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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