalent and concurrent studies will also take place in soil-grown wheat as the model plant with PET and PVC at 10 mg/kg and EPs at 1 mg/kg (arsenic, chromium, lead and boscalid) and 50 ug/kg (PFOS). 2. Development and use of Py-GCMS method for analysis and quantification of MNPs: In Year 3, we will utilize the digestion, extraction, and Py-GCMS methods previously developed and optimized for PS and PVC MNPs, to optimized methods for isolation and quantification of cryomilled and size fractionated PET PM10 MNPs in plant tissues and intestinal cell lysates and media samples. Quality controls will include test analyses of procedural blanks, spiked blanks, replicate samples, and spiked produce samples along with the original samples, as was done in year 2 for optimization of PVC MNP sample analysis. We will use the optimized methods to analyze root, stem and shoot samples generated in Aim 1 plant studies as well as cell lysate and media samples generated in Aim 3 gastrointestinal digestion and small intestinal epithelium models to enable quantificatoin of PET PM10 MNP uptake and translocation in each experimental system. ?Aim 3 In year 3 we will finish the studies on PET and complete inhibitor studies of MNP uptake mechanisms, using the panel of inhibitors described in the proposal to determine the relative roles of active and passive uptake, and of specific active uptake mechanisms, including phagocytosis, macropinocytosis, clathrin mediated endocytosis (CME), Fast endophilin mediated endocytosis (FEME), and clathrin-independent carrier glycosylphosphatidylinositol-anchored protein-enriched early endocytic compartment (CLIC/GEEC) endocytosis. RNA sequencing of epithelial cell lysates (previously exposed to MNP-EPs for 24 h) will be conducted to investigate gene regulations responsible for EP translocation.
Impacts
What was accomplished under these goals?
Aim 1 1. Generation of incinerated MNPs: In year 2, we utilized the INEXS platform to generate sufficient quantities incinerated PVC PM0.1 MNPs (PVC-I) for plant and intestinal epithelium studies. 2. Generation of cryomilled and UV- weathered MNPs:We used combined cryo-milling and UV aging to generate sufficient quantities of PVC-C, PVC-CW and PET-CW reference MNPs for plant and intestinal epithelium studies. Size separation: Because size is a critical determinant of fate and bioactivityof MNPs, we developed a method of sequential dry and wet sieving for size fractionation, to isolate the smallest particles possible for our studies. Dry sieving was employed to obtain a PM53 size fraction, which was processed by wet sieving in ethanol (which acts as a surfactant to disperse agglomerates), to obtain a PM10 size fraction. Ethanol was then exchanged with water in a rotary evaporator. Synthesis of cryomilled and weathered PVC and PET MNPs: PVC and PE powders were cooled -196 °C and cryomilled in a mechanical mill for 2 h (PVC) or 6 h (PET). Thepowders were size fractionated by dry and wet sieving. A portion of the PM10 fractions were subjected to UV weathering to obtain cryomilled/UV weatherd PVC PM10 and PET PM10. 3. Physicochemical and morphological characterization of model MNPs: Characterizaiton included X-ray photoelectron spectroscopy (XPS) for surface elemental analysis, Fourier-transform infrared spectroscopy (FTIR) to analyze surface functional groups, scanning electron microscopy (SEM) to assess size and morphology, MALD to determine size distributions in suspensions, and contact angle measurement to assess hydrophobicity. 4. Investigation of interactions between MNPs and EPs: The interactions between PVC MNPs and EPs (Cr, As, Pb, PFOS, and boscalid) were investigated to assess sorption of EPs by MNPs in water. Each PVC MNP was suspended at a 1mg/mL in water spiked with EPs at regulatory limit concentrations. The mixtures were incubated at 200 rpm at room temperature for 48 hto equilibrate. The resulting suspension was filtered using 0.2 µm cellulose filters. EP concentrations measured in filtrates were used to calculate the percentage of each EP sorped by MNPs and the partition coefficients for each MNP-EP interaction. Quantitative analysis of toxic metals (Cr, As, Pb) was conducted using ICP-MS at CAES. PFAS and boscalid were analyzed via LC-MS at CAES. Our study revealed that unweathered, weathered, and incinerated PVC can sorb EPs. This suggests that micro-nano PVC MNPs can act as carriers, transporting pollutants, potentially leading to bioaccumulation and harm to ecosystems and human health. Supporting our hypothesis, the rate of EP sorption in water was determined to be contingent upon the chemical properties of the pollutants, degradation scenario, and size of PVC. Aim 2 1. Studies of MNP and EP impact and accumulation in plants: In year 2, three co-exposure experiments were conducted to evaluate effects of unweathered and weathered PVC PM10 on accumulation of EPs in two plants (lettuce and wheat). In hydroponic experiments, lettuce plants were co-exposed to MNPs at 5 mg/L,0.5 mg/L As, Cr, Pb and boscalid,and 5 ug/L PFOS. In a soil study, MNPs were at 10 mg/kg and EPs at 1 mg/kg (As, Cr, Pb and boscalid) and 50 ug/kg (PFOS). Results showed that only EPs exposure decreased lettuce biomass, and the same effect was observed in PVC-EPs irrespective of ageing. Importantly, aged-PVC significantly increase the accumulation of toxic elements and boscalid in lettuce shoots. Similarly, the relative chlorophyll content in lettuce was significantly reduced by both forms of PVCs in combination with EPs. Findings to date show that co-exposure of MNPs with EPs can significantly impact EP accumulation and toxicity, presenting an unknown risk to humans. Wheat plants were also grown in soil under co-exposure conditions (experiment 3). Plants have been harvested and analyses are underway. 2. Development of a pyrolysis gas chromatography-mass spectrometry (Py-GCMS) method for analysis and quantification of MNPs: In year 2, we utilized the Py-GCMS method developed and optimized for PS MNPs in year 1 to optimized methods for analysis of PVC MNPs, including cryomilled PVC PM10 PET, cryomilled and weathered PVC PM10, and incinerated PVC PM0.1, in plant tissues and intestinal cell lysates and media. Methods were optimized for each PVC MNP from each sample type. Quality controls included procedural blanks, spiked blanks, replicate samples, and spiked produce samples along with the original samples; triplicate measurements to assess the precision and sensitiviey, and deuterated standards to ensure polymer identification and subsequent quantification. We used the optimized methods to analyze 135 samples from plant and intestinal studies. Alkaline digestion (10% KOH) was effective and non-selective at breaking down cell lysates to isolate the PVC MNPs. We pefromed quantitative analysis of concentrated PVC MNP extracts by pyrolysis GC-MS, and completed data analysis of the samples. Aim 3 1. Fate of MNP-sorbed EPs across the digestive tract and effect of MNPs on bioaccessibility of EPs. The fate of MNPs (unweathered and weathered PM10 PVC, incinerated PM0.1 PVC, and weathered PET)-sorbed EPs (Cr, As, Pb, PFOS, and boscalid) across the GI tract and the effect of MNPs on EP bioaccessibility were assessed in a three-phase simulated digestion model. Each MNP suspended at 7.2 mg/mL and 2.4 mg/mL in water spiked with EPs at regulatory limit concentration was incubated in an orbital shaker at 200 rpm for 48 hours at room temperature. The resulting suspension was subjected to 3-phase simulated digestion. Samples collected after each phase were analyzed by MALD or DLS to assess size distribution. Quantitative analysis of EPs in 0.2 µm filtrates of suspensions was conducted by CAES for calculation of EP bioaccessibility in each phase. Results suggested that unweathered PM10, weathered PM10, and incinerated PM0.1 PVC MNPs are not broken down. The bioaccessibility of EPs was dependent on the chemical properties, degradation scenario, and size of PVC. 2. Reciprocal effects of EPs and MNPs on toxicity, uptake, and translocation in the triculture model: In year 2, we studied reciprocal effects of EPs and PVC MNPs across the life cycle of plastic (PVC-C, PVC-CW, and PVC-I) on uptake, translocation, and toxicity in the triculture intestinal epithelial model. PVC-C, PVC-CW, and PVC-I caused no significant change in TEER, LDH release, or dextran permeability, however 6-hour exposure of EP+PVC-CW (50 µg/mL) and EP+PVC-I (50 µg/mL) increased ROS production. Supporting our hypothesis, we found that PVC-CW promoted EP translocation more than PVC-C or PVC-I. Translocation was EP-specific and MNP concentration-dependent. No significant changes were observed in the uptake of MNPs and EP-sorped MNPs. Only EP-PVC-CW at 50 µg/mL showed higher translocation, suggesting that translocation of PVC-CW is EP- and concentration-dependent. 3. Toxicity and absorption of micro-nanoplastics in healthy and Crohn's disease human duodenum-chip models: A duodenum-on-a-chip model developed from human intestinal epithelial organoids from healthy and Crohn's disease donors and human intestinal microvascular endothelial cells was used to evaluate toxicity and mechanisms underlying uptake of 25 nm polystyrene shell-gold core tracer MNPs (AuPS25). RNAseq analysis of epithelial cells identified 9 dysregulated genes, including downregulation of IFI6, which has antiviral and immunosuppressive functions in the intestine. Inhibitor studies revealed that AuPS25 uptake in the IOC occurred by passive diffusion and active actin- and dynamin-dependent mechanisms. 4. EP analysis methods Methods were developed at CAES for the analysis of As, Cr, Pb, boscalid and PFOS in different matrices (water, nutrient solution, cell lysates).
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Bui TH, Zuverza-Mena N, Kendrick E, Tamez C, Kendrick E, Tamez C, Yadav M, Alotaibi S, Dimkpa C, DeLoid G, Sadik O, Demokritou P, White JC;. 2024. Micro-nanoscale polystyrene co-exposure impacts the uptake and translocation of arsenic and boscalid by lettuce (Lactuca sativa), NanoImpact
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Kharaghani D, DeLoid GM, Bui TH, Zuverza-Mena N, Tamez C, Musante C, White JC, Demokritou P; 2025. Ingested polystyrene micro-nanoplastics increase absorption of co-ingested arsenic in an in vitro triculture small intestinal epithelium model, Microplastics
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Kharaghani D, DeLoid GM, He P, Swenor B, Bui TH, Zuverza-Mena N, Tamez C, Musante C, Verzi M, White JC, Demokritou P; 2025. Toxicity and absorption of polystyrene micro-nanoplastics in healthy and Crohns disease human duodenum-chip models., Journal of Hazardous Materials
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Progress 03/01/23 to 02/29/24
Outputs
Target Audience:The target audience includes : 1) environmental heath risk assessors; 2) the lay public; 3) lawmakers; 4) the chemical and plastics industries; and 5) the agricultural community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Aim 1 Aim 1 of this project facilitated the training of a number of postdoctoral fellows and students, who have acquired proficiency in cryo-milling and UV weathering system method development, and thermal degradation of plastics while also expanding their skill set in particle characterization techniques. Aim 2 The CAES post-doc for plant studies was just brought on staff in January of 2023; as such, no training or professional development was initiated in year 1. The NJIT Py-GCMS project allowed the training of one postdoctoral fellow who has gained expertise in Py-GCMS methods development and sample preparation for plants. We have also recently brought in a PhD student. Aim 3 The in vitro triculture Aim 3 project allowed the training of two postdoctoral fellows and a number of undergraduate students, who have gained expertise in performing simulated digestion, preparation and use of the in vitro triculture small intestinal epithelial model, and physicochemical characterization of MNPs. How have the results been disseminated to communities of interest?Dr. Nubia Zuverza-Mena, senior key person on the project, attended the 2023 ACS Fall Meetings in San Francisco (August 13-17, 2023) and gave a presentation entitled "Micro/nano plastics influence on the uptake of environmental pollutants by lettuce" in the Division of Environmental Chemistry Special symposium "Processes and Risks of Micro-& Nano-Plastics in the Environment." Dr. Sadik delivered the Wallace H. Coulter Lecture at Pittcon 2024 entitled Sustainable Nanomaterials for Sensing Human Health and the Environment, San Diego Convention Center, February 24-28, 2024. Sarah Alotaibi, a PhD student on the Aim 2 CAES plant studies project, also presented a poster at Pittcon 2024 titled: Comparative Sample Preparation and Quantitation of Microplastics in Plants using Pyrolysis Gas Chromatography - Mass Spectrometry, Pittcon 2024, San Diego Convention Center, February 24 - 28, 2024. We have also completed a manuscript that describes the work in the presentation; that paper is currently under review at NanoImpact. Dr. Demokritou presented a number of invited lectures on micronanoplastics and potential health effects at national meetings including the Annual AAAS meeting in 2023 and lectures at Universities (NYU, FIU, etc). We have also published 3 scientific papers including a review paper of current knowledge about the toxicology of MNPs in Nanoimpact, a manuscript describing our MNP genotoxicity studies in Nanomaterials, as well as a manuscript describing our study of polystyrene MNP uptake in the triculture system in the Journal of Hazardous Materials. Several additonal manuscripts are in preparation and under review. What do you plan to do during the next reporting period to accomplish the goals?Aim 1 In Year 2, we will utilize the both INEXS platform and combined cryo-milling and UV aging method to generate substantial quantities of environmentally relevant reference MNPs for AIM 2 and AIM 3 of the project (PVC and PET). In addition to synthesis and characterization of MNPs, we will investigate the interactions between (MNPs) and (EPs), including Organic Chemical Pollutants (OCPs) and toxic metals. The primary goal is to assess whether the presence of MNPs affects the bioaccessibility and bioavailability of EPs in drinking water or food, compared to EPs alone. Experimental Setup: Model MNPs will be exposed to a cocktail of selected EPs at environmentally relevant concentrations for 48 hours to allow sorption equilibrium. The EP cocktail includes PFAS (specifically PFOS), pesticides (boscalid), and toxic metals (Cr, As, Pb) at regulatory limit concentrations. Assessment of Sorption: MNPs will be suspended in freshwater and spiked with EPs. After incubation for 48 h in an orbital shaker at room temperature, the suspension will be filtered using 3 kDa centrifugal filters, and EP concentrations measured in filtrates and starting EP concentrations will be used to calculate partition coefficients. Quantitative analysis of toxic metals will be conducted using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) at CAES. PFAS and boscalid will be analyzed via Liquid Chromatography - Mass Spectrometry (LC-MS) at CAES. Aim 2 1. Studies of MNP and EP impacts and accumulation in plants: In year 2 of this project, a significant amount of work is planned under Aim 2. We will conduct investigations of uptake/translocation of environmental pollutants (EP) in hydroponically grown lettuce upon exposure to PET and PVC micro-nano plastics (MNP). We hypothesize that (a) MNP co-exposure will impact EP translocation to lettuce shoots, (b) MNP type will impact EP translocation to lettuce shoots, and (c) MNP aging will impact EP translocation to lettuce shoots. We will use a hydroponic design initially to facilitate understanding of the mechanisms of analyte interaction, as well as fate and effects. The MNPs will be either PET or PVC cryo-milled-/UV aged (conc. 5 mg/L; size- PM3.0 [≤ 3 µm]); the EP cocktail will include Arsenic (V), Cr (VI), Pb (II) to be used at 0.5 mg/L each, as well as PFOS (5 µg/L) and boscalid (0.5 mg/L each). Additional studies will involve a similar experiment with wheat, as well as other work with smaller size MNPs. Equivalent studies are planned in soil at the end of year 2, although those may not occur until year 3 depending on the completion of the hydroponic studies. 2. Development and use of Py-GCMS method for analysis and quantification of MNPs: In Year 2, we will utilize the quantitative Py-GCMS method that had been developed for polystyrene for the other MNPs, including PET and PVC in plant tissues and cell samples. We will optimize the methods for the tissue samples following appropriate digestion. We will use Py-GCMS to analyze root, stem and shoot samples generated in Aim 1 plant studies as well as cell lysate and media samples generated in Aim 3 in vitro gastrointestinal digestion and triculture small intestinal epithelium model to enable assessment of MNP uptake and translocation in each experimental system. For quality control, we will perform test analyses of procedural blanks, spiked blanks, replicate samples, and spiked produce samples along with the original samples. For spiked samples, a mixture of standards for all six polymers (PE, PET, PVC, PS, PU, PP) will be spiked into the original samples at low concentrations. Replicate measurements will be conducted to assess the precision, sensitivity, and distribution of polymers throughout the sample. We will perform procedural blanks using analytical grade MilliQ® water control for background contamination during sample preparation and analysis. Blanks will undergo the same sample pre-treatment and analytical steps as the original samples. The limits of detection and quantification will be calculated for all samples based on standard operating procedures. Lastly, to ensure polymer identification and subsequent quantification, deuterated standard materials will be employed to determine specific products of polymer degradation. The following steps will be performed for each sample and control: 1. Collection and washing; 2. Extraction: Wet peroxide oxidation (WPO) using 30% hydrogen peroxide and 0.05 M Fe (II) catalyst will be used for the digestion of samples. Digested samples will be pre-concentrated by ultracentrifugation; and 3. Identification and quantification: Samples will be placed in the pyrolyzer and heated in "single-shot" mode and then in "double-shot" mode. Aim 3 In year 2 we will complete the assessment of MNP, EP, and MNP-EP toxicity and translocation using three-phase simulated digestion and an in vitro transwell triculture model of the small intestinal epithelium. As detailed in the proposal, reference MNPs (cryomilled and cryomilled/UV-aged PVC , incinerated PVC, and cryomilled/UV-aged PET, EPs (As, Pb, Cr, boscalid, PFOS) alone, or MNP-EP mixtures will be subjected to three-phase simulated digestion, and tricultures will be treated for 4 or 24 h with the final small intestinal digestas to assess effects of MNPs on toxicity, uptake, and translocation of EPs, as well as effects of EPs on toxicity, uptake, and translocation of MNPs. Toxicological endpoints will include dextran permeability to assess barrier integrity, LDH quantification to assess cytotoxicity, measurement of ROS to assess oxidative stress, and analysis of cytokines and chemokines to asses inflammation. Uptake and translocation of MNPs will be assessed by Py-GCMS analysis at NJIT of cell lysates and triculture transwell basolateral fluid, respectively. Uptake and translocation of EPs will be assessed by ICP-MS (As, Pb, Cr) or LC-MS (boscalid, PFOS) analysis at CAES of cell lysates and basolateral fluid. Uptake and localization of MNPs will also be visualized by enhanced darkfield hyperspectral microscopy (EDHM). In addition, in year 2 we will begin inhibitor studies of MNP uptake mechanisms, using panel of inhibitors described in the proposal to determine the relative roles of active and passive uptake, and of specific active uptake mechanisms, including phagocytosis, macropinocytosis, clathrin mediated endocytosis (CME), Fast endophilin mediated endocytosis (FEME), and clathrin-independent carrier glycosylphosphatidylinositol-anchored protein enriched early endocytic compartment (CLIC/GEEC) endocytosis. We plan to complete most of the inhibitor studies in year 2.
Impacts
What was accomplished under these goals?
Aim 1 1. Generation of incineration-derived MNPs: We used our Integrated Exposure Generation System (INEX) thermal degradation platform to generate incinerated MNPs. This platform is coupled with aerosol instrumentation and particle sampling systems for size fractionation of the released MNPs. As part of method development, we incinerated six pristine plastics: Acrylonitrile butadiene styrene (ABS), High-density polyethylene (HDPE), Polycarbonate (PC), Polyethylene terephthalate (PET), Polypropylene (PP), and Polyvinyl chloride (PVC). PM0.1 was collected for all plastics, and PM0.1 - 2.5 was collected for HDPE. Comprehensive physicochemical and biological characterization was performed on all generated MNPs, and small amounts of each were generated for toxicological and plant studies. 2. Generation of cryomilled and UV-aged/weathered MNPs: Mechanical degradation of plastics: We devised a method for simulating mechanical degradation of plastics using our cryogenic milling platform (Retsch Mixer Mill MM 400). Plastic pellets were cooled to liquid nitrogen temperature to embrittle them, allowing mechanical fragmentation into a fine polydisperse powder. We studied the effect of cryomilling energy/time on fragmentation of the plastic materials. The powder was size fractionated using an ultrasonic size sorter (VariSifter, Advanced Mfg.). Size separation: For risk assessment studies, it is important to have size sorted MNPs to assess their behaviour in biological and environmental systems (size defines fate and transport in environmental and biological systems), therefore we have developed a method to perform size separation of mechanically generated MNPs. Due to their hydrophobic nature, MNPs agglomerate in powder form, making "dry seiving" impossible. To address this issue, we suspended the MNP powder in 70% ethanol, leveraging ethanol's surfactant properties to break down the agglomerates via bath sonication. The ethanol suspension was wet sieved to collect MNPs smaller than 10 µm (PM10), and ethanol was then exchanged with water by rotary evaporation. Weathering of MNPS: The aqueous PM10 suspension was subjected to photo-aging using an accelerated weathering system (Q-SUN Xe-1 Xenon Test Chamber, Q-Lab), following ASTM standard protocols, and the impact of UV weathering on physicochemical and morphological changes as a function of exposure time was studied (see below). The above method was utilized to generate unweathered and weathered PM10 PVC MNPs for toxicological and plant studies. 3. Physicochemical and morphological (PCM) characterization of reference model MNPs: Characterizaiton included X-ray photoelectron spectroscopy (XPS) for surface elemental analysis, Fourier-transform infrared spectroscopy (FTIR) to analyze surface functional groups, scanning electron microscopy (SEM) to assess size and morphology, MALD to determine size distributions in suspensions, and contact angle measurement to assess hydrophobicity. Aim 2 1. Initial studies of MNP and EP impact and accumulation in plants: Under Aim 2, assessment of the accumulation and impact of MNPs and EPs in plants under hydroponic and greenhouse soil conditions was begun using a simplistic model MNP (polystyrene) to fine-tune the methodology. In year 1, three co-exposure experiments were conducted to evaluate if exposure to MNPs (polystyrene, PS; 20 or 1000 nm) had an impact on the accumulation of other EPs (arsenic and boscalid, As and Bos) in lettuce. In two hydroponic experiments, plants were co-exposed to MNPs at 10 or 50 mg/L, and to 1 mg/L of each EP. In a soil study, the MNPs and EPs were at 50 and 10 mg/kg, respectively. The mixture of As and Bos significantly reduced shoot biomass and/or length by 59.2% and 8.6%, respectively, and increased phytotoxicity (photosynthetic parameters) compared to single contaminant exposure and the control. Phytotoxicity was enhanced by PS under both growth conditions. Nanoscale PS had a greater impact than microscale PS on As uptake, including increased As translocation into edible tissues. Localization of MNPs within plant tissues was demonstrated by fluorescent microscopy and pyrolysis-gas chromatography-mass spectrometry (Py-GCMS). Findings to date demonstrate that co-exposure of MNPs with other EPs can significantly impact co-contaminant accumulation and toxicity, presenting an unknown risk to humans and other receptors. 2. Development of a pyrolysis gas chromatography-mass spectrometry (Py-GCMS) method for analysis and quantification of MNPs: We set up the method to identify and quantify MNPs in both plants and cell lysates. In year 1, using the plants grown in hydroponics and soil that had been exposed to PS, we developed a method to identify and quantify polystyrene MNPs in plant tissues with and without EPs. Various digestion procedures for plant tissues were tested and optimized for the project. We subsequently analyzed the digested plant tissues with Py-GCMS, and polystyrene was identified in almost all samples tested. We then conducted quantitation of polystyrene in the samples. LOD and LOQ were determined for PS as 0.6-0.9 mg/g and 1.8-2-9 mg/g, respectively. PS nanoplastics were quantified in root, shoot, and leaf tissues. The percent recoveries were found to be in the range of 85-95, 72-98, and 78-105 for root, shoot, and leaf, respectively. The PS content in the plant samples suggested that the uptake of nanoplastics (PS with 20 nm size) is greater and is, therefore, present more in shoot tissues than root in comparison to microplastic (PS 1000 nm). Another observation is that in the presence of environmental pollutants, the uptake of micro-nanoplastics significantly increases. The findings of this work suggest that the uptake of nanoplastics is greater than microplastics. In addition, the presence of environmental pollutants, such as Arsenic and Boscalid, significantly increases the uptake of micro-nanoplastics. A paper describing this study is currently under review. Aim 3 1. Pilot Study of polylstyrene MNP uptake mechanisms to fine-tune methods to be used in year 2. We employed in vitro digestion, the triculture model, and the inhibitors described in Aim 3 to assess possible mechanisms of absorption of 26 nm carboxylated polystyrene (PS26C) MNPs. Our results suggested that uptake occurred by passive diffusion as well as phatocytosis, clathrin mediated endocytosis (CME), and fast endophilin-mediated endocytosis (FEME). A paper describing this study was accepted and is awaiting publication in the Journal of Hazardous Materials. 2. Initial studies of the effects of polystyrene MNPs on arsenic bioavailability: We investigaed the interactions between 25 and 1000 nm polystyrene MNPs (PS25C and PS1KC) and arsenic (As), and the effects of coingestion of these MNPs and As on toxicity, uptake, and translocation of As in the triculture model. Arsenic sorption by PS MNPs was size dependent, with PS25C sorbing 13% of As compared to 2% sorbed by PS1KC. Exposure of triclutres to digestas of 100 ppb As, 1 mg/mL PS25C or PS1KC, or combined As and MNPs for 24 h had no effects on cytotoxicity, ROS production, or dextran permeability. PS25C significantly increased uptake of As, with 6% of As found in cell lysates compared to 0% without PS25C. Translocation of As was significantly increased by PS25C, with 10% translocated compared to 5% without MNPs. Uptake of As was also increased two-fold by PS25C and PS1KC. A manuscript describing this study is in preparation. 3. Study of MNP Genotoxicity: The genotoxicity of MNPs was assessed in the triculture model using the CometChip assay. Suspensions of PS25C and PS1KC, and incinerated polyethylene (PEI, PM0.1) PS25C and PS1KC induced DNA damage in a time- and concentration-dependent manner. These findings suggest that ingestion of high concentrations of MNPs could have serious genotoxic consequences in the small intestinal epithelium. A paper describing this study has been published in Nanomaterials.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Yang Z, DeLoid GM, Zarbl H, Baw J, Demokritou P, Micro-and nanoplastics (MNPs) and their potential toxicological outcomes: State of science, knowledge gaps and research needs, NanoImpact. 2023 Oct;32:100481
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Yang Z, DeLoid GM, Baw J, Zarbl H, Demokritou P., Assessment of Ingested Micro- and Nanoplastic (MNP)-Mediated Genotoxicity in an In Vitro Model of the Small Intestinal Epithelium (SIE), Nanomaterials (Basel). 2024 May 6;14(9).
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2024
Citation:
DeLoid GM, Yang Z, Bazina L, Kharaghani D, Sadrieh F, Demokritou P. Mechanisms of ingested polystyrene micro-nanoplastics (MNPs) uptake and translocation in an in vitro tri-culture small intestinal epithelium, Journal of Hazardous Materials. 2024
- Type:
Journal Articles
Status:
Submitted
Year Published:
2024
Citation:
Bui TH, Kendrick E, Tamez C, Yadav M, Alotaibi S, Nason S, Dimkpa C, DeLoid G, Sadik O, Demokritou P, White JC; Zuverza-Mena N. 2024. Micro-nanoscale polystyrene co-exposure impacts the uptake and translocation of arsenic and boscalid by lettuce (Lactuca sativa), Environmental Science and Technology, in review
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