Chemical and Colloidal Dynamics of MnO2 Nanosheets in Biological Media Relevant for Nanosafety Assessment.

Many layered crystal phases can be exfoliated or assembled into ultrathin 2D nanosheets with novel properties not achievable by particulate or fibrous nanoforms. Among these 2D materials are manganese dioxide (MnO2 ) nanosheets, which have applications in batteries, catalysts, and biomedical probes. A novel feature of MnO2 is its sensitivity to chemical reduction leading to dissolution and Mn2+ release. Biodissolution is critical for nanosafety assessment of 2D materials, but the timing and location of MnO2 biodissolution in environmental or occupational exposure scenarios are poorly understood. This work investigates the chemical and colloidal dynamics of MnO2 nanosheets in biological media for environmental and human health risk assessment. MnO2 nanosheets are insoluble in most aqueous phases, but react with strong and weak reducing agents in biological fluid environments. In vitro, reductive dissolution can be slow enough in cell culture media for MnO2 internalization by cells in the form of intact nanosheets, which localize in vacuoles, react to deplete intracellular glutathione, and induce cytotoxicity that is likely mediated by intracellular Mn2+ release. The results are used to classify MnO2 nanosheets within a new hazard screening framework for 2D materials, and the implications of MnO2 transformations for nanotoxicity testing and nanosafety assessment are discussed.

Chronic and pulse exposure effects of silver nanoparticles on natural lake phytoplankton and zooplankton.

The increasing use of silver nanoparticles (AgNPs) in consumer products raises concerns regarding the environmental exposure and impact of AgNPs on natural aquatic environments. Here, we investigated the effects of environmentally relevant AgNP concentrations on the natural plankton communities using in situ enclosures. Using twelve lake enclosures, we tested the hypotheses that AgNP concentration, dosing regimen, and capping agent (poly-vinyl pyrrolidone (PVP) vs. citrate) exhibit differential effects on plankton communities. Each of the following six treatments was replicated twice: control (no AgNPs added), low, medium, and high chronic PVP treatments (PVP-capped AgNPs added continuously, with target nominal concentrations of 4, 16, and 64 µg/L, respectively), citrate treatment (citrate-capped AgNPs added continuously, target nominal concentrations of 64 µg/L), and pulse treatment (64 µg/L PVP-AgNPs added as a single dose). Although Ag accumulated in the phytoplankton, no statistically significant treatment effect was found on phytoplankton community structure or biomass. In contrast, as AgNP exposure rate increased, zooplankton abundance generally increased while biomass and species richness declined. We also observed a shift in the size structure of zooplankton communities in the chronic AgNP treatments. In the pulse treatments, zooplankton abundance and biomass were reduced suggesting short periods of high AgNP concentrations affect zooplankton communities differently than chronic exposures. We found no evidence that capping agent affected AgNP toxicity on either community. Overall, our study demonstrates variable AgNP toxicity between trophic levels with stronger AgNP effects on zooplankton. Such effects on zooplankton are troubling and indicate that AgNP contamination could affect aquatic food webs.

Extraction and analysis of silver and gold nanoparticles from biological tissues using single particle inductively coupled plasma mass spectrometry.

Expanded use of engineered nanoparticles (ENPs) in consumer products increases the potential for environmental release and unintended biological exposures. As a result, measurement techniques are needed to accurately quantify ENP size, mass, and particle number distributions in biological matrices. This work combines single particle inductively coupled plasma mass spectrometry (spICPMS) with tissue extraction to quantify and characterize metallic ENPs in environmentally relevant biological tissues for the first time. ENPs were extracted from tissues via alkaline digestion using tetramethylammonium hydroxide (TMAH). Method development was performed using ground beef and was verified in Daphnia magna and Lumbriculus variegatus . ENPs investigated include 100 and 60 nm Au and Ag stabilized by polyvynylpyrrolidone (PVP). Mass- and number-based recovery of spiked Au and Ag ENPs was high (83-121%) from all tissues tested. Additional experiments suggested ENP mixtures (60 and 100 nm Ag ENPs) could be extracted and quantitatively analyzed. Biological exposures were also conducted to verify the applicability of the method for aquatic organisms. Size distributions and particle number concentrations were determined for ENPs extracted from D. magna exposed to 98 µg/L 100 nm Au and 4.8 µg/L 100 nm Ag ENPs. The D. magna nanoparticulate body burden for Au ENP uptake was 613 ± 230 µg/kgww, while the measured nanoparticulate body burden for D. magna exposed to Ag ENPs was 59 ± 52 µg/kgww. Notably, the particle size distributions determined from D. magna tissues suggested minimal shifts in the size distributions of ENPs accumulated, as compared to the exposure media.

Single particle inductively coupled plasma-mass spectrometry: a performance evaluation and method comparison in the determination of nanoparticle size.

Sizing engineered nanoparticles in simple, laboratory systems is now a robust field of science; however, application of available techniques to more complex, natural systems is hindered by numerous challenges including low nanoparticle number concentrations, polydispersity from aggregation and/or dissolution, and interference from other incidental particulates. A new emerging technique, single particle inductively coupled plasma-mass spectrometry (spICPMS), has the potential to address many of these analytical challenges when sizing inorganic nanoparticles in environmental matrices. However, to date, there is little beyond the initial feasibility studies that investigates the performance characteristics and validation of spICPMS as a nanoparticle sizing technique. This study compares sizing of four silver nanoparticle dispersions (nominal diameters of 40, 60, 80, and 100 nm) by spICPMS to four established sizing techniques: dynamic light scattering, differential centrifugal sedimentation, nanoparticle tracking analysis, and TEM. Results show that spICPMS is able to size silver nanoparticles, across different sizes and particle number concentrations, with accuracy similar to the other commercially available techniques. Furthermore, a novel approach to evaluating particle coincidence is presented. In addition, spICPMS size measurements were successfully performed on nanoparticles suspended in algal growth media at low concentrations. Overall, while further development of the technique is needed, spICPMS yields important advantages over other techniques when sizing nanoparticles in environmentally relevant media.

Manganese dioxide nanosheets induce mitochondrial toxicity in fish gill epithelial cells.

The development and production of engineered 2D nanomaterials are expanding exponentially, increasing the risk of their release into the aquatic environment. A recent study showed 2D MnO2 nanosheets, under development for energy and biomedical applications, dissolve upon interaction with biological reducing agents, resulting in depletion of intracellular glutathione levels within fish gill cells. However, little is known concerning their toxicity and interactions with subcellular organelles. To address this gap, we examined cellular uptake, cytotoxicity and mitochondrial effects of 2D MnO2 nanosheets using an in vitro fish gill cell line to represent a target tissue of rainbow trout, a freshwater indicator species. The data demonstrate cellular uptake of MnO2 nanosheets into lysosomes and potential mechanisms of dissolution within the lysosomal compartment. MnO2 nanosheets induced severe mitochondrial dysfunction at sub-cytotoxic doses. Quantitative, single cell fluorescent imaging revealed mitochondrial fission and impaired mitochondrial membrane potential following MnO2 nanosheet exposure. Seahorse analyses for cellular respiration revealed that MnO2 nanosheets inhibited basal respiration, maximal respiration and the spare respiratory capacity of gill cells, indicating mitochondrial dysfunction and reduced cellular respiratory activity. MnO2 nanosheet exposure also inhibited ATP production, further supporting the suppression of mitochondrial function and cellular respiration. Together, these observations indicate that 2D MnO2 nanosheets impair the ability of gill cells to respond to energy demands or prolonged stress. Finally, our data demonstrate significant differences in the toxicity of the 2D MnO2 nanosheets and their microparticle counterparts. This exemplifies the importance of considering the unique physical characteristics of 2D nanomaterials when conducting safety assessments.

Biodissolution and Cellular Response to MoO3 Nanoribbons and a New Framework for Early Hazard Screening for 2D Materials.

Two-dimensional (2D) materials are a broad class of synthetic ultra-thin sheet-like solids whose rapid pace of development motivates systematic study of their biological effects and safe design. A challenge for this effort is the large number of new materials and their chemical diversity. Recent work suggests that many 2D materials will be thermodynamically unstable and thus non-persistent in biological environments. Such information could inform and accelerate safety assessment, but experimental data to confirm the thermodynamic predictions is lacking. Here we propose a framework for early hazard screening of nanosheet materials based on biodissolution studies in reactive media, specially chosen for each material to match chemically feasible degradation pathways. Simple dissolution and in vitro tests allow grouping of nanosheet materials into four classes: A, potentially biopersistent; B: slowly degradable (>24-48 hours); C, biosoluble with potentially hazardous degradation products; and D, biosoluble with low-hazard degradation products. The proposed framework is demonstrated through an experimental case study on MoO3 nanoribbons, which have a dual 2D / 1D morphology and have been reported to be stable in aqueous stock solutions. The nanoribbons are shown to undergo rapid dissolution in biological simulant fluids and in cell culture, where they elicit no adverse responses up to 100µg ml-1 dose. These results place MoO3 nanoribbons in Class D, and assigns them a low priority for further nanotoxicology testing. We anticipate use of this framework could accelerate the risk assessment for the large set of new powdered 2D nanosheet materials, and promote their safe design and commercialization.

Comparing the effects of nanosilver size and coating variations on bioavailability, internalization, and elimination, using Lumbriculus variegatus.

As the production and applications of silver nanoparticles (AgNPs) increase, it is essential to characterize fate and effects in environmental systems. Nanosilver materials may settle from suspension; therefore, the authors' objective was to utilize environmentally relevant bioassays and study the impact, bioaccumulation, tissue distribution, uptake, and depuration of AgNPs on a sediment-dwelling invertebrate, Lumbriculus variegatus. Hydrodynamic diameters of uncoated 30-nm, 80-nm, and 1500-nm AgNP powders and a polyvinyl pyrrolidone (PVP) AgNP suspension were measured utilizing dynamic light scattering in freshwater media (0-280 µS/cm). Aggregation for 30 nm, 80 nm, and 1500 nm silver increased with conductivity but was minimal for PVP silver. Lumbriculus variegatus were exposed to AgNPs or silver nitrate (AgNO3 ) spiked into sediment (nominally 100 mg/kg) and water (PVP 30 nm and 70 nm Ag, nominally 5 mg/L). Uptake was assessed through inductively coupled plasma mass spectroscopy (ICP-MS) and hyperspectral imaging. Particle sizes were examined through field flow fractionation-ICP-MS (FFF-ICP-MS) and ICP-MS in single particle mode (SP-ICP-MS). Lumbriculus variegatus were also depurated for 6 h, 8 h, 24 h, and 48 h to determine gut clearance. Bioaccumulation factors of sediment-exposed L. variegatus were similar regardless of particle size or coatings. The FFF-ICP-MS and SP-ICP-MS detected AgNPs for up to 48 h post depuration. The present study provides information on bioaccumulation and interactions of AgNPs within biological systems.