FAIR assessment of nanosafety data reusability with community standards.

Nanomaterials hold great promise for improving our society, and it is crucial to understand their effects on biological systems in order to enhance their properties and ensure their safety. However, the lack of consistency in experimental reporting, the absence of universally accepted machine-readable metadata standards, and the challenge of combining such standards hamper the reusability of previously produced data for risk assessment. Fortunately, the research community has responded to these challenges by developing minimum reporting standards that address several of these issues. By converting twelve published minimum reporting standards into a machine-readable representation using FAIR maturity indicators, we have created a machine-friendly approach to annotate and assess datasets' reusability according to those standards. Furthermore, our NanoSafety Data Reusability Assessment (NSDRA) framework includes a metadata generator web application that can be integrated into experimental data management, and a new web application that can summarize the reusability of nanosafety datasets for one or more subsets of maturity indicators, tailored to specific computational risk assessment use cases. This approach enhances the transparency, communication, and reusability of experimental data and metadata. With this improved FAIR approach, we can facilitate the reuse of nanosafety research for exploration, toxicity prediction, and regulation, thereby advancing the field and benefiting society as a whole.

Investigation of the effects and mechanisms of manganese-based NMs on rice growth.

Manganese-based (Mn-based) nanomaterials (NMs) have great potential as alternatives to conventional Mn fertilizers. Yet, its environmental risks and effects on plant growth are not completely well understood. This study investigated the physiological effects of manganese dioxide (MnO2) and manganese tetroxide (Mn3O4) NMs on inter-root exposure (0-500 mg/L) of hydroponically grown rice. The results showed that on inter-root exposure, 50 mg/L Mn-based NMs promoted the uptake of mineral elements and enhanced the enzymatic activities of antioxidant systems (CAT and SOD) in rice, whereas 500 mg/L Mn3O4 NMs disrupted the mineral element homeostasis and led to phytotoxicity. The promotion effect of MnO2 NMs was better, firstly because MnO2 NMs treatment had lower Mn content in the plant than Mn3O4 NMs. In addition, MnO2 NMs are more transported and absorbed in the plant in ionic form, while Mn3O4 NMs exist in granular form. MnO2 NMs and Mn3O4 NMs both can be used as nano-fertilizers to improve the growth of rice by inter-root application, but the doses should be carefully selected.

Effects of dissolved organic matter on the environmental behavior and toxicity of metal nanomaterials: A review.

Metal nanomaterials (MNMs) have been released into the environment during their usage in various products, and their environmental behaviors directly impact their toxicity. Numerous environmental factors potentially affect the behaviors and toxicity of MNMs with dissolved organic matter (DOM) playing the most essential role. Abundant facts showing contradictory results about the effects of DOM on MNMs, herein the occurrence of DOM on the environmental process change of MNMs such as dissolution, dispersion, aggregation, and surface transformation were summarized. We also reviewed the effects of MNMs on organisms and their mechanisms in the environment such as acute toxicity, oxidative stress, oxidative damage, growth inhibition, photosynthesis, reproductive toxicity, and malformation. The presence of DOM had the potential to reduce or enhance the toxicity of MNMs by altering the reactive oxygen species (ROS) generation, dissolution, stability, and electrostatic repulsion of MNMs. Furthermore, we summarized the factors that affected different toxicity including specific organisms, DOM concentration, DOM types, light conditions, detection time, and production methods of MNMs. However, the more detailed mechanism of interaction between DOM and MNMs needs further investigation.

Recent advances on environmental behavior of Cu-based nanomaterials in soil-plant system: A review.

In recent years, copper-based nanomaterials (Cu-based NMs) have shown great potential in promoting agriculture development due to their special physicochemical characteristics. With the mass production and overuse of Cu-based NMs, there are potential effects on the soil-plant environment. Soil organisms, especially soil microorganisms, play a significant part in terrestrial or soil ecosystems; plants, as indirect organisms with soil-related Cu-based NMs, may affect human health through plant agricultural products. Understanding the accumulation and transformation of Cu-based NMs in soil-plant systems, as well as their ecotoxicological effects and potential mechanisms, is a prerequisite for the scientific assessment of environmental risks and safe application. Therefore, based on the current literature, this review: (i) introduces the accumulation and transformation behaviors of Cu-based NMs in soil and plant systems; (ii) focuses on the ecotoxicological effects of Cu-based NMs on a variety of organisms (microorganisms, invertebrates, and plants); (iii) reveals their corresponding toxicity mechanisms. It appears from studies hitherto made that both Cu-based NMs and released Cu2+ may be the main reasons for toxicity. When Cu-based NMs enter the soil-plant environment, their intrinsic physicochemical properties, along with various environmental factors, could also affect their transport, transformation, and biotoxicity. Therefore, we should push for intensifying the multi-approach research that focuses on the behaviors of Cu-based NMs in terrestrial exposure environments, and mitigates their toxicity to ensure the promotion of Cu-based NMs.

Emerging concern of nano-pollution in agro-ecosystem: Flip side of nanotechnology.

Nanomaterials (NMs) have proven to be a game-changer in agriculture, showcasing their potential to boost plant growth and safeguarding crops. The agricultural sector has widely adopted NMs, benefiting from their small size, high surface area, and optical properties to augment crop productivity and provide protection against various stressors. This is attributed to their unique characteristics, contributing to their widespread use in agriculture. Human exposure from various components of agro-environmental sectors (soil, crops) NMs residues are likely to upsurge with exposure paths may stimulates bioaccumulation in food chain. With the aim to achieve sustainability, nanotechnology (NTs) do exhibit its potentials in various domains of agriculture also have its flip side too. In this review article we have opted a fusion approach using bibliometric based analysis of global research trend followed by a holistic assessment of pros and cons i.e. toxicological aspect too. Moreover, we have also tried to analyse the current scenario of policy associated with the application of NMs in agro-environment.

Mechanisms of surface groups regulating developmental toxicity of graphene-based nanomaterials via glycerophospholipid metabolic pathway.

Surface modification of graphene-based nanomaterials (GBNs) may occur in aquatic environment and during intentional preparation. However, the influence of the surface groups on the developmental toxicity of GBNs has not been determined. In this study, we evaluated the developmental toxicity of three GBNs including GO (graphene oxide), RGO (reduced GO) and RGO-N (aminated RGO) by employing zebrafish embryos at environmentally relevant concentrations (1-100 µg/L), and the underlying metabolic mechanisms were explored. The results showed that both GO and RGO-N disturbed the development of zebrafish embryos, and the adverse effect of GO was greater than that of RGO-N. Furthermore, the oxygen-containing groups of GBNs play a more important role in inducing developmental toxicity compared to size, defects and nitrogen-containing groups. Specifically, the epoxide and hydroxyl groups of GBNs increased their intrinsic oxidative potential, promoted the generation of ROS, and caused lipid peroxidation. Moreover, a significant decrease in guanosine and abnormal metabolism of multiple glycerophospholipids were observed in all three GBN-treated groups. Nevertheless, GO exposure triggered more metabolic activities related to lipid peroxidation than RGO or RGO-N exposure, and the disturbance intensity of the same metabolite was greater than that of the other two agents. These findings reveal underlying metabolic mechanisms of GBN-induced developmental toxicity.

Exposure to nanoplastics and nanomaterials either single and combined affects the gill-associated microbiome of the Antarctic soft-shelled clam Laternula elliptica.

Nanoplastics and engineering nanomaterials (ENMs) are contaminants of emerging concern (CECs), increasingly being detected in the marine environment and recognized as a potential threat for marine biota at the global level including in polar areas. Few studies have assessed the impact of these anthropogenic nanoparticles in the microbiome of marine invertebrates, however combined exposure resembling natural scenarios has been overlooked. The present study aimed to evaluate the single and combined effects of polystyrene nanoparticles (PS NP) as proxy for nanoplastics and nanoscale titanium dioxide (nano-TiO2) on the prokaryotic communities associated with the gill tissue of the Antarctic soft-shell clam Laternula elliptica, a keystone species of marine benthos Wild-caught specimens were exposed to two environmentally relevant concentrations of carboxylated PS NP (PS-COOH NP, ∼62 nm size) and nano-TiO2 (Aeroxide P25, ∼25 nm) as 5 and 50 µg/L either single and combined for 96h in a semi-static condition.Our findings show a shift in microbiome composition in gills of soft-shell clams exposed to PS NP and nano-TiO2 either alone and in combination with a decrease in the relative abundance of OTU1 (Spirochaetaceae). In addition, an increase of gammaproteobacterial OTUs affiliated to MBAE14 and Methylophagaceae (involved in ammonia denitrification and associated with low-quality water), and the OTU Colwellia rossensis (previously recorded in polluted waters) was observed. Our results suggest that nanoplastics and nano-TiO2 alone and in combination induce alterations in microbiome composition by promoting the increase of negative taxa over beneficial ones in the gills of the Antarctic soft-shell clam. An increase of two low abundance OTUs in PS-COOH NPs exposed clams was also observed. A predicted gene function analysis revealed that sugar, lipid, protein and DNA metabolism were the main functions affected by either PS-COOH NP and nano-TiO2 exposure. The molecular functions involved in the altered affiliated OTUs are novel for nano-CEC exposures.

Building species trait-specific nano-QSARs: Model stacking, navigating model uncertainties and limitations, and the effect of dataset size.

A strong need exists for broadly applicable nano-QSARs, capable of predicting toxicological outcomes towards untested species and nanomaterials, under different environmental conditions. Existing nano-QSARs are generally limited to only a few species but the inclusion of species characteristics into models can aid in making them applicable to multiple species, even when toxicity data is not available for biological species. Species traits were used to create classification- and regression machine learning models to predict acute toxicity towards aquatic species for metallic nanomaterials. Afterwards, the individual classification- and regression models were stacked into a meta-model to improve performance. Additionally, the uncertainty and limitations of the models were assessed in detail (beyond the OECD principles) and it was investigated whether models would benefit from the addition of more data. Results showed a significant improvement in model performance following model stacking. Investigation of model uncertainties and limitations highlighted the discrepancy between the applicability domain and accuracy of predictions. Data points outside of the assessed chemical space did not have higher likelihoods of generating inadequate predictions or vice versa. It is therefore concluded that the applicability domain does not give complete insight into the uncertainty of predictions and instead the generation of prediction intervals can help in this regard. Furthermore, results indicated that an increase of the dataset size did not improve model performance. This implies that larger dataset sizes may not necessarily improve model performance while in turn also meaning that large datasets are not necessarily required for prediction of acute toxicity with nano-QSARs.

Gene expression profiles and protein-protein interaction networks in THP-1 cells exposed to metal-based nanomaterials.

We analyzed gene expression in THP-1 cells exposed to metal-based nanomaterials (NMs) [TiO2 (NM-100), ZnO (NM-110), SiO2 (NM-200), Ag (NM-300 K)]. A functional enrichment analysis of the significant differentially expressed genes (DEGs) identified the key modulated biological processes and pathways. DEGs were used to construct protein-protein interaction networks. NM-110 and NM-300 K induced changes in the expression of genes involved in oxidative and genotoxic stress, immune response, alterations of cell cycle, detoxification of metal ions and regulation of redox-sensitive pathways. Both NMs shared a number of highly connected protein nodes (hubs) including CXCL8, ATF3, HMOX1, and IL1B. NM-200 induced limited transcriptional changes, mostly related to the immune response; however, several hubs (CXCL8, ATF3) were identical with NM-110 and NM-300 K. No effects of NM-100 were observed. Overall, soluble nanomaterials NM-110 and NM-300 K exerted a wide variety of toxic effects, while insoluble NM-200 induced immunotoxicity; NM-100 caused no detectable changes on the gene expression level.

Unveiling key mechanisms: Transcriptomic meta-analysis of diverse nanomaterial applications addressing biotic and abiotic stresses in Arabidopsis Thaliana.

The potential applications of nanomaterials in agriculture for alleviating diverse biotic and abiotic stresses have garnered significant attention. The reported mechanisms encompass promoting plant growth and development, alleviating oxidative stress, inducing defense responses, modulating plant-microbe interactions, and more. However, individual studies may not fully uncover the common pathways or distinguish the effects of different nanostructures. We examined Arabidopsis thaliana transcriptomes exposed to biotic, abiotic, and metal or carbon-based nanomaterials, utilizing 24 microarray chipsets and 17 RNA-seq sets. The results showed that: 1) from the perspective of different nanostructures, all metal nanomaterials relieved biotic/abiotic stresses via boosting metal homeostasis, particularly zinc and iron. Carbon nanomaterials induce hormone-related immune responses in the presence of both biotic and abiotic stressors. 2) Considering the distinct features of various nanostructures, metal nanomaterials displayed unique characteristics in seed priming for combating abiotic stresses. In contrast, carbon nanomaterials exhibited attractive features in alleviating water deprivation and acting as signaling amplifiers during biotic stress. 3) For shared pathway analysis, response to hypoxia emerges as the predominant and widely shared regulatory mechanism governing diverse stress responses, including those induced by nanomaterials. By deciphering shared and specific pathways and responses, this research opens new avenues for precision nano-agriculture, offering innovative strategies to optimize plant resilience, improve stress management, and advance sustainable crop production practices.

Molecular dynamics simulations suggest the potential toxicity of fluorinated graphene to HP35 protein via unfolding the α-helix structure.

Fluorinated graphene, a two-dimensional nanomaterial composed of three atomic layers, a central carbon layer sandwiched between two layers of fluorine atoms, has attracted considerable attention across various fields, particularly for its potential use in biomedical applications. Nonetheless, scant effort has been devoted to assessing the potential toxicological implications of this nanomaterial. In this study, we scrutinize the potential impact of fluorinated graphene on a protein model, HP35 by utilizing extensive molecular dynamics (MD) simulation methods. Our MD results elucidate that upon adsorption to the nanomaterial, HP35 undergoes a denaturation process initiated by the unraveling of the second helix of the protein and the loss of the proteins hydrophobic core. In detail, substantial alterations in various structural features of HP35 ensue, including alterations in hydrogen bonding, Q value, and RMSD. Subsequent analyses underscore that hydrophobic and van der Waals interactions (predominant), alongside electrostatic energy (subordinate), exert influence over the adsorption of HP35 on the fluorinated graphene surface. Mechanistic scrutiny attests that the unrestrained lateral mobility of HP35 on the fluorinated graphene nanomaterial primarily causes the exposure of HP35's hydrophobic core, resulting in the eventual structural denaturation of HP35. A trend in the features of 2D nanostructures is proposed that may facilitate the denaturation process. Our findings not only substantiate the potential toxicity of fluorinated graphene but also unveil the underlying molecular mechanism, which thereby holds significance for the prospective utilization of such nanomaterials in the field of biomedicine.

Hemocompatibility studies in nanotoxicology: Hemolysis or eryptosis? (A review).

Hemocompatibility evaluation is an important step in nanotoxicological studies. It is generally accepted that nanomaterials promote lysis of erythrocytes, blood clotting, alter phagocytosis, and upregulate pro-inflammatory cytokines. However, there are no standardized guidelines for testing nanomaterials hemocompatibility despite the fact that nanomaterials enter the bloodstream and interact with blood cells. In this review, the current knowledge on the ability of nanomaterials to induce distinct cell death modalities of erythrocytes is highlighted primarily focusing on hemolysis and eryptosis. This review aims to summarize the molecular mechanisms underlying erythrotoxicity of nanomaterials and critically compare the sensitivity and efficiency of hemolysis or eryptosis assays for nanomaterials blood compatibility testing. The list of eryptosis-inducing nanomaterials is growing, but it is still difficult to generalize how physico-chemical properties of nanoparticles affect eryptosis degree and molecular mechanisms involved. Thus, another aim of this review is to raise the awareness of eryptosis as a nanotoxicological tool to encourage the corresponding studies. It is worthwhile to consider adding eryptosis to in vitro nanomaterials hemocompatibility testing protocols and guidelines.

Inhibitory effects of black phosphorus nanosheets on tumor cell proliferation through downregulation of ADIPOQ and downstream signaling pathways.

Exposure to environmental pollutants, including nanomaterials, has a significant impact on tumor progression. The increased demand for black phosphorus nanosheets (BPNSs), driven by their exceptional properties, raises concerns about potential environmental contamination. Assessing their toxicity on tumor growth is essential. Herein, we employed a range of biological techniques, including cytotoxicity measurement, bioinformatics tools, proteomics, target gene overexpression, Western blot analysis, and apoptosis detection, to investigate the toxicity of BPNSs across A549, HepG-2, MCF-7, and Caco-2 cell lines. Our results demonstrated that BPNSs downregulated the expression of ADIPOQ and its associated downstream pathways, such as AMP-activated protein kinase (AMPK), nuclear factor erythroid 2-related factor 2 (Nrf2), and other unidentified pathways. These downregulated pathways ultimately led to mitochondria-dependent apoptosis. Notably, the specific downstream pathways involved varied depending on the type of tumors. These insightful findings not only confirm the consistent inhibitory effects of BPNSs across different tumor cells, but also elucidate the cytotoxicity mechanisms of BPNSs in different tumors, providing valuable information for their safe application and health risk assessment.

Nanomaterials-induced programmed cell death: Focus on mitochondria.

Nanomaterials are widely utilized in several domains, such as everyday life, societal manufacturing, and biomedical applications, which expand the potential for nanomaterials to penetrate biological barriers and interact with cells. Multiple studies have concentrated on the particular or improper utilization of nanomaterials, resulting in cellular death. The primary mode of cell death caused by nanotoxicity is programmable cell death, which includes apoptosis, ferroptosis, necroptosis, and pyroptosis. Based on our prior publications and latest research, mitochondria have a vital function in facilitating programmed cell death caused by nanomaterials, as well as initiating or transmitting death signal pathways associated with it. Therefore, this review takes mitochondria as the focal point to investigate the internal molecular mechanism of nanomaterial-induced programmed cell death, with the aim of identifying potential targets for prevention and treatment in related studies.

Paradoxical effects of nanomaterials on plants: Phytohormonal perspective exposes hidden risks amidst potential benefits.

The rapid growth of nanotechnology has led to the production of a significant amount of engineered nanomaterials (NMs), raising concerns about their impact on various domains. This study investigates the negative interactions between NMs and phytohormones in plants, revealing the changes in signaling crosstalk, integrated responses and ecological repercussions caused by NM pollution. Phytohormones, which include auxins, cytokinins, gibberellins, abscisic acid, ethylene, jasmonic acid, salicylic acid and brassinosteroids are essential for plant growth, development, and stress responses. This review examines the intricate relationships between NMs and phytohormones, highlighting disruptions in signaling crosstalk, integrated responses, and ecological consequences in plants due to NM pollution. Various studies demonstrate that exposure to NMs can lead to alterations in gene expression, enzyme functions, and ultimately affect plant growth and stress tolerance. Exposure to NMs has the capacity to affect plant phytohormone reactions by changing their levels, biosynthesis, and signaling mechanisms, indicating a complex interrelation between NMs and phytohormone pathways. The complexity of the relationships between NMs and phytohormones necessitates further research, utilizing modern molecular techniques, to unravel the intricate molecular mechanisms and develop strategies to mitigate the ecological consequences of NM pollution. This review provides valuable insights for researchers and environmentalists concerned about the disruptive effects of NMs on regulating phytohormone networks in plants.

Ageing influences the toxicity of two innovative nanofertilizers to the soil invertebrates Enchytraeus crypticus and Folsomia candida.

The increasing global food demand is threatening the sustainability of agrifood production systems. The intensification of agricultural practices, with inadequate use of pesticides and fertilizers, poses major challenges to the good functioning of agroecosystems and drastically degrades the soil quality. Nanotechnology is expected to optimize the current farming practices and mitigate some associated impacts. Layered double hydroxides (LDHs) are a class of nanomaterials with high potential for use in agricultural productions, mostly due to their sustained release of nutrients. Considering its novelty and lack of studies on the terrestrial ecosystem, it is essential to assess potential long-term harmful consequences to non-target organisms. Our study aimed to evaluate the effect of Zn-Al-NO3 LDH and Mg-Al-NO3 LDH ageing on the survival and reproduction of two soil invertebrate species Enchytraeus crypticus and Folsomia candida. We postulated that the toxicity of nanomaterials to soil invertebrates would change with time, such that the ageing of soil amendments would mediate their impacts on both species. Our results showed that the toxicity of LDHs was species-dependent, with Zn-Al-NO3 LDH being more toxic to E. crypticus, while Mg-Al-NO3 LDH affected more F. candida, especially in the last ageing period, where reproduction was the most sensitive biological parameter. The toxicity of both nanomaterials increased with ageing time, as shown by the decrease of the EC50 values over time. The influence of LDH dissolution and availability of Zn and Mg in the soil pore water was the main factor related to the toxicity, although we cannot rule out the influence of other structural constituents of LDHs (e.g., nitrates and aluminium). This study supports the importance of incorporating ageing in the ecotoxicity testing of nanomaterials, considering their slow release, as effects on soil organisms can change and lead to more severe impacts on the ecosystem functioning.

Developing a Selection Framework for Zinc Ion-Based Biomaterial Design: Guided by the Biosafety Assessment of ZIF-8 and ZnO.

In recent years, nanomaterials have gained widespread use in the biomedical field, with ZIF-8 and ZnO emerging as promising candidates due to their remarkable performance in osteogenesis, angiogenesis, and antimicrobial therapy. However, before advancing these nanomaterials for clinical applications, it is imperative to evaluate their biocompatibility. In particular, comparing nanomaterials with similar biomedical functions is crucial for identifying the most suitable nanomaterials for further development and market entry. Our study aimed to compare the biocompatibility of nano-ZIF-8 and nano-ZnO under the same conditions. We found that nano-ZIF-8 exhibited lower toxicity both in vitro and in vivo compared to nano-ZnO. To gain insights into the underlying mechanisms responsible for this difference, we conducted further experiments to investigate lysosome damage, mitochondrial change, and the occurrence of ferroptosis. Additionally, we performed transcriptome sequencing to analyze the expression of relevant genes, thereby providing robust validation for our findings. In summary, our study highlighted the importance of evaluating nanomaterials with similar biomedical effects. Through this comparative study, we have not only shed light on the superior biocompatibility of nano-ZIF-8 over nano-ZnO, but also contributed valuable insights and methodological references for future material screening endeavors. Ultimately, our study served as a stepping stone toward the development of safer and more effective nanomaterials for various biomedical applications.

Safe and sustainable by design Ag nanomaterials: A case study to evaluate the bio-reactivity in the environment using a soil model invertebrate.

Safe-and-sustainable-by-design (SSbD) nanomaterials (NMs) or NM-containing products are a priority. Silver (Ag) NMs have a vast array of applications, including biomedical and other products, even as nanopesticides. Thus, their release to the environment is expected to increase. The aim of the present study was to assess the ecotoxicity of the SSbD Ag NM to the soil model species Enchytraeus crypticus (Oligochaeta). The Ag NM tested consists in a SSbD Ag with biomedical applications, a hydroxyethyl cellulose (HEC) coated Ag NMs (AgHEC) and its toxicity was compared to the naked Ag NMs (Ag-Sigma), an Ag-based biomedical product (PLLA-Ag: Poly l-Lactide microfibers doped with Ag), and AgNO3. Effects were assessed both in soil and aqueous media, following the standard OECD guideline in soil (28 days) and the OECD extension (56 days), and short-term pulse (5 days) in aqueous media: reconstituted water (ISO water) and soil:water (S:W) extracts, followed by a 21-days recovery period in soil. Ag materials were thoroughly characterized as synthesized and during the test in media and animals. Results in S:W showed AgHEC was more toxic than Ag-Sigma (ca. 150 times) and PLLA-Ag (ca. 2.5 times), associated with a higher Ag uptake. Higher toxicity was related to a smaller hydrodynamic size and higher suspension stability, which in turn resulted in a higher bioavailability of Ag NMs and released ions, particularly in S:W. Toxicity was correlated with the main physicochemical features, providing useful prediction of AgNMs bioactivity. The ability to test E. crypticus in a range of media with different and/or increasing complexity (water, S:W extracts, soil) provided an excellent source to interpret results and is here recommended.

Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices.

The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP.

MoS2 2D materials induce spinal cord neuroinflammation and neurotoxicity affecting locomotor performance in zebrafish.

MoS2 nanosheets belong to an emerging family of nanomaterials named bidimensional transition metal dichalcogenides (2D TMDCs). The use of such promising materials, featuring outstanding chemical and physical properties, is expected to increase in several fields of science and technology, with an enhanced risk of environmental dispersion and associated wildlife and human exposures. In this framework, the assessment of MoS2 nanosheets toxicity is instrumental to safe industrial developments. Currently, the impact of the nanomaterial on the nervous tissue is unexplored. In this work, we use as in vivo experimental model the early-stage zebrafish, to investigate whether mechano-chemically exfoliated MoS2 nanosheets reach and affect, when added in the behavioral ambient, the nervous system. By high throughput screening of zebrafish larvae locomotor behavioral changes upon exposure to MoS2 nanosheets and whole organism live imaging of spinal neuronal and glial cell calcium activity, we report that sub-acute and prolonged ambient exposures to MoS2 nanosheets elicit locomotor abnormalities, dependent on dose and observation time. While 25 µg mL-1 concentration treatments exerted transient effects, 50 µg mL-1 ones induced long-lasting changes, correlated to neuroinflammation-driven alterations in the spinal cord, such as astrogliosis, glial intracellular calcium dysregulation, neuronal hyperactivity and motor axons retraction. By combining integrated technological approaches to zebrafish, we described that MoS2 2D nanomaterials can reach, upon water (i.e. ambient) exposure, the nervous system of larvae, resulting in a direct neurological damage.