Size-Dependent Uptake and Depuration of Nanoplastics in Tilapia (Oreochromis niloticus) and Distinct Intestinal Impacts.

Nanoplastics (NPs, <1 µm) are of great concern worldwide because of their high potential risk toward organisms in aquatic systems, while very little work has been focused on their tissue-specific toxicokinetics due to the limitations of NP quantification for such a purpose. In this study, NPs with two different sizes (86 and 185 nm) were doped with palladium (Pd) to accurately determine the uptake and depuration kinetics in various tissues (intestine, stomach, liver, gill, and muscle) of tilapia (Oreochromis niloticus) in water, and subsequently, the corresponding toxic effects in the intestine were explored. Our results revealed uptake and depuration constants of 2.70-378 L kg-1 day-1 and 0.138-0.407 day-1 for NPs in tilapia for the first time, and the NPs in tissues were found to be highly dependent on the particle size. The intestine exhibited the greatest relative accumulation of both sizes of NPs; the smaller NPs caused more severe damage than the larger NPs to the intestinal mucosal layer, while the larger NPs induced a greater impact on microbiota composition. The findings of this work explicitly indicate the size-dependent toxicokinetics and intestinal toxicity pathways of NPs, providing new insights into the ecological effects of NPs on aquatic organisms.

Nanoplastic Exposure at Environmental Concentrations Disrupts Hepatic Lipid Metabolism through Oxidative Stress Induction and Endoplasmic Reticulum Homeostasis Perturbation.

In this study, we investigated the mechanism underlying the perturbation of hepatic lipid metabolism in response to micro/nanoplastic (MP/NP) exposure at environmentally relevant concentrations. Polystyrene (PS) MPs/NPs with different sizes (0.1, 0.5, and 5.0 µm) were studied for their effects on the homeostasis and function of Nile tilapia (Oreochromis niloticus) liver. Results showed that PS MPs/NPs were readily internalized and accumulated in various internal organs/tissues, especially in fish liver and muscle. Smaller-sized NPs caused more severe toxicity than larger MPs, including hepatic steatosis, inflammatory response, and disturbed liver function. Mechanistically, PS NPs with a particle size of 100 nm perturbed protein homeostasis in the endoplasmic reticulum (ER) by inhibiting the expression of chaperone proteins and genes involved in ER-associated degradation. This led to the activation of the PERK-eIF2α pathway, which caused dysfunction of hepatic lipid metabolism. Induction of oxidative stress and activation of the Nrf2/Keap1 pathway were also involved in the PS NP-induced hepatic lipid accumulation. These findings highlight the potential adverse effects of environmental MPs/NPs on aquatic organisms, raising concerns about their ecotoxicity and food safety.

Protein Corona-Mediated Extraction for Quantitative Analysis of Nanoplastics in Environmental Waters by Pyrolysis Gas Chromatography/Mass Spectrometry.

There is a growing concern about the effects of nanoplastics on biological safety and human health because of their global ubiquity in the environment. Methodologies for quantitative analysis of nanoplastics are important for the critical evaluation of their possible risks. Herein, a sensitive yet simple and environmentally friendly extraction approach mediated by protein corona is developed and coupled to pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) for nanoplastic determination in environmental waters. The developed methodology involved the formation of protein corona by addition of bovine serum albumin (BSA) to samples and protein precipitation via salting out. Then, the resulting extract was directly introduced to Py-GC/MS for nanoplastic mass quantification. Taking 50 nm polystyrene (PS) particles as a model, the highest extraction efficiency for nanoplastics was achieved under the extraction conditions of BSA concentration of 20 mg/L, equilibration time of 5 min, pH 3.0, 10% (w/v) NaCl, incubation temperature of 80 °C, and incubation period of 15 min. The extraction was confirmed to be mediated by the protein corona by transmission electron microscopy (TEM) analysis of the extracted nanoplastics. In total, 1.92 and 2.82 µg/L PS nanoplastics were detected in river water and the influent of wastewater treatment plant (WWTP), respectively. Furthermore, the feasibility of the present methodology was demonstrated by applying to extract PS and poly(methyl methacrylate) (PMMA) nanoplastics from real waters with recoveries of 72.1-98.9% at 14.2-50.4 µg/L spiked levels. Consequently, our method has provided new insights and possibilities for the investigation of nanoplastic pollution and its risk assessment in the environment.

Sequential Isolation of Microplastics and Nanoplastics in Environmental Waters by Membrane Filtration, Followed by Cloud-Point Extraction.

Respective detection of microplastics (MPs) and nanoplastics (NPs) is of great importance for their different environmental behaviors and toxicities. Using spherical polystyrene (PS) and poly(methyl methacrylate) (PMMA) plastics as models, the efficiency for sequential isolation of MPs and NPs by membrane filtration and cloud-point extraction was evaluated. After filtering through a glass membrane (1 µm pore size), over 90.7% of MPs were trapped on the membrane, whereas above 93.0% of NPs remained in the filtrate. The collected MPs together with the glass membrane were frozen in liquid nitrogen, ground, and suspended in water (1 mL) and subjected to pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) determination. The NPs in the filtrate were concentrated by cloud-point extraction, heated at 190 °C to degrade the extractant, and then determined by Py-GC/MS. For MPs and NPs spiked in pure water, the method detection limits are in the range of 0.05-1.9 µg/L. The proposed method is applied to analyze four real water samples, with the detection of 1.6-7.6 µg/L PS MPs and 0.6 µg/L PMMA MPs in three samples, and spiked recoveries of 75.0-102% for MPs and 67.8-87.2% for NPs. Our method offers a novel sample pretreatment approach for the respective determination of MPs and NPs.

Quantitative Analysis of Polystyrene and Poly(methyl methacrylate) Nanoplastics in Tissues of Aquatic Animals.

Micro- and nanoplastics unavoidably enter into organisms and humans as a result of widespread exposures through drinking waters, foods, and even inhalation. However, owing to the limited availability of quantitative analytical methods, the effect of nanoplastics inside animal bodies is poorly understood. Herein, we report a sensitive and robust method to determine the chemical composition, mass concentration, and size distribution of nanoplastics in biological matrices. This breakthrough is based on a novel procedure including alkaline digestion and protein precipitation to extract nanoplastics from tissues of aquatic animals, followed by quantitative analysis with pyrolysis gas chromatography-mass spectrometry. The optimized procedure exhibited good reproducibility and high sensitivity with the respective detection limits of 0.03 µg/g for polystyrene (PS) nanoplastics and 0.09 µg/g poly(methyl methacrylate) (PMMA) nanoplastics. This method also preserved the original morphology and size of nanoplastics. Furthermore, to demonstrate the feasibility of the proposed method, 14 species of aquatic animals were collected, and PS nanoplastics in a concentration range of 0.093-0.785 µg/g were detected in three of these animals. Recovery rates of 73.0-89.1% were further obtained for PS and PMMA nanospheres when they were spiked into the tissues of Zebra snail and Corbicula fluminea at levels of 1.84-2.12 µg/g. Consequently, this method provides a powerful tool for tracking nanoplastics in animals.

Cloud-Point Extraction Combined with Thermal Degradation for Nanoplastic Analysis Using Pyrolysis Gas Chromatography-Mass Spectrometry.

The contamination of micro- and nanoplastics in marine systems and freshwater is a global issue. Determination of micro- and nanoplastics in the aqueous environment is of high priority to fully assess the risk that plastic particles will pose. Although microplastics have been detected in a variety of aquatic ecosystems, the analysis of nanoplastics remains an unsolved challenge. Herein, for the first time, a Triton X-45 (TX-45)-based cloud-point extraction (CPE) was proposed to preconcentrate trace nanoplastics in environmental waters. Under the optimum extraction conditions, an enrichment factor of 500 was obtained for two types of nanoplastics with different compositions, polystyrene (PS) and poly(methyl methacrylate) (PMMA), without disturbing their original morphology and sizes. Additionally, following thermal treatment at 190 °C for 3 h, the CPE-obtained extract could be submitted to pyrolysis gas chromatography-mass spectrometry (Py-GC/MS) analysis for mass quantification of nanoplastics. Taking 66.2 nm PS nanoplastics and 86.2 nm PMMA nanoplastics as examples, the proposed method showed excellent reproducibility, and high sensitivity with respective detection limits of 11.5 and 2.5 fM. Feasibility of the proposed approach was verified by application of the optimized procedure to four real water samples. Recoveries of 84.6-96.6% at a spiked level of 88.6 fM for PS nanoplastics and 76.5-96.6% at a spiked level of 50.4 fM for PMMA nanoplastics were obtained. Consequently, this work provides an efficient approach for nanoplastic analysis in environmental waters.

Polyvinylidene Fluoride Micropore Membranes as Solid-Phase Extraction Disk for Preconcentration of Nanoparticulate Silver in Environmental Waters.

Efficient separation and preconcentration of trace nanoparticulate silver (NAg) from large-volume environmental waters is a prerequisite for reliable analysis and therefore understanding the environmental processes of silver nanoparticles (AgNPs). Herein, we report the novel use of polyvinylidene fluoride (PVDF) filter membrane for disk-based solid phase extraction (SPE) of NAg in 1 L of water samples with the disk-based SPE system, which consists of a syringe pump and a syringe filter holder to embed the filter membrane. While the PVDF membrane can selectively adsorb NAg in the presence of Ag+, aqueous solution of 2% (m/v) FL-70 is found to efficiently elute NAg. Analysis of NAg is performed following optimization of filter membrane and elution conditions with an enrichment factor of 1000. Additionally, transmission electron microscopy (TEM), UV-vis spectroscopy, and size-exclusion chromatography coupled with ICP-MS (SEC-ICP-MS) analysis showed that the extraction gives rise to no change in NAg size or shape, making this method attractive for practical applications. Furthermore, feasibility of the protocol is verified by applying it to extract NAg in four real waters with recoveries of 62.2-80.2% at 0.056-0.58 µg/L spiked levels. This work will facilitate robust studies of trace NAg transformation and their hazard assessments in the environment.

Speciation Analysis of Labile and Total Silver(I) in Nanosilver Dispersions and Environmental Waters by Hollow Fiber Supported Liquid Membrane Extraction.

Hollow fiber supported liquid membrane (HFSLM) extraction was coupled with ICP-MS for speciation analysis of labile Ag(I) and total Ag(I) in dispersions of silver nanoparticles (AgNPs) and environmental waters. Ag(I) in aqueous samples was extracted into the HFSLM of 5%(m/v) tri-n-octylphosphine oxide in n-undecane, and stripped in the acceptor of 10 mM Na2S2O3 and 1 mM Cu(NO3)2 prepared in 5 mM NaH2PO4-Na2HPO4 buffer (pH 7.5). Negligible depletion and exhaustive extraction were conducted under static and 250 rpm shaking to extract the labile Ag(I) and total Ag(I), respectively. The extraction equilibration was reached in 8 h for both extraction modes. The extraction efficiency and detection limit were (2.97 ± 0.25)% and 0.1 µg/L for labile Ag(I), and (82.3 ± 2.0)% and 0.5 µg/L for total Ag(I) detection, respectively. The proposed method was applied to determine labile Ag(I) and total Ag(I) in different sized AgNP dispersions and real environmental waters, with spiked recoveries of total Ag(I) in the range of 74.0-98.1%. With the capability of distinguishing labile and total Ag(I), our method offers a new approach for evaluating the bioavailability and understanding the fate and toxicity of AgNPs in aquatic systems.

In Vitro Toxicity and Modeling Reveal Nanoplastic Effects on Marine Bivalves.

Nanoplastics (NPs) represent a growing concern for global environmental health, particularly in marine ecosystems where they predominantly accumulate. The impact of NPs on marine benthic organisms, such as bivalves, raises critical questions regarding ecological integrity and food safety. Traditional methods for assessing NP toxicity are often limited by their time-intensive nature and ethical considerations. Herein, we explore the toxicological effects of NPs on the marine bivalve Ruditapes philippinarum, employing a combination of in vitro cellular assays and advanced modeling techniques. Results indicate a range of adverse effects at the organismal level, including growth inhibition (69.5-108%), oxidative stress, lipid peroxidation, and DNA damage in bivalves, following exposure to NPs at concentrations in the range of 1.6 × 109-1.6 × 1011 particles/mL (p/mL). Interestingly, the growth inhibition predicted by models (54.7-104%), based on in vitro cellular proliferation assays, shows strong agreement with the in vivo outcomes of NP exposure. Furthermore, we establish a clear correlation between cytotoxicity observed in vitro and the toxicological responses at the organismal level. Taken together, this work suggests that the integration of computational modeling with in vitro toxicity assays can predict the detrimental effects of NPs on bivalves, offering insightful references for assessing the environmental risk assessment of NPs in marine benthic ecosystems.

Surface-Charge-Driven Ferroptosis and Mitochondrial Dysfunction Is Involved in Toxicity Diversity in the Marine Bivalve Exposed to Nanoplastics.

Nanoplastics (NPs) pervade daily life, posing serious threats to marine ecosystems. Despite the crucial role that surface charge plays in NP effects, there is a substantial gap in our understanding of how surface charge influences NP toxicity. Herein, by exposing Ruditapes philippinarum (R. philippinarum) to both positively charged NPs (p-NPs) and negatively charged NPs (n-NPs) at environmentally relevant particle number levels for a duration of 35 days, we unequivocally demonstrate that both types of NPs had discernible impacts on the clams depending on their surface charge. Through transcriptomic and proteomic analyses, we unveiled the primary mechanisms behind p-NP toxicity, which stem from induced mitochondrial dysfunction and ferroptosis. In contrast, n-NPs predominantly stimulated innate immune responses, influencing salivary secretion and modulating the complement and coagulation cascades. Furthermore, in vitro tests on clam immune cells confirmed that internalized p-NPs triggered alterations in mitochondrial morphology, a decrease in membrane potential, and the initiation of ferroptosis. Conversely, n-NPs, to a certain extent, moderated the expression of genes related to immune responses, thus mitigating their adverse effects. Taken together, these findings indicate that the differential surface-charge-driven ferroptosis and mitochondrial dysfunction in clams play a critical role in the toxicity profile of NPs, providing an insightful reference for assessing the ecological toxicity associated with NPs.