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Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse effects, including hepatotoxicity, developmental toxicity and immunotoxicity. So far, little information is available about the mechanisms underlying the toxicity of PFASs, including those related to their immunotoxicity. Reported immunotoxic effects of PFASs include decreased antibody responses in experimental animals and humans, indicating that PFASs may, among others, affect B cell function. In the present study, we first assessed the effects of PFOA on the transcriptome of the human Namalwa B cell line using RNA seq analysis. Gene expression changes, analyzed using Ingenuity Pathway Analysis, pointed to various cellular processes affected by PFOA, including ‘B cell development’ and ‘Primary immunodeficiency signaling’. Interestingly, PFOA decreased the expression of RAG1 and RAG2, genes involved in immunoglobulin and T cell receptor V(D)J recombination. As a next step, time- and concentration-dependent changes in the expression of RAG1 and RAG2 upon exposure to PFOA, PFNA, PFHxS and PFOS were studied through RT-qPCR analysis. Analysis with the concentration–response modeling software PROAST resulted in the following potency ranking: PFNA > PFOA > PFOS > PFHxS. Altogether, the present in vitro study provides insights into the effects of selected PFASs on B cells, identifying RAG1 and RAG2 expression as possible relevant targets that may play a role in the immunotoxicity of PFASs.

There is a global research interest in metal nanoparticles (MNPs) due to their diverse applications, rapidly increasing use, and increased presence in the aquatic environment. Currently, most MNPs in the environment are at levels unlikely to cause overt toxicity. Sub-lethal effects that MNPs may induce, notable immunotoxicity, could however have significant health implications. Thus, deciphering the immunological interactions of MNPs with aquatic organisms constitutes a much-needed area of research. In this article, we critically assess the evidence for immunotoxic effects of MNPs in bivalves and fish, as key wildlife sentinels with widely differing ecological niches that are used as models in ecotoxicology. The first part of this review details the properties, fate, and fundamental physicochemical behavior of MNPs in the aquatic ecosystem. We then consider the toxicokinetics of MNP uptake, accumulation, and deposition in fish and bivalves. The main body of the review then focuses on immune reactions in response to MNPs exposure in bivalves and fish illustrating their immunotoxic potential. Finally, we identify major knowledge gaps in our current understanding of the implications of MNPs exposure for immunological functions and the associated health consequences for bivalves and fish, as well as the general lessons learned on the immunotoxic properties of the emerging class of nanoparticulate contaminants in fish and bivalves.

The continuous release of nanoparticles and nanoplastics into the marine environment necessitates the examination of their combined effects in marine organisms. Natural Organic Matter (NOM) can significantly influence the behavior of nanomaterials in the marine environment. The present study explores the effects of algal Extracellular Polymeric Substances (EPS) in reducing the combined toxic effects of three different polystyrene nanoplastics (PSNPs)— aminated (NH2-PSNPs), carboxylated (COOH-PSNPs), and plain PSNPs — and P25 titanium dioxide nanoparticles (Nano-TiO2) towards the marine alga, Chlorella sp. Two doses (0.25 and 2.5 mg/L) of nano-TiO2 mixed with the PSNPs (1 mg/L) were employed. The COOH-PSNPs with 2.5 mg/L nano-TiO2 exhibited higher growth inhibition toward algal cells. Addition of algal EPS to the mixture of NMs decreased the negative effect significantly. The mean hydrodynamic diameter increased significantly from 666 to 797 nm and 1248 to 3589 nm at concentrations 0.25 and 2.5 mg/L, respectively when the mixtures of nano-TiO2 and COOH-PSNPs were incubated with the algal EPS. In comparison to the pristine NMs, the EPS-NMs were found to significantly reduce the superoxide and hydroxyl radical production. The results were further validated with the estimation of lipid peroxidation (LPO), esterase activity, photosynthetic efficiency, and membrane permeability in the cells. The major outcomes from this study highlight the role of algal EPS in significantly reducing the toxic impact of binary mixture of NMs in marine organisms.

Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse health effects, including hepatotoxicity, developmental toxicity, and immunotoxicity. The aim of the present work was to assess whether human HepaRG liver cells can be used to obtain insight into differences in hepatotoxic potencies of a series of PFASs. Therefore, the effects of 18 PFASs on cellular triglyceride accumulation (AdipoRed assay) and gene expression (DNA microarray for PFOS and RT-qPCR for all 18 PFASs) were studied in HepaRG cells. BMDExpress analysis of the PFOS microarray data indicated that various cellular processes were affected at the gene expression level. From these data, ten genes were selected to assess the concentration–effect relationship of all 18 PFASs using RT-qPCR analysis. The AdipoRed data and the RT-qPCR data were used for the derivation of in vitro relative potencies using PROAST analysis. In vitro relative potency factors (RPFs) could be obtained for 8 PFASs (including index chemical PFOA) based on the AdipoRed data, whereas for the selected genes, in vitro RPFs could be obtained for 11–18 PFASs (including index chemical PFOA). For the readout OAT5 expression, in vitro RPFs were obtained for all PFASs. In vitro RPFs were found to correlate in general well with each other (Spearman correlation) except for the PPAR target genes ANGPTL4 and PDK4. Comparison of in vitro RPFs with RPFs obtained from in vivo studies in rats indicate that best correlations (Spearman correlation) were obtained for in vitro RPFs based on OAT5 and CXCL10 expression changes and external in vivo RPFs. HFPO-TA was found to be the most potent PFAS tested, being around tenfold more potent than PFOA. Altogether, it may be concluded that the HepaRG model may provide relevant data to provide insight into which PFASs are relevant regarding their hepatotoxic effects and that it can be applied as a screening tool to prioritize other PFASs for further hazard and risk assessment.

Per: and polyfluoroalkyl substances (PFASs) have been associated with increased blood lipids in humans. Perfluorooctanoic acid (PFOA) has been also linked with elevated alanine transferase (ALT) serum levels in humans, and in rodents the liver is a main target organ for many PFASs. With the focus on New Approach Methodologies, the chronic oral equivalent effect doses were calculated for PFOA, PFNA (perfluorononanoic acid), PFHxS (perfluorohexanesulfonic acid) and PFOS (perfluorooctane sulfonic acid) based on in vitro effects measured in the HepaRG cell line. Selected in vitro readouts were considered biomarkers for lipid disturbances and hepatotoxicity. Concentration-response data obtained from HepaRG cells on triglyceride (TG) accumulation and expression changes of 12 selected genes (some involved in cholesterol homeostasis) were converted into corresponding human dose-response data, using physiologically based kinetic (PBK) model-facilitated reverse dosimetry. Next to this, the biokinetics of the chemicals were studied in the cell system. The current European dietary PFASs exposure overlaps with the calculated oral equivalent effect doses, indicating that the latter may lead to interference with hepatic gene expression and lipid metabolism. These findings illustrate an in vitro-in silico methodology, which can be applied for more PFASs, to select those that should be prioritized for further hazard characterization.

Risk assessment of chemical substances is always a challenging process. It can be supported by using the potential of the in silico methods such as the read-across approach. Several frameworks and methodologies can be found, e.g. the Read-across Assessment Framework (RAAF) developed by ECHA, which describes how the analysis is carried out using the read-across approach. However, they are focused on classical chemical substances, not nanomaterials. Thus, our goal was to evaluate publicly available read-across frameworks in the context of ENM. Especially, in view of the recent update of the REACH regulations (Annex VI), which introduced the concept of “nanoform” of the substance. We examined the possibilities as well as the challenges for nanomaterials when applying available frameworks by carrying out readacross case studies for selected nanoforms of nano-SiO2. Structural properties of five ENMs and data related to their ecotoxicity were extracted from the JRC Repository characterization dossier on nanoSiO2 amorphous materials and the corresponding NanoReg2 H2020 project deliverable. From all endpoints available, toxicity results towards the Carp leucocyte cell line were considered as the most appropriate. For the purposes of the case study, we decided to treat one of the nano-SiO2 as a target (NM200) and the four others (NM 201- 204) as source analogues. The analysis consisted of several steps: i) identification and characterization of all nanoforms; ii) development of grouping hypothesis; iii) assignment to groups; iv) data gathering; v) applicability assessment; vi) filling data gaps. After passing through all the stages we were able to estimate the toxicity of target ENM. The formulated hypothesis of the read-across approach for the assessment of ecotoxicity was as follows: SiO2 nanoforms can be separated into two distinct groups based on how the following properties influenced cytotoxicity in fish cells: i) surface area, ii) coating mass, iii) size distribution in stock and media solutions, iv) polydispersity in stock and media solutions. This leads to the follow-up hypothesis of novel SiO2 ENMs with similar physicochemical/structural parameters inducing similar toxicological activities in fish cells. Subsequently, we employed similarity analysis in the space of the mentioned properties. Based on the calculated Euclidean distances, the target nanoform (NM200), has been placed within the group of toxic source analogues (NM201 and 204). Therefore, according to the worst-case approach, one can assume that the target nanoform will be highly toxic to fish cells. The results and lessons learned from this exercise will be discussed further in the context of the work carried out in the PATROLS project.

Increasing knowledge of the impacts of pesticides on soil ecological communities is fundamental to a comprehensive understanding of the functional changes in the global agroecosystem industry. In this study, we examined microbial community shifts in the gut of the soil-dwelling organism Enchytraeus crypticus and functional shifts in the soil microbiome (bacteria and viruses) after 21 d of exposure to difenoconazole, one of the main fungicides in intensified agriculture. Our results demonstrated reduced body weight and increased oxidative stress levels of E. crypticus under difenoconazole treatment. Meanwhile, difenoconazole not only altered the composition and structure of the gut microbial community, but also interfered with the soil-soil fauna microecology stability by impairing the abundance of beneficial bacteria. Using soil metagenomics, we revealed that bacterial genes encoding detoxification and viruses encoding carbon cycle genes exhibited a dependent enrichment in the toxicity of pesticides via metabolism. Taken together, these findings advance the understanding of the ecotoxicological impact of residual difenoconazole on the soil-soil fauna micro-ecology, and the ecological importance of virus-encoded auxiliary metabolic genes under pesticide stress.

In recent years, various ecotoxicological test guidelines and (technical) guidance documents have been evaluated and updated with regard to their applicability to nanomaterials (NMs). Several of these have currently reached official regulatory status. Ensuring their harmonized implementation with previously recognized methods for ecotoxicity testing of chemicals is a crucial next step towards effective and efficient regulation of NMs. In the present study, we evaluated the feasibility of assessing multigenerational effects in the first generation of offspring derived from exposed Daphnia magna whilst maintaining test conditions in accordance with regulatory test guidelines and guidance documents for NMs. To do so, we integrated the recommendations for ecotoxicological testing of NMs as defined in OECD Guidance Document 317 into an extended long-term D. magna reproduction test method (OECD Test Guideline 211) and assessed effects of two poorly soluble NMs (nTiO2 and nCeO2). Our results show adverse effects on life-history parameters of D. magna exposed to the selected nanomaterials within the range of reported environmental concentrations. We argue that conforming to OECD test guidelines and accompanying guidance for nanomaterials is feasible when performing D. magna reproduction tests and can minimize unnecessary duplication of similar experiments, even when extensions to the standardized test setup are added.

The goal of the present study was to develop an online web-based toolbox that contains generic physiologically based kinetic (PBK) models for rats and humans, including underlying calculation tools to predict plasma protein binding and tissue:plasma distribution, to be used for quantitative in-vitro-to-in-vivo extrapolations (QIVIVE). The PBK models within the toolbox allow first estimations of internal plasma and tissue concentrations of chemicals to be made, based on the logP and pKa of the chemicals and values for intestinal uptake and intrinsic hepatic clearance. As a case study, the toolbox was used to predict oral equivalent doses of in vitro ToxCast bioactivity data for the food additives methylparaben, propyl gallate, octyl gallate, and dodecyl gallate. These oral equivalent doses were subsequently compared with human exposure estimates, as a low tier assessment allowing prioritization for further assessment. The results revealed that daily intake levels of especially propyl gallate can lead to internal plasma concentrations that are close to in vitro biological effect concentrations, particularly with respect to the inhibition of human thyroid peroxidase (TPO). Estrogenic effects were not considered likely to be induced by the food additives, as daily exposure levels of the different compounds remained 2 orders of magnitude below the oral equivalent doses for in vitro estrogen receptor activation. Overall, the results of the study show how the toolbox, which is freely accessible through www.qivivetools.wur.nl, can be used to obtain initial internal dose estimates of chemicals and to prioritize chemicals for further assessment, based on the comparison of oral equivalent doses of in vitro biological activity data with human exposure levels.

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by β-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine. Electronic supplementary material The online version of this article (10.1007/s00204-020-02953-6) contains supplementary material, which is available to authorized users.

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.

Functionalized high-aspect-ratio silver nanowires (AgNWs) have been recognized as one of the most promising alternatives for fabricating products, with their use ranging from electronic devices to biomedical fields. Given concerns on the safety of AgNWs, there is an urgent need to investigate the relation between intrinsic properties of AgNWs and their toxicity. In this study, lettuce was exposed for either 6 or 18 d to different AgNWs to determine how the size/aspect ratio and coating of AgNWs affect the contributions of the dissolved and particulate Ag to the overall phytotoxicity and uptake kinetics. We found that the uncoated AgNW (39 nm diameter × 8.4 µm length) dissolved fastest of all AgNWs investigated. The phytotoxicity, uptake rate constants, and bioaccumulation factors of the PVP-coated AgNW (43 nm diameter × 1.8 µm length) and the uncoated AgNW (39 nm diameter × 8.4 µm length) were similar, and both were higher than that of the PVP-coated AgNW with the larger diameter(65 nm diameter × 4.4 µm length). These results showed that the diameter of the AgNWs predominantly affected toxicity and Ag accumulation in plants. Particulate Ag was found to be the predominant driver/descriptor of overall toxicity and Ag accumulation in the plants rather than dissolved Ag for all AgNWs tested. The relative contribution of dissolved versus particulate Ag to the overall effects was influenced by the exposure concentration and the extent of dissolution of AgNWs. This work highlights inherent particulate-dependent effects of AgNWs in plants and suggests that toxicokinetics should explicitly be considered for more nanomaterials and organisms, consequently providing more realistic input information for their environmental risk assessment.

Background The EU-project GRACIOUS developed an Integrated Approach to Testing and Assessment (IATA) to support grouping high aspect ratio nanomaterials (HARNs) presenting a similar inhalation hazard. Application of grouping reduces the need to assess toxicity on a case-by-case basis and supports read-across of hazard data from substances that have the data required for risk assessment (source) to those that lack such data (target). The HARN IATA, based on the fibre paradigm for pathogenic fibres, facilitates structured data gathering to propose groups of similar HARN and to support read-across by prompting users to address relevant questions regarding HARN morphology, biopersistence and inflammatory potential. The IATA is structured in tiers, allowing grouping decisions to be made using simple in vitro or in silico methods in Tier1 progressing to in vivo approaches at the highest Tier3. Here we present a case-study testing the applicability of GRACIOUS IATA to form an evidence-based group of multiwalled carbon nanotubes (MWCNT) posing a similar predicted fibre-hazard, to support read-across and reduce the burden of toxicity testing. Results The case-study uses data on 15 different MWCNT, obtained from the published literature. By following the IATA, a group of 2 MWCNT was identified (NRCWE006 and NM-401) based on a high degree of similarity. A pairwise similarity assessment was subsequently conducted between the grouped MWCNT to evaluate the potential to conduct read-across and fill data gaps required for regulatory hazard assessment. The similarity assessment, based on expert judgement of Tier 1 assay results, predicts both MWCNT are likely to cause a similar acute in vivo hazard. This result supports the possibility for read-across of sub-chronic and chronic hazard endpoint data for lung fibrosis and carcinogenicity between the 2 grouped MWCNT. The implications of accepting the similarity assessment based on expert judgement of the MWCNT group are considered to stimulate future discussion on the level of similarity between group members considered sufficient to allow regulatory acceptance of a read-across argument. Conclusion This proof-of-concept case-study demonstrates how a grouping hypothesis and IATA may be used to support a nuanced and evidence-based grouping of ‘similar’ MWCNT and the subsequent interpolation of data between group members to streamline the hazard assessment process.

Humans are chronically exposed to the mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1), as indicated by their widespread presence in foods and occasional exposure in the workplace. This exposure is confirmed by human biomonitoring (HBM) studies on (metabolites of) these mycotoxins in human matrices. We evaluated the exposure-health relationship of the mycotoxins in humans by reviewing the available literature. Since human studies did not allow the identification of unequivocal chronic health effects upon exposure to DON and FB1, the adverse outcome pathway (AOP) framework was used to structure additional mechanistic evidence from in vitro and animal studies on the identified adverse effects. In addition to a preliminary AOP for DON resulting in the adverse outcome (AO) 'reduced body weight gain', we developed a more elaborated AOP for FB1, from the molecular initiating event (MIE) 'inhibition of ceramide synthases' leading to the AO 'neural tube defects'. The mechanistic evidence from AOPs can be used to support the limited evidence from human studies, to focus FB1- and DON-related research in humans to identify related early biomarkers of effect. In order to establish additional human exposure-health relationships in the future, recommendations are given to maximize the information that can be obtained from HBM. Humans are chronically exposed to the mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1), as indicated by their widespread presence in foods and occasional exposure in the workplace. This exposure is confirmed by human biomonitoring (HBM) studies on (metabolites of) these mycotoxins in human matrices. We evaluated the exposure-health relationship of the mycotoxins in humans by reviewing the available literature. Since human studies did not allow the identification of unequivocal chronic health effects upon exposure to DON and FB1, the adverse outcome pathway (AOP) framework was used to structure additional mechanistic evidence from in vitro and animal studies on the identified adverse effects. In addition to a preliminary AOP for DON resulting in the adverse outcome (AO) 'reduced body weight gain', we developed a more elaborated AOP for FB1, from the molecular initiating event (MIE) 'inhibition of ceramide synthases' leading to the AO 'neural tube defects'. The mechanistic evidence from AOPs can be used to support the limited evidence from human studies, to focus FB1- and DON-related research in humans to identify related early biomarkers of effect. In order to establish additional human exposure-health relationships in the future, recommendations are given to maximize the information that can be obtained from HBM.

Various modern products have metallic nanoparticles (MNPs) embedded to enhance products performance. Technological advances enable nowadays even multiple hybrid nanoparticles. Consequently, the future co-release of multiple MNPs will inevitably result in the presence of MNP mixtures in the environment. An important question is if the responses of mixtures of MNPs can be dealt with in a similar way as with the responses of biota to mixtures of metal salts. Moreover, natural organic matter (NOM) is an important parameter affecting the behavior and effect of MNPs. Herein, we determined the joint toxicity and accumulation of copper nanoparticles (CuNPs) and zinc oxide nanoparticles (ZnONPs) in Daphnia magna in the absence and presence of Suwannee River natural organic matter (SR-NOM), compared to the joint toxicity and accumulation of corresponding metal salts. The results of toxicity testing showed that the joint toxicity of CuNPs + ZnONPs was greater than the single toxicity of CuNPs or ZnONPs. The joint toxic action of CuNPs + ZnONPs was additive or more-than-additive for D. magna. A similar pattern was found in the toxicity of the mixtures of Cu- and Zn-salts from the literature data. The presence of SR-NOM had no significant impact on the joint toxicity of CuNPs + ZnONPs. The calculated component-specific contribution to overall toxicity indicated that SR-NOM increased the relative contribution of dissolved ions released from the MNPs to the toxicity of the binary mixtures at high-effect concentrations of individual MNPs. Moreover, dissolved Zn-ions released from the ZnONPs were found to dominate the joint toxicity of CuNPs + ZnONPs in the presence of SR-NOM. Furthermore, the results of the accumulation experiment displayed that the presence of SR-NOM significantly enhanced the accumulation of either CuNPs or ZnONPs in D. magna exposed to the MNP mixtures.

Pyrrolizidine alkaloids (PAs) are produced by various plant species and have been detected as contaminants in food and feed. Monitoring programmes should include PAs that are present in relevant matrices and that exhibit a high toxic potential. The aim of the present study was to use a bioassay-directed analysis approach to identify relevant PAs not yet included in monitoring programmes. To that end, extracts of Heliotropium europaeum and H. popovii were prepared and analysed with LC–MS/MS for the presence of 35 PAs included in monitoring programmes, as well as for genotoxic activity in the HepaRG/γH2AX assay. Europine, heliotrine and lasiocarpine were found to be the most abundant PAs. The extracts showed a higher γH2AX activity than related artificial mixtures of quantified known PAs, which might point to the presence of unknown toxic PAs. The H. europaeum extract was fractionated and γH2AX activities of individual fractions were determined. Fractions were further analysed applying LC–Orbitrap-MS analysis and Compound Discoverer software, identifying various candidate PAs responsible for the non-explained genotoxic activity. Altogether, the results obtained show that bioassay-directed analysis allows identification of candidate PAs that can be included in monitoring programmes.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAsʼ pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

In this study five types of herbal teas were used to quantify the effect of comminution of the leaves on resulting PA exposure. Results show that PA levels extracted from intact leaves were consistently lower than from comminuted tea leaves. The Margin of Exposure (MOE) approach was applied to evaluate the consequences of this difference for the associated risks in the scenario of lifetime exposure. Furthermore, we considered medicinal use of these teas for shorter-than-lifetime exposure scenarios, and also analysed the risks of shorter-than-lifetime use of eight herbal medicines and 19 previously analysed plant food supplements. This analysis revealed that shorter-than-lifetime use resulted in MOE values < 10,000 upon use for 40–3450 weeks during a lifetime, with for only a limited number of herbal teas and medicines use of two weeks a year (150 weeks during a 75 year lifetime) would still raise a concern. It is concluded that taking more realistic conditions into account markedly reduces the concerns raised for these herbal preparations. These results also illustrate the need for development of a generally accepted method for taking short term exposure into account in risk assessment of compounds that are genotoxic and carcinogenic.

With the rapid development of nanotechnology in agriculture, there is increasing urgency to assess the impacts of nanoparticles (NPs) on the soil environment. This study merged raw high-throughput sequencing (HTS) data sets generated from 365 soil samples to reveal the potential ecological effects of NPs on soil microbial community by means of metadata analysis and machine learning methods. Metadata analysis showed that treatment with nanoparticles did not have a significant impact on the alpha diversity of the microbial community, but significantly altered the beta diversity. Unfortunately, the abundance of several beneficial bacteria, such as Dyella, Methylophilus, Streptomyces, which promote the growth of plants, and improve pathogenic resistance, was reduced under the addition of synthetic nanoparticles. Furthermore, metadata demonstrated that nanoparticles treatment weakened the biosynthesis ability of cofactors, carriers, and vitamins, and enhanced the degradation ability of aromatic compounds, amino acids, etc. This is unfavorable for the performance of soil functions. Besides the soil heterogeneity, machine learning uncovered that a) the exposure time of nanoparticles was the most important factor to reshape the soil microbial community, and b) long-term exposure decreased the diversity of microbial community and the abundance of beneficial bacteria. This study is the first to use a machine learning model and metadata analysis to investigate the relationship between the properties of nanoparticles and the hazards to the soil microbial community from a macro perspective. This guides the rational use of nanoparticles for which the impacts on soil microbiota are minimized.

Associations between per- and polyfluoroalkyl substances (PFASs) and increased blood lipids have been repeatedly observed in humans, but a causal relation has been debated. Rodent studies show reverse effects, i.e. decreased blood cholesterol and triglycerides, occurring however at PFAS serum levels at least 100-fold higher than those in humans. This paper aims to present the main issues regarding the modulation of lipid homeostasis by the two most common PFASs, PFOS and PFOA, with emphasis on the underlying mechanisms relevant for humans. Overall, the apparent contrast between human and animal data may be an artifact of dose, with different molecular pathways coming into play upon exposure to PFASs at very low versus high levels. Altogether, the interpretation of existing rodent data on PFOS/PFOA-induced lipid perturbations with respect to the human situation is complex. From a mechanistic perspective, research on human liver cells shows that PFOS/PFOA activate the PPAR alpha pathway, whereas studies on the involvement of other nuclear receptors, like PXR, are less conclusive. Other data indicate that suppression of the nuclear receptor HNF4 alpha signaling pathway, as well as perturbations of bile acid metabolism and transport might be important cellular events that require further investigation. Future studies with human-relevant test systems would help to obtain more insight into the mechanistic pathways pertinent for humans. These studies shall be designed with a careful consideration of appropriate dosing and toxicokinetics, so as to enable biologically plausible quantitative extrapolations. Such research will increase the understanding of possible perturbed lipid homeostasis related to PFOS/ PFOA exposure and the potential implications for human health.

Analytical limitations considerably hinder our understanding of the impacts of the physicochemical properties of nanomaterials (NMs) on their biological fate in organisms. Here, using a fit-for-purpose analytical workflow, including dosing and emerging analytical techniques, NMs present in organisms are characterized and quantified across an aquatic food chain. The size and shape of gold (Au)-NMs are shown to control the number of Au-NMs attached to algae that were exposed to an equal initial concentration of 2.9 × 1011 particles mL−1. The Au-NMs undergo size/shape-dependent dissolution and agglomeration in the gut of the daphnids, which determines the size distribution of the NMs accumulated in fish. The biodistribution of NMs in fish tissues (intestine, liver, gills, and brain) also depends on NM size and shape, although the highest particle numbers per unit of mass are almost always present in the fish brain. The findings emphasize the importance of physicochemical properties of metallic NMs in their biotransformations and tropic transfers., Biological fate of nanomaterials in organisms is an important topic, however, limitations of analytical techniques has hampered understanding. Here, the authors report on a study into the fate of model, gold nanoparticles in an aquatic food chain using an analytical workflow and range of analytical methods.

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by β-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.

10 pages, 2 figures, 3 tables, Chronic toxicity of copper (Cu) at sublethal levels is associated with ionoregulatory disturbance and oxidative stress. These factors were considered in a toxicokinetic-toxicodynamic model in the present study. The ionoregulatory disturbance was evaluated by the activity of the Na+/K+-ATPase enzyme (NKA), while oxidative stress was presented by lipid peroxidation (LPO) and glutathione-S-transferase (GST) activity. NKA activity was related to the binding of Cu2+ and Na + to NKA. LPO and GST activity were linked with the simulated concentration of unbound Cu. The model was calibrated using previously reported data and empirical data generated when zebra mussels were exposed to Cu. The model clearly demonstrated that Cu might inhibit NKA activity by reducing the number of functional pump sites and the limited Cu-bound NKA turnover rate. An ordinary differential equation was used to describe the relationship between the simulated concentration of unbound Cu and LPO/GST activity. Although this method could not explain the fluctuations in these biomarkers during the experiment, the measurements were within the confidence interval of estimations. Model simulation consistently shows non-significant differences in LPO and GST activity at two exposure levels, similar to the empirical observation, This research was financed by the Deutsche Forschungsgemeinschaft (DFG), Germany (LE 3716/2-1)

The existing limitations in analytical techniques for characterization and quantification of nanoscale plastic debris (NPD) in organisms hinder understanding of the parental and trophic transfer of NPD in organisms. Herein, we used iron oxide-doped polystyrene (PS) NPD (Fe-PS-NPD) of 270 nm and Europium (Eu)-doped PS-NPD (Eu-PS-NPD) of 640 nm to circumvent these limitations and to evaluate the influence of particle size on the trophic transfer of NPD along an algae-daphnids food chain and on the reproduction of daphnids fed with NPD-exposed algae. We used Fe and Eu as proxies for the Fe-PS-NPD and Eu-Ps-NPD, respectively. The algae cells (Pseudokirchinella subcapitata) were exposed to 4.8 × 1010 particles/L of Fe-PS-NPD or Eu-PS-NPD for 72 h. A high percentage (>60%) of the NPD was associated with algal cells. Only a small fraction (

An approach to solve the emerging need of prediction of the toxicity of mixtures of engineered nanoparticles (ENPs) is presented. The integration of classic approaches to mixture toxicity assessmen...

Global production of fibrous material is significantly growing reaching an expected 145 million metric tons in 2030. Fiber production includes mostly synthetic polymers, cotton and man-made cellulose (viscose). The main uses are in clothing, household and furnishing, industrial construction, automotive and other. Increasing consumption of fabric material causes the accumulation of single fibers into the natural environment. Significant numbers are discharged via wastewater from washing clothes, deposition from atmosphere or by other ways of transport. Fibers are now the most prevalent type of anthropogenic particles found by microplastic pollution surveys around the world. Substantial fiber concentrations are found in surface water, deep-sea and fresh water ecosystems. Consequently, fibers are present in food, drinking water, human lungs and digestive tracts of aquatic animals. Currently, there is great concern for the release of plastic nano- and micro fibers and microparticles (microplastics) to the natural environment for which nobody knows, so far, the ultimate consequences for health and ecological homeostasis. The potential risk introduced by the presence of fibers in the environment induces significant interest.These challenges were the source of inspiration for organizing our workshop . A group of scientists from different parts of the world met on Nov 4/5 2019 at Wetsus, European Centre of Excellence for Sustainable Water Technology in Leeuwarden, The Netherlands, to discuss all known aspects of synthetic nano- and microfibers. This included morphology, physicochemical properties, production and origin of nano/micro fibers entering the atmosphere, water and food chain; the potential consequences of inhalation and ingestion for human health; exposure and ingress via life cycle for aquatic biota; analytical and measurement methods; techniques to clean air and water, and protection means against inhalation or other ways to enter the human body.

Due to the large amount of chemical substances on the market, fast and reproducible screening is essential to prioritize chemicals for further evaluation according to highest concern. We here evaluate the performance of structural similarity models that are developed to identify potential substances of very high concern (SVHC) based on structural similarity to known SVHCs. These models were developed following a systematic analysis of the performance of 112 different similarity measures for varying SVHC-subgroups. The final models consist of the best combinations of fingerprint, similarity coefficient and similarity threshold, and suggested a high predictive performance (≥80%) on an internal dataset consisting of SVHC and non-SVHC substances. However, the application performance on an external dataset was not evaluated. Here, we evaluated the application performance of the developed similarity models with a 'pseudo-external assessment' on a set of substances (n = 60-100 for the varying SVHC-subgroups) that were putatively assessed as SVHC or non-SVHC based upon consensus scoring using expert elicitations (n = 30 experts). Expert scores were direct evaluations based on structural similarity to the most similar SVHCs according to the similarity models, and did not consider an extensive evaluation of available data. The use of expert opinions is particularly suitable as this is exactly the intended purpose of the chemical similarity models: a quick, reproducible and automated screening tool that mimics the expert judgement that is frequently applied in various screening applications. In addition, model predictions were analyzed via qualitative approaches and discussed via specific examples, to identify the model's strengths and limitations. The results indicate a good statistical performance for carcinogenic, mutagenic or reprotoxic (CMR) and endocrine disrupting (ED) substances, whereas a moderate performance was observed for (very) persistent, (very) bioaccumulative and toxic (PBT/vPvB) substances when compared to expert opinions. For the PBT/vPvB model, particularly false positive substances were identified, indicating the necessity of outcome interpretation. The developed similarity models are made available as a freely-accessible online tool. In general, the structural similarity models showed great potential for screening and prioritization purposes. The models proved to be effective in identifying groups of substances of potential concern, and could be used to identify follow-up directions for substances of potential concern.

Per- and polyfluoroalkyl substances (PFASs) are omnipresent in the environment, food chain, and humans. Epidemiological studies have shown a positive association between serum levels of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), and increased serum cholesterol and, in some cases, also triglyceride levels. However, causality has been questioned, as animal studies, as well as a human trial, showed a decrease in serum cholesterol and no effects or a decrease in plasma triglycerides. To obtain more insight into the effects of PFASs on these processes, the present study investigated the effects of PFOA, PFOS, and perfluorononanoic acid (PFNA) on intracellular triglyceride and cholesterol levels in human HepaRG liver cells. DNA microarray analyses were performed to provide insight into underlying mechanisms. All PFASs induced an increase in cellular triglyceride levels, but had no effect on cholesterol levels. Gene set enrichment analysis (GSEA) of the microarray data indicated that gene sets related to cholesterol biosynthesis were repressed by PFOA, PFOS, and PFNA. Other gene sets commonly affected by all PFAS were related to PERK/ATF4 signaling (induced), tRNA amino-acylation (induced), amino acid transport (induced), and glycolysis/gluconeogenesis (repressed). Moreover, numerous target genes of peroxisome proliferator-activated receptor α (PPARα) were found to be upregulated. Altogether, the present study shows that PFOA, PFOS, and PFNA increase triglyceride levels and inhibit cholesterogenic gene expression in HepaRG cells. In addition, the present study indicates that PFASs induce endoplasmic reticulum stress, which may be an important mechanism underlying some of the toxic effects of these chemicals. Electronic supplementary material The online version of this article (10.1007/s00204-020-02808-0) contains supplementary material, which is available to authorized users.

Titanium dioxide nanoparticles (nano-TiO

Made available in DSpace on 2020-12-12T01:06:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-03-15 We systematically investigated how the combinations of size, shape and the natural organic matter (NOM)-ecocorona of gold (Au) engineered nanoparticles (ENPs) influence the attachment of the particles to algae and physical toxicity to the cells. Spherical (10, 60 and 100 nm), urchin-shaped (60 nm), rod-shaped (10 × 45, 40 × 60 and 50 × 100 nm), and wire-shaped (75 × 500, 75 × 3000 and 75 × 6000 nm) citrate-coated and NOM-coated Au-ENPs were used. Among the spherical particles only the spherical 10 nm Au-ENPs caused membrane damage to algae. Only the rod-shaped 10 × 45 nm induced membrane damage among the rod-shaped Au-ENPs. Wire-shaped Au-ENPs caused no membrane damage to the algae. NOM ecocorona decreased the membrane damage effects of spherical 10 nm and rod-shaped 10 × 45 nm ENPs. The spherical Au-ENPs were mostly loosely attached to the cells compared to other shapes, whereas the wire-shaped Au-ENPs were mostly strongly attached compared to particles with other shapes. NOM ecocorona determined the strength of Au-ENPs attachment to the cell wall, leading to the formation of loose rather than strong attachment of Au-ENPs to the cells. After removal of the loosely and strongly attached Au-ENPs, some particles remained anchored to the surface of the algae. The highest concentration was detected for spherical 10 nm Au-ENPs followed by rod-shaped 10 × 45 nm Au-ENPs, while the lowest concentration was observed for the wire-shaped Au-ENPs. The combined effect of shape, size, and ecocorona controls the Au-ENPs attachment and physical toxicity to cells. Institute of Environmental Sciences (CML) Leiden University Institute of Hydrobiology and Soil & Water Research Infrastructure Biology Centre CAS Department of Physics and Chemistry School of Engineering São Paulo State University (UNESP) School of Geography Earth and Environmental Science University of Birmingham Center for Safety of Substances and Products National Institute of Public Health and the Environment (RIVM) Department of Physics and Chemistry School of Engineering São Paulo State University (UNESP)

Silver nanoparticles (AgNPs) are widely used because of their excellent antibacterial properties. They are, however, easily discharged into the water environment, causing potential adverse environmental effects. Meta-transcriptomic analyses are helpful to study the transcriptional response of prokaryotic and eukaryotic aquatic microorganisms to AgNPs. In the present study, microcosms were used to investigate the toxicity of AgNPs to a natural aquatic microbial community. It was found that a 7-day exposure to 10 μg L-1 silver nanoparticles (AgNPs) dramatically affected the structure of the microbial community. Aquatic micro eukaryota (including eukaryotic algae, fungi, and zooplankton) and bacteria (i.e., heterotrophic bacteria and cyanobacteria) responded differently to the AgNPs stress. Meta-transcriptomic analyses demonstrated that eukaryota could use multiple cellular strategies to cope with AgNPs stress, such as enhancing nitrogen and sulfur metabolism, over-expressing genes related to translation, amino acids biosynthesis, and promoting bacterial-eukaryotic algae interactions. By contrast, bacteria were negatively affected by AgNPs with less signs of detoxification than in case of eukaryota; various pathways related to energy metabolism, DNA replication and genetic repair were seriously inhibited by AgNPs. As a result, eukaryotic algae (mainly Chlorophyta) dominated over cyanobacteria in the AgNPs treated microcosms over the 7-d exposure. The present study helps to understand the effects of AgNPs on aquatic microorganisms and provides insights into the contrasting AgNPs toxicity in eukaryota and bacteria.

There are various types of hepatic steatosis of which non-alcoholic fatty liver disease, which may be caused by exposure to chemicals and environmental pollutants is the most prevalent, representing a potential major health risk. QSAR modelling has the potential to provide a rapid and cost-effective method to identify compounds which may trigger steatosis. Although models exist to predict key molecular initiating events of steatosis such as nuclear receptor binding, we are aware of no models to predict the apical effect steatosis. In this study, we describe the development of a QSAR model to predict steatosis using freely available machine learning tools. It was built using a dataset of 207 pharmaceuticals and pesticides which were identified as steatotic or non-steatotic from existing data from in vivo human and animal studies. The best performing model developed using the linear discriminant analysis module in TANAGRA, based on four chemical descriptors, had an accuracy of 70 %, a sensitivity of 66% and a specificity of 74 %. The expansion of the steatosis dataset to other chemical types, to enable the development of further models, would be of benefit in the identification of compounds with a range of mechanisms of action contributing to steatosis.

Whether toxicity of silver nanoparticles (AgNPs) to organisms originates from the nanoparticles themselves or from the dissolved Ag-ions is still debated, with the majority of studies claiming that extracellular release of Ag-ions is the main cause of toxicity. The objective of this study was to determine the contributions of both particles and dissolved ions to toxic responses, and to better understand the underlying mechanisms of toxicity. In addition, the pathways of AgNPs exposure to plants might play an important role and therefore are explicitly studied as well. We systematically assessed the phytotoxicity, internalization, biodistribution, and antioxidant responses in lettuce (Lactuca sativa) following root or foliar exposure to AgNPs and ionic Ag at various concentrations. For each endpoint the relative contribution of the particle-specific versus the ionic form was quantified. The results reveal particle-specific toxicity and uptake of AgNPs in lettuce as the relative contribution of particulate Ag accounted for more than 65% to the overall toxicity and the Ag accumulation in whole plant tissues. In addition, particle toxicity is shown to originate from the accumulation of Ag in plants by blocking nutrient transport, while ion toxicity is likely due to the induction of excess ROS production. Root exposure induced higher toxicity than foliar exposure at comparable exposure levels. Ag was found to be taken up and subsequently translocated from the exposed parts of plants to other portions regardless of the exposure pathway. These findings suggest particle related toxicity, and demonstrate that the accumulation and translocation of silver nanoparticles need to be considered in assessment of environmental risks and of food safety following consumption of plants exposed to AgNPs by humans.

Soybean toxin (SBTX) is a protein isolated from soybean seeds and composed of two polypeptide subunits (17 and 27 kDa). SBTX has in vitro activity against phytopathogenic fungi such as Cercospora sojina, Aspergillus niger, and Penicillium herguei, and yeasts like Candida albicans, C. parapsilosis, Kluyveromyces marxiannus, and Pichia membranifaciens. The present study aimed to analyze in vitro whether SBTX causes any side effects on non-target bacterial and mammalian cells that could impede its potential use as a novel antifungal agent. SBTX at 100 mu g/mL and 200 mu g/mL did not hinder the growth of the bacteria Salmonella enterica (subspecies enterica serovar choleraesuis), Bacillus subtilis (subspecies spizizenii) and Staphylococcus aureus. Moreover, SBTX at concentrations up to 500 mu g/mL did not significantly affect the viability of erythrocytes, neutrophils, and human intestinal Caco-2 cells. To study whether SBTX could induce relevant alterations in gene expression, in vitro DNA microarray experiments were conducted in which differentiated Caco-2 cells were exposed for 24 h to 100 mu g/mL or 200 mu g/mL SBTX. SBTX up-regulated genes involved in cell cycle and immune response pathways, but downregulated genes that play a role in cholesterol biosynthesis and platelet degranulation pathways. Thus, although SBTX did not affect bacteria, nor induced cytotoxity in mammalian cells, it affected some biological pathways in the human Caco-2 cell line that warrants further investigation.

Metal-based nanoparticles exhibiting antimicrobial activity are of emerging concern to human and environmental health. In addition to their direct adverse effects to plants and animals, indirect effects resulting from disruption of beneficial host-microbiota interactions may contribute to the toxicity of these particles. To explore this hypothesis, we compared the acute toxicity of silver and zinc oxide nanoparticles (nAg and nZnO) to zebrafish larvae that were either germ-free or colonized by microbiota. Over two days of exposure, germ-free zebrafish larvae were more sensitive to nAg than microbially colonized larvae, whereas silver ion toxicity did not differ between germ-free and colonized larvae. Using response addition modeling, we confirmed that the protective effect of colonizing microbiota against nAg toxicity was particle-specific. Nearly all mortality among germ-free larvae occurred within the first day of exposure. In contrast, mortality among colonized larvae increased gradually over both exposure days. Concurrent with this gradual increase in mortality was a marked reduction in the numbers of live host-associated microbes, suggesting that bactericidal effects of nAg on protective microbes resulted in increased mortality among colonized larvae over time. No difference in sensitivity between germ-free and colonized larvae was observed for nZnO, which dissolved rapidly in the exposure medium. At sublethal concentrations, these particles moreover did not exert detectable bactericidal effects on larvae-associated microbes. Altogether, our study shows the importance of taking host-microbe interactions into account in assessing toxic effects of nanoparticles to microbially colonized hosts, and provides a method to screen for microbiota interference with nanomaterial toxicity.

Humans are exposed to pesticide residues through various food products. As these residues can occur in mixtures, there is a need to investigate possible mixture effects on human health. Recent exposure studies revealed the preponderance of imazalil, thiacloprid, and clothianidin in food diets. In this study, we assessed their toxicity alone and in binary mixtures in a 28-day gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg bw/day) ranging from a typical toxicological reference value to a clear effect dose were applied. Data show that the liver was a target organ of all pesticides and their mixtures. Increases in liver weight were observed and histopathological examination revealed centrilobular hepatocellular hypertrophy and cytoplasm degeneration for all treatment conditions. No accumulation of hepatic triglycerides was reported. Tissue residue analysis showed altered pesticide residues in the liver and the kidney when being in mixture as compared to the levels of pesticide residues for the single compound treatment, indicating possible toxicokinetic interactions. Overall, all mixtures appeared to follow the additivity concept, even though quantitative analysis was limited for some endpoints due to the semi-quantitative nature of the data, raising no specific concern for the risk assessment of the examined pesticides.

Non-animal methods for toxicokinetics, such as in vitro hepatic metabolic clearance studies, play an important role in chemical risk evaluations. To gain regulatory acceptance of such clearance data, the development of a test guideline for performing in vitro hepatic clearance studies is crucial. The aim of the present study was to obtain insight in the experimental conditions of clearance studies that influence obtained intrinsic clearance (CLint) values. To that end, in vitro hepatic CLint data obtained with rat or human hepatocytes and methodological aspects of the experiments, were collected from 42 different suitable studies published between 1995 and 2018. The CLint values for the majority of chemicals differed by more than one order of magnitude. We estimated the systematic effect of different experimental setups on the CLint values using a random forest regression analysis, revealing that 'hepatocyte concentration', 'species' (rat or human hepatocytes) and 'culture medium' have the largest impact. Calculating unbound CLint (CLint,u) values slightly reduced the variation for most chemicals. Given that in vivo clearance is in general underpredicted based on in vitro clearance data, a harmonized protocol is preferably based on a protocol that provides relatively high in vitro CLint values.

A full-factorial test design was applied to systematically investigate the contribution and significance of water chemistry parameters (pH, divalent cations and dissolved organic carbon (DOC) concentration) and their interactions on the behavior and fate of copper nanoparticles (CuNPs). The total amount of Cu remaining in the water column after 48 h of incubation was mostly influenced by divalent cation content, DOC concentration and the interaction of divalent cations and DOC. DOC concentration was the predominant factor influencing the dissolution of CuNPs, which was far more important than the effect of pH in the range from 6 to 9 on the dissolution of the CuNPs. The addition of DOC at concentrations ranging from 5 to 50 mg C/L resulted in a 3–5 fold reduction of dissolution of CuNPs after 48 h of incubation, as compared to the case without addition of DOC. Divalent cation content was found to be the most influential factor regarding aggregation behavior of the particles, followed by DOC concentration and the interaction of divalent cations and DOC. In addition, the aggregation behavior of CuNPs rather than particulate dissolution explained most of the variance in the sedimentation profiles of CuNPs. These results are meaningful for improved understanding and prediction of the behavior and fate of metallic NPs in aqueous environments.

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish.

Wheat is one of several cereals that is capable of accumulating higher amounts of Cd in plant tissues. It is important to understand the Cd2+ transport processes in roots that result in excess Cd accumulation. Traditional destructive technologies have limited capabilities in analyzing root samples due to methodological limitations, and sometimes may result in false conclusions. The mechanisms of Cd2+ uptake into the roots of wheat seedlings (Triticum aestivum L.) were investigated by assessing the impact of various inhibitors and channel blockers on Cd accumulation as well as the real-time net Cd2+ flux at roots with the non-destructive scanning ion-selective electrode technique. The P-type ATPase inhibitor Na3VO4 (500 μM) had little effect on Cd uptake (p

Microbial reduction of selenium (Se) oxyanions to elemental Se is a promising technology for bioremediation and treatment of Se wastewaters. But a fraction of biogenic nano-Selenium (nano-Seb) form...

Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.

Suitable methods and fit-for-purpose techniques are required to allow characterization of carbon-based nanomaterials (CB-NMs) in complex matrices. In this study, two methods were developed; a method for extraction and characterization of CB-NMs in biological media and a method for fractionation of natural organic matter (NOM) coated CB-NMs in environmental matrices. The former method was developed by extracting carbon nanotubes (CNTs: sized 0.75 × 3000 nm) and nanoplastics (sized 60, 200 and 600 nm) from eggshells and characterizing the extracted CB-NMs in terms of particle size distribution using asymmetrical flow field-flow fractionation (AF4) coupled with multi-angle light scattering (MALS). The latter method was developed using AF4-MALS to fraction NOM-coated CNT (sized 0.75 × 3000 nm) and nanoplastics (sized 60, 200 and 300 nm) in a simulated natural surface water and provide information about the size distribution of the CB-NM-NOM complexes. The developed AF4-MALS method successfully fractioned the CB-NM-NOM complexes based on hydrodynamic size and provided the size distribution of the complexes. The NOM corona did not shift significantly the median size of the CB-NMs. It influenced however the size distribution of the nanoplastics and CNTs. The sample preparation method failed to extract the CNTs (recovery 60%). The AF4-MALS fractogram showed that the extraction method did not significantly influence the size distribution of the nanoplastics of 60 and 200 nm size, whereas the peak of 600 nm nanoplastics shifted towards a smaller hydrodynamic size. In conclusion, the developed sample preparation method followed by the developed AF4-MALS method can be applied for extraction, separation and characterization of CB-NMs in biological and environmental matrices. Thus, the methods have a high potential to be methods of choice to investigate CB-NMs in future studies.

There is a strong demand for early stage identification of potential Substances of Very High Concern (SVHC). SVHCs are substances that are classified as carcinogenic, mutagenic or reprotoxic (CMR); persistent, bioaccumulative and toxic (PBT) or very persistent and very bioaccumulative (vPvB); or as substances with an equivalent level of concern, like endocrine disruption (ED). The endeavor to improve the identification of potential SVHCs is also acknowledged by the European Commission, in their long-term vision towards a non-toxic environment. However, it has been shown difficult to identify substances as potentially harmful.With this goal in mind, we have developed a methodology that predicts whether a substance is a potential SVHC based on chemical similarity to chemicals already identified as SVHC. The approach is based on the structural property principle, which states that structurally similar chemicals are likely to have similar properties.We systematically analyzed the predictive performance of 112 similarity measures (i.e. all different combinations of 16 binary fingerprints and 7 similarity coefficients) classifying the substances in the dataset as (potential) SVHC or non-SVHC. The outcomes were analyzed for 546 substances that we collected within the Dutch SVHC database – with identified CMR, PBT/vPvB and/or ED properties – and 411 substances that lack these hazardous properties. The best similarity measures showed a high predictive performance with a balanced accuracy of 85% correct identifications for the whole dataset of SVHC substances, and 80% for CMR, 95% for PBT/vPvB and 99% for ED subgroups.This effective screening methodology showed great potential for early stage identification of potential SVHCs. This model can be applied within regulatory frameworks and safe-by-design trajectories, and hence can contribute to the EU goal of achieving a non-toxic environment.

Pyrrolizidine alkaloids (PAs) are naturally occurring genotoxic compounds, and PA-containing plants can pose a risk to humans through contaminated food sources and herbal products. Upon metabolic activation, PAs can form DNA adducts, DNA and protein cross links, chromosomal aberrations, micronuclei, and DNA double-strand breaks. These genotoxic effects may induce gene mutations and play a role in the carcinogenesis of PAs. This study aims to predict in vivo genotoxicity for two well-studied PAs, lasiocarpine and riddelliine, in rat using in vitro genotoxicity data and physiologically based kinetic (PBK) modelling-based reverse dosimetry. The phosphorylation of histone protein H2AX was used as a quantitative surrogate endpoint for in vitro genotoxicity of lasiocarpine and riddelliine in primary rat hepatocytes and human HepaRG cells. The in vitro concentration–response curves obtained from primary rat hepatocytes were subsequently converted to in vivo dose–response curves from which points of departure (PoDs) were derived that were compared to available in vivo genotoxicity data. The results showed that the predicted PoDs for lasiocarpine and riddelliine were comparable to in vivo genotoxicity data. It is concluded that this quantitative in vitro-in silico approach provides a method to predict in vivo genotoxicity for the large number of PAs for which in vivo genotoxicity data are lacking by integrating in vitro genotoxicity assays with PBK modelling-facilitated reverse dosimetry.

There is a lack of appropriate models to delineate the toxicity of rare earth elements (REEs) while taking into account the factors that affect bioavailability. Here, standardized wheat (Triticum aestivum L.) root elongation tests were conducted to examine the impact of exposure conditions (i.e., varying Ca, Mg, Na, K and pH levels) on Y and Ce toxicity. Cation competition and electrostatic theory were examined for their applicability in explaining the observed variations in toxicity. Only Ca2+ and Mg2+ significantly alleviated the toxicity of Y3+ and Ce3+, while Na+, K+ and H+ showed no significant effects. Based on the cation competition, the derived binding constants for the hypothetical biotic ligands of wheat logKCaBL, logKMgBL, logKYBL, and logKCeBL were 3.87, 3.59, 6.70, and 6.48, respectively. The biotic ligand model (BLM) succeeded in predicting toxicities of Y and Ce, with more than 93% of the variance in toxicity explained. Given the BLM requires large data sets for deriving model parameters, attempts were further made to explore a simpler electrostatic based model to quantify REEs toxicity. The results demonstrated that the predictive capacity of the electrostatic approach, which considers ion activities at the plasma membrane surface, was comparable to that of BLM with at least 87% of the variations in toxicity explained. This suggested that the electrostatic theory can serve as a surrogate to BLM in modeling Y and Ce toxicities. Therefore, we recommend the BLM and electrostatic-based model as effective approaches to incorporate bioavailability in quantifying REEs toxicity in the presence of various levels of other major cations.