A template wizard for the cocreation of machine-readable data-reporting to harmonize the evaluation of (nano)materials.

Making research data findable, accessible, interoperable and reusable (FAIR) is typically hampered by a lack of skills in technical aspects of data management by data generators and a lack of resources. We developed a Template Wizard for researchers to easily create templates suitable for consistently capturing data and metadata from their experiments. The templates are easy to use and enable the compilation of machine-readable metadata to accompany data generation and align them to existing community standards and databases, such as eNanoMapper, streamlining the adoption of the FAIR principles. These templates are citable objects and are available as online tools. The Template Wizard is designed to be user friendly and facilitates using and reusing existing templates for new projects or project extensions. The wizard is accompanied by an online template validator, which allows self-evaluation of the template (to ensure mapping to the data schema and machine readability of the captured data) and transformation by an open-source parser into machine-readable formats, compliant with the FAIR principles. The templates are based on extensive collective experience in nanosafety data collection and include over 60 harmonized data entry templates for physicochemical characterization and hazard assessment (cell viability, genotoxicity, environmental organism dose-response tests, omics), as well as exposure and release studies. The templates are generalizable across fields and have already been extended and adapted for microplastics and advanced materials research. The harmonized templates improve the reliability of interlaboratory comparisons, data reuse and meta-analyses and can facilitate the safety evaluation and regulation process for (nano) materials.

Safe(r) by design guidelines for the nanotechnology industry.

Expectations for safer and sustainable chemicals and products are growing to comply with the United Nations and European strategies for sustainability. The application of Safe(r) by Design (SbD) in nanotechnology implies an iterative process where functionality, human health and safety, environmental and economic impact and cost are assessed and balanced as early as possible in the innovation process and updated at each step. The EU H2020 NanoReg2 project was the first European project to implement SbD in six companies handling and/or manufacturing nanomaterials (NMs) and nano-enabled products (NEP). The results from this experience have been used to develop these guidelines on the practical application of SbD. The SbD approach foresees the identification, estimation, and reduction of human and environmental risks as early as possible in the development of a NM or NEP, and it is based on three pillars: (i) safer NMs and NEP; (ii) safer use and end of life and (iii) safer industrial production. The presented guidelines include a set of information and tools that will help deciding at each step of the innovation process whether to continue, apply SbD measures or carry out further tests to reduce uncertainty. It does not intend to be a prescriptive protocol where all suggested steps have to be followed to achieve a SbD NM/NEP or process. Rather, the guidelines are designed to identify risks at an early state and information to be considered to identify those risks. Each company adapts the approach to its specific needs and circumstances as company decisions influence the way forward.

Nanopharmaceuticals (Au-NPs) after use: Experiences with a complex higher tier test design simulating environmental fate and effect.

The current environmental hazard assessment is based on the testing of the pristine substance. However, it cannot be excluded that (nano)pharmaceuticals are excreted into sewage during the use phase followed by entry into wastewater treatment plants (WWTPs). Sorption to sewage sludge or release via effluent can result in modified ecotoxicological effects which possibly can only be detected with a modified test approach. The objective of our study was to investigate a realistic exposure scenario for metallic nanoparticles (NPs) in pharmaceutical products, excreted into effluent, and released into the environment after treatment in WWTPs. The test approach was illustrated by using gold (Au) NPs. Effluent from model WWTPs were investigated in aquatic tests (Daphnia magna, fish cell lines). Sewage sludge was used as a sole food source (Eisenia fetida) or mixed with soil and used as test medium (soil microorganisms, Folsomia candida, Enchytraeus crypticus). To cover the aspect of regulation, the test systems described in OECD-test guidelines (OECD TG 201, 211, 220, 232, 249, 317) were applied. Modifications and additional test approaches were included to meet the needs arising out of the testing of nanomaterials and of the exposure scenarios. The results were assessed regarding the suitability of the test design and the toxicity of Au-NPs. Except for activated sludge as a sole food source for E.fetida, the selected test approach is suitable for the testing of nanomaterials. Additional information can be gained when compared to the common testing of the pristine nanomaterials in the standardized test systems. Effects of Au-NPs were observed in concentrations exceeding the modeled environmental.

Comparison of uptake and elimination kinetics of metallic oxide nanomaterials on the freshwater microcrustacean Daphnia magna.

The widespread use and release of nanomaterials (NMs) in aquatic ecosystems is a concerning issue as well as the fate and behavior of the NMs in relation to the aquatic organisms. In this work, the freshwater microcrustacean Daphnia magna was exposed to 12 different and well-known NMs under the same conditions for 24 h and then placed in clean media for 120 h, in order to determine their different uptake and elimination behaviors. The results showed that most of the tested NMs displayed a fast uptake during the first hours arriving to a plateau by the end of the uptake phase. The elimination behavior was determined by a fast loss of NMs during the first hours in the clean media, mainly stimulated by the presence of food. Remaining NMs concentrations can still be found at the end of the elimination phase. Two NMs had a different profile (i) ZnO-NM110 exhibited increase and loss during the uptake phase, and (ii) SiO2-NM204 did not show any uptake. A toxicokinetic model was applied and the uptake and elimination rates were found along with the dynamic bioconcentration factors. These values allowed to compare the NMs, to cluster them by their similar rates, and to determine that the TiO2-NM102 is the one that has the fastest uptake and elimination behavior, SiO2-NM204 has the slowest uptake and CeO2 <10 nm has the slowest elimination. The present work represents a first attempt to compare different NMs based on their uptake and elimination behaviors from a perspective of the nano-bio interactions influence.

Plastic additives: challenges in ecotox hazard assessment.

The risk of plastic debris, and specifically micro(nano)plastic particles, to ecosystems remains to be fully characterized. One particular issue that warrants further characterization is the hazards associated with chemical additives within micro(nano)plastic as they are not chemically bound within the polymers and can be persistent and biologically active. Most plastics contain additives and are therefore potential vectors for the introduction of these chemicals into the environment as they leach from plastic, a process that can be accelerated through degradation and weathering processes. There are knowledge gaps on the ecotoxicological effects of plastic additives and how they are released from parent plastic materials as they progressively fragment from the meso to micro and nano scale. This review summarizes the current state of knowledge of the ecotoxicity of plastic additives and identifies research needs to characterize the hazard they present to exposed biota. The potential ecological risk of chemical additives is of international concern so key differences in governance between the European Union and New Zealand to appropriately characterize their risk are highlighted.

Towards FAIR nanosafety data.

Nanotechnology is a key enabling technology with billions of euros in global investment from public funding, which include large collaborative projects that have investigated environmental and health safety aspects of nanomaterials, but the reuse of accumulated data is clearly lagging behind. Here we summarize challenges and provide recommendations for the efficient reuse of nanosafety data, in line with the recently established FAIR (findable, accessible, interoperable and reusable) guiding principles. We describe the FAIR-aligned Nanosafety Data Interface, with an aggregated findability, accessibility and interoperability across physicochemical, bio-nano interaction, human toxicity, omics, ecotoxicological and exposure data. Overall, we illustrate a much-needed path towards standards for the optimized use of existing data, which avoids duplication of efforts, and provides a multitude of options to promote safe and sustainable nanotechnology.

Integrative approach in a safe by design context combining risk, life cycle and socio-economic assessment for safer and sustainable nanomaterials.

Moving towards safe and sustainable innovations is an international policy ambition. In the on-hand manuscript, a concept combining safe by design and sustainability was implemented through the integration of human and environmental risk assessment, life cycle assessment as well as an assessment of the economic viability. The result is a nested and iterative process in form of a decision tree that integrates these three elements in order to achieve sustainable, safe and competitive materials, products or services. This approach, embedded into the stage-gate-model for safe by design, allows to reduce the uncertainty related to the assessment of risks and impacts by improving the quality of the data collected along each stage. In the second part of the manuscript, the application is shown for a case study dealing with the application of nanoparticles for Li-Ion batteries. One of the general conclusions out of this case study is that data gaps are a key aspect in view of the reliability of the results.

Characterization of the nano-bio interaction between metallic oxide nanomaterials and freshwater microalgae using flow cytometry.

Since nanomaterials (NMs) are particulate contaminants, their first contact with organisms is a physical encounter ruled by physic-chemical processes that can determinate the potential NMs accumulation, toxicity, and trophic transfer. Freshwater ecosystems often become a final depository for NMs, so they can get in contact with the biota, especially primary organisms as algae. There are almost none comparative studies of this interaction using various NMs in the same conditions. This work identifies, analyzes, and compares the algae-NMs interaction by flow cytometry after a short-term contact test in which three freshwater algae (Raphidocelis subcapitata, Desmodesmus subspicatus, and Chlorella vulgaris) interact individually with a set of twelve metallic oxide NMs. Dose-response profiles and differences in the algae-NMs interaction were found according to each algae species (C. vulgaris had the most affinity, starting the interaction from 0.5 mg/L and D. subspicatus had the less affinity starting at 5 mg/L). Flow cytometry results were confirmed by optical microscopy. Some NMs characteristics were identified as key-factors that govern the algae-NMs interaction: NMs composition (no interaction for SiO2 NMs), surface electric charge (higher interaction for the positively charged NMs and lower interaction for the negatively charged ones) and crystalline form (for TiO2 NMs). The presented method can be useful for a rapid determination of the interaction between free cells organisms as microalgae and (nano)particulate substances.

Investigating the Impact of Manufacturing Processes on the Ecotoxicity of Carbon Nanofibers: A Multi-Aquatic Species Comparison.

Manufactured nanomaterial production is outpacing the ability to investigate environmental hazard using current regulatory paradigms, causing a backlog of materials requiring testing. To ameliorate this issue, regulatory bodies have proposed integrating safety into the production of novel nanomaterials, allowing for hazards to be identified early in development rather than aftermarket release. In addition, there is a growing interest in short-term ecotoxicity testing to rapidly identify environmental hazards. In this sense, the present study investigated 3 carbon nanofibers (CNFs), created with different production methods, using short-term in vitro and in vivo exposures on fish cell lines, mussel hemocytes, crustacea, and algae. The present study investigated if differences in ecotoxicity hazard between the CNFs could be identified and, if so, which product could be considered less hazardous. A major challenge in assessing the potential hazards posed by manufactured nanomaterials is standardizing the preparation for testing. Standardized operating protocols have been proposed using protein to facilitate the preparation of stable stock suspension, which is not environmentally representative. As such, the study also assessed the potential impacts these standardized protocols (with or without the use of protein) could have on the interpretation of environmental hazard. The results demonstrated that there were clear differences between the 3 CNFs and that the dispersion protocol influenced the interpretation of hazard, demonstrating a need for caution when interpreting ecotoxicity in a regulatory context. Environ Toxicol Chem 2019;38:2314-2325. © 2019 SETAC.

Investigating a transcriptomic approach on marine mussel hemocytes exposed to carbon nanofibers: An in vitro/in vivo comparison.

Manufactured nanomaterials are an ideal test case of the precautionary principle due to their novelty and potential environmental release. In the context of regulation, it is difficult to implement for manufactured nanomaterials as current testing paradigms identify risk late into the production process, slowing down innovation and increasing costs. One proposed concept, namely safe(r)-by-design, is to incorporate risk and hazard assessment into the design process of novel manufactured nanomaterials by identifying risks early. When investigating the manufacturing process for nanomaterials, differences between products will be very similar along key physicochemical properties and biological endpoints at the individual level may not be sensitive enough to detect differences whereas lower levels of biological organization may be able to detect these variations. In this sense, the present study used a transcriptomic approach on Mytilus edulis hemocytes following an in vitro and in vivo exposure to three carbon nanofibers created using different production methods. Integrative modeling was used to identify if gene expression could be in linked to physicochemical features. The results suggested that gene expression was more strongly associated with the carbon structure of the nanofibers than chemical purity. With respect to the in vitro/in vivo relationship, results suggested an inverse relationship in how the physicochemical impact gene expression.

Towards the development of a high throughput screening approach for Mytilus edulis hemocytes: A case study on silicon-based nanomaterials.

To have an understanding of potential mechanistic effects, sublethal endpoints able to discriminate between nanomaterials with similar physical and chemical features need to be used. In this sense, quantitative PCR was used to measure a battery of genes linked to a wide array of different cellular processes. Gene expression was measured in Mytilus edulis hemocytes following an in vitro and in vivo exposure to pure silicon (40 nm) and carbon-coated silicon (40 and 75 nm) after 24 h. Partial least squares discriminant analysis and correlation analysis were used to develop an integrative model, describing the relationship between genes, to identify which genes were important in describing responses to engineered nanomaterial exposure. The results suggested that some discriminations could be made based on the presence of a carbon coating or the alteration of size which could inform industrial patterns on ways to reduce the ecotoxicological impact of their product. The results also indicate that HTS on Mytilus hemocytes may be integrated into a safer-by-design approach but additional characterization of nanomaterial behavior in media is required to determine if it is a suitable alternative to in vivo testing.

Environmental release of engineered nanomaterials from commercial tiles under standardized abrasion conditions.

The study presented here focuses on commercial antibacterial tiles whose emissivity of (nano) particles due to abrasion has yet barely been investigated. The tiles have been characterized regarding their surface properties and composition throughout their chain-of-use, i.e. from their state of commercialization until the experimental end-of-service life. In contrast to plane standard tiles, their surfaces form hilly surfaces. In the depressions, titanium dioxide is found at the surface, thus theoretically protected by the hilly areas against abrasion on the tile's surface. Furthermore, a deposition technique has been put in place by producers allowing for coating the before mentioned commercial tiles with titanium dioxide, thus being similar to those commercially available. It consists in depositing titanium dioxide on the surface, latter one allowing fixing the first. This development allows for better understanding the future options for product formulation and thus improvement with respect to particle release. The tests reveal the aerosolization from commercial antibacterial tiles of micronic and submicronic particles in the inhalable region or particles that can subjected to be released in the environment (<10µm). The aersolization of the particles from the coated tiles was found to be significantly higher compared to the non coated tiles.

The comet assay in higher terrestrial plant model: Review and evolutionary trends.

The comet assay is a sensitive technique for the measurement of DNA damage in individual cells. Although it has been primarily applied to animal cells, its adaptation to higher plant tissues significantly extends the utility of plants for environmental genotoxicity research. The present review focuses on 101 key publications and discusses protocols and evolutionary trends specific to higher plants. General consensus validates the use of the percentage of DNA found in the tail, the alkaline version of the test and root study. The comet protocol has proved its effectiveness and its adaptability for cultivated plant models. Its transposition in wild plants thus appears as a logical evolution. However, certain aspects of the protocol can be improved, namely through the systematic use of positive controls and increasing the number of nuclei read. These optimizations will permit the increase in the performance of this test, namely when interpreting mechanistic and physiological phenomena.

Adapting OECD Aquatic Toxicity Tests for Use with Manufactured Nanomaterials: Key Issues and Consensus Recommendations.

The unique or enhanced properties of manufactured nanomaterials (MNs) suggest that their use in nanoenabled products will continue to increase. This will result in increased potential for human and environmental exposure to MNs during manufacturing, use, and disposal of nanoenabled products. Scientifically based risk assessment for MNs necessitates the development of reproducible, standardized hazard testing methods such as those provided by the Organisation of Economic Cooperation and Development (OECD). Currently, there is no comprehensive guidance on how best to address testing issues specific to MN particulate, fibrous, or colloidal properties. This paper summarizes the findings from an expert workshop convened to develop a guidance document that addresses the difficulties encountered when testing MNs using OECD aquatic and sediment test guidelines. Critical components were identified by workshop participants that require specific guidance for MN testing: preparation of dispersions, dose metrics, the importance and challenges associated with maintaining and monitoring exposure levels, and the need for reliable methods to quantify MNs in complex media. To facilitate a scientific advance in the consistency of nanoecotoxicology test results, we identify and discuss critical considerations where expert consensus recommendations were and were not achieved and provide specific research recommendations to resolve issues for which consensus was not reached. This process will enable the development of prescriptive testing guidance for MNs. Critically, we highlight the need to quantify and properly interpret and express exposure during the bioassays used to determine hazard values.

Soil genotoxicity assessment--results of an interlaboratory study on the Vicia micronucleus assay in the context of ISO standardization.

The Vicia micronucleus assay was standardized in an international protocol, ISO 29200, "Assessment of genotoxic effects on higher plants-Vicia faba micronucleus test," for soil or soil materials (e.g., compost, sludge, sediment, waste, and fertilizing materials). The aim of this interlaboratory study on the Vicia micronucleus assay was to investigate the robustness of this in vivo assay in terms of its applicability in different countries where each participant were asked to use their own seeds and reference soil, in agreement with the ISO 29200 standard. The ISO 29200 standard protocol was adopted for this study, and seven laboratories from three countries (France, Italy, and Brazil) participated in the study. Negative and positive controls were correctly evaluated by 100 % of the participants. In the solid-phase test, the micronucleus frequency (number of micronuclei/1,000 cells) varied from 0.0 to 1.8 for the negative control (i.e., Hoagland's solution) and from 5.8 to 85.7 for the positive control (i.e., maleic hydrazide), while these values varied from 0.0 to 1.7 for the negative control and from 14.3 to 97.7 for the positive control in the case of liquid-phase test. The variability in the data obtained does not adversely affect the robustness of the protocol assessed, on the condition that the methodology described in the standard ISO 29200 is strictly respected. Thus, the Vicia micronucleus test (ISO 29200) is appropriate for complementing prokaryotic or in vitro tests cited in legislation related to risk assessment of genotoxicity potential.

The use of soil mites in ecotoxicology: a review.

Mites, and especially soil-inhabiting ones, have been less studied than the other invertebrates used in bio-assays for the assessment of soil quality and the hazards of chemicals, although these organisms are included in the regulatory assessment scheme of pesticides. The recent advances in the development of test methods for soil mites groups have provided more information on their sensitivities towards chemicals, which needs to be presented for a more robust assessment of the current trends in soil mite ecotoxicology. Moreover, interestingly mite is the only taxa for which test methods were developed and standardized on predatory organisms. This review summarizes the different protocols for the assessment of chemicals using soil-inhabiting mites, including laboratory, semi-field and field studies. Among the data found in the literature, most of the chemicals assessed with mites were pesticides, while a few environmental samples were assessed with these organisms. Their sensitivities towards chemicals were then compared and discussed regarding other soil invertebrates. Finally, we conclude on the usefulness of soil mites in ecotoxicology, and provide future research trail in this area.

Ecotoxicological assessment of organic wastes spread on land: Towards a proposal of a suitable test battery.

The land spreading of organic wastes in agriculture is a common practice in Europe, under the regulation of the Directive 86/278/EEC. One of the objectives of this Directive is to prevent harmful effects of organic wastes on soil, plants and animals. Despite this regulatory framework, there is still a lack of harmonized ecotoxicological test strategy to assess the environmental hazard of such wastes. The aim of this study was to provide a first step towards the a priori ecotoxicological assessment of organic wastes before their land use. For that purpose, nine different organic wastes were assessed using direct (i.e. terrestrial tests) and indirect (i.e. tests on water eluates) approaches, for a total of thirteen endpoints. Then, multivariate analyzes were used to discriminate the most relevant test strategy, among the application rates and bioassays used. From our results, a draft of test strategy was proposed, using terrestrial bioassays (i.e. earthworms and plants) and a concentration range between one and ten times the recommended application rates of organic wastes.

Selecting chemical and ecotoxicological test batteries for risk assessment of trace element-contaminated soils (phyto)managed by gentle remediation options (GRO).

During the past decades a number of field trials with gentle remediation options (GRO) have been established on trace element (TE) contaminated sites throughout Europe. Each research group selects different methods to assess the remediation success making it difficult to compare efficacy between various sites and treatments. This study aimed at selecting a minimum risk assessment battery combining chemical and ecotoxicological assays for assessing and comparing the effectiveness of GRO implemented in seven European case studies. Two test batteries were pre-selected; a chemical one for quantifying TE exposure in untreated soils and GRO-managed soils and a biological one for characterizing soil functionality and ecotoxicity. Soil samples from field studies representing one of the main GROs (phytoextraction in Belgium, Sweden, Germany and Switzerland, aided phytoextraction in France, and aided phytostabilization or in situ stabilization/phytoexclusion in Poland, France and Austria) were collected and assessed using the selected test batteries. The best correlations were obtained between NH4NO3-extractable, followed by NaNO3-extractable TE and the ecotoxicological responses. Biometrical parameters and biomarkers of dwarf beans were the most responsive indicators for the soil treatments and changes in soil TE exposures. Plant growth was inhibited at the higher extractable TE concentrations, while plant stress enzyme activities increased with the higher TE extractability. Based on these results, a minimum risk assessment battery to compare/biomonitor the sites phytomanaged by GROs might consist of the NH4NO3 extraction and the bean Plantox test including the stress enzyme activities.

Ecotoxicity of non-aged and aged CeO2 nanomaterials towards freshwater microalgae.

The ecotoxicity of artificially alterated cerium dioxide nanoparticles (nano-CeO2) suspensions was determined using the freshwater microalgae growth inhibition test. The agglomeration or aggregation state of the alterated suspensions was followed because it represents one of the obvious modifications when nanoparticles reached the environment. In addition, its influence on the ecotoxicity of nanoparticles is currently not well-addressed. Our results showed that the suspensions were stable within the first 24 h and then agglomerate up to 10 µm after 3 and 30 days. The inhibitory effect on the growth of exposed algae was however similar whatever the tested suspension. This supports the fact that the agglomeration state of nano-CeO2, in our conditions, has few influences on the ecotoxicity toward these organisms. The EC50 values were 5.6; 4.1 and 6.2 mg L(-1), after exposure to non aged, 3 and 30 days aged suspensions respectively. The interaction between algal cells and nano-CeO2 was also addressed.

Improvement of the Caenorhabditis elegans growth and reproduction test to assess the ecotoxicity of soils and complex matrices.

A growth and reproduction test using the nematode Caenorhabditis elegans was recently standardized by the International Organization for Standardization (ISO). Performing the ISO 10872 protocol (2010) revealed some drawbacks when applied to soil or soil mixed with complex matrices. The authors propose some modifications to the current protocol to normalize the test conditions. An appropriate range of moisture conditions was determined as a percentage of the water-holding capacity (WHC) of the soil. According to the authors' results, C. elegans tests can be performed in the range of 60% to 100% WHC. To ensure that the modifications of the protocol did not affect the organisms' recovery, extraction ratios for the juveniles were subsequently estimated. The modified protocol was found to be as reliable as the standard one concerning recovery of juveniles (over 80%). The protocol was also applied to several chemicals to investigate their potential as reference chemicals for soil toxicity tests. Boric acid, copper chloride, and nickel sulfate showed deleterious effects in a concentration-dependent manner for the growth and reproduction of C. elegans. Finally, the modified protocol was used to assess the growth and reproduction of C. elegans in soil amended with a limed sewage sludge. The authors conclude that the C. elegans modified protocol is a promising tool for the assessment of soil toxicity as well as the toxicity of mixtures with complex matrices.