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Due to the wide range of applications of cerium oxide nanoparticles (CeO2NPs), a risk assessment of their biological effects using environmentally relevant species becomes highly important. There are contradictory reports on the effects of CeO2NPs, which may be related to the use of different types of nanoparticles (NPs) and coatings. CeO2NPs may act as an oxidant causing toxicity or as an antioxidant able to scavenge free radicals. As a consequence of such complexity, the toxicological behaviour of these NPs is still poorly understood. Moreover, little is known about the internalization process of CeO2NPs in algae. There is evidence of CeO2NP-internalization by the green alga Chlamydomonas reinhardtii, but the mechanism and route of uptake are still unknown. In this study, we used uncoated and different polyvinylpyrrolidone (PVP)-coated CeO2NPs with the aim of identifying their toxicological mechanisms to C. reinhardtii and exploring their possible internalization. Our results showed that PVP coated-CeO2NPs significantly increased the formation of reactive oxygen species in exposed cells, indicating that oxidative stress is an important toxicity mechanism for these particles. Direct contact and damage of the cellular membrane was identified as the mechanism causing the toxicity of uncoated NPs. From experiments with endocytosis inhibitors, clathrin-dependent endocytosis was revealed as the main internalization route for all NPs. However, as uncoated CeO2NPs led to severe cellular membrane damage, direct passage of NPs through membrane holes could not be discarded. To our knowledge, this is the first report with evidence of direct linking between NP internalization and a specific endocytic pathway. The results presented here will help to unravel the toxicological mechanism and behaviour of CeO2NPs and provide input information for the environmental health and safety assessment of CeO2NPs.

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1–100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5–100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization.

ACEnano is an EU-funded project which aims at developing, optimising and validating methods for the detection and characterisation of nanomaterials (NMs) in increasingly complex matrices to improve confidence in the results and support their use in regulation. Within this project, several interlaboratory comparisons (ILCs) for the determination of particle size and concentration have been organised to benchmark existing analytical methods. In this paper the results of a number of these ILCs for the characterisation of NMs are presented and discussed. The results of the analyses of pristine well-defined particles such as 60 nm Au NMs in a simple aqueous suspension showed that laboratories are well capable of determining the sizes of these particles. The analysis of particles in complex matrices or formulations such as consumer products resulted in larger variations in particle sizes within technologies and clear differences in capability between techniques. Sunscreen lotion sample analysis by laboratories using spICP-MS and TEM/SEM identified and confirmed the TiO2 particles as being nanoscale and compliant with the EU definition of an NM for regulatory purposes. In a toothpaste sample orthogonal results by PTA, spICP-MS and TEM/SEM agreed and stated the TiO2 particles as not fitting the EU definition of an NM. In general, from the results of these ILCs we conclude that laboratories are well capable of determining particle sizes of NM, even in fairly complex formulations.

A central challenge for the safe design of nanomaterials (NMs) is the inherent variability of NM properties, both as produced and as they interact with and evolve in, their surroundings. This has led to uncertainty in the literature regarding whether the biological and toxicological effects reported for NMs are related to specific NM properties themselves, or rather to the presence of impurities or physical effects such as agglomeration of particles. Thus, there is a strong need for systematic evaluation of the synthesis and processing parameters that lead to potential variability of different NM batches and the reproducible production of commonly utilized NMs. The work described here represents over three years of effort across 14 European laboratories to assess the reproducibility of nanoparticle properties produced by the same and modified synthesis routes for four of the OECD priority NMs (silica dioxide, zinc oxide, cerium dioxide and titanium dioxide) as well as amine-modified polystyrene NMs, which are frequently employed as positive controls for nanotoxicity studies. For 46 different batches of the selected NMs, all physicochemical descriptors as prioritized by the OECD have been fully characterized. The study represents the most complete assessment of NMs batch-to-batch variability performed to date and provides numerous important insights into the potential sources of variability of NMs and how these might be reduced.

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure’s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

Metal nanoparticles (NPs) released into the aquatic environment will likely accumulate in sediment and be available for sediment-dwelling invertebrates that serve as food for other organisms, such as fish. The aim of the present study was to investigate trophic transfer of copper oxide (CuO) NPs and dissolved Cu (CuCl2) from natural sediment to the sediment-dwelling worm Tubifex tubifex, and then to the predatory fish Gasterosteus aculeatus (three-spined stickleback). Cu enriched in the stable isotope 65Cu was used to increase detection and discriminate newly added/assimilated Cu from background Cu levels. Worms were exposed to sediment spiked with 65CuCl2 or 65CuO NPs (~20 nm) at environmentally relevant concentrations for 7 days and subsequently fed to fish for 7 days. Worms accumulated 65Cu during sediment exposure to both 65CuCl2 and 65CuO NPs (0.7 and 1.1 µg 65Cu g-1 dw tissue, respectively), resulting in 65Cu body burdens significantly different from control. Furthermore, significantly more 65Cu was released from the sediment into the overlying water in the 65CuO NP exposures compared to the 65CuCl2 exposures. 65Cu accumulation in fish feeding on 65CuCl2 and 65CuO NP-exposed worms was limited (intestinal tissue: 80 and 65 ng g-1 dw; fish carcass: 7 and 10 ng g-1 dw, and liver: 50 and 10 ng g-1 dw, respectively). Glutathione peroxidase (gpx) mRNA expression was significantly higher in fish feeding on 65CuCl2-exposed worms compared to 65CuO NP-exposed worms (though 65Cu tissue concentrations were similar). No significant effects were detected for the other investigated genes (ctr1, mta, gcl, gr, sod, cat, zo-1). Our results show that NP-derived Cu can enter freshwater food webs, but bioaccumulation and trophic transfer under environmentally realistic exposures is low (detectable with a tracer) and not different from that of dissolved Cu.

Transformation of nutrients to their nano-form, such as selenium (Se) engineered nanonutrients (Se-ENNs), is expected to enhance the absorption of the nutrients into fish and increase the efficiency of the feed. However, dissolution, aggregation, and release of ENNs from the feed matrix may decrease the efficiency of the Se-ENNs. In this study, we provided fish feed supplemented with Se-ENNs which do not aggregate or dissolve and the particles are also not released from the feed matrix. As a proof of principle, we compared the toxicity of a diet containing Se-ENNs of two different sizes (60 nm and 120 nm) with diets containing ionic Se. The adverse effects were measured by monitoring the survival rate, acetylcholinesterase (AChE) levels and swimming behavior of zebrafish over 21 days of feeding with either the Se-ENNs or ionic Se supplemented fish diets. The number size distribution of the 60 nm Se-ENNs in the diet was similar to that in MilliQ water, while the size distribution of the 120 nm Se-ENNs in the diet was slightly wider. Ion and particle release from Se-ENNs containing diets in the exposure media was not observed, indicating the stability of the particles in the feed matrices. To determine toxicity, zebrafish (Danio rerio) were nourished using a control diet (without Se and Se-ENNs), Se (sodium selenite) containing diets (with 2.4 or 240 mg Se per kg feed) and Se-ENNs containing diets (with 2.4 or 240 mg Se-ENNs of 60 or 120 nm per kg feed) for 21 days. Both sizes of Se-ENNs were taken up in the fish, however only the 120 nm Se-ENNs were detected in the brains of fish. Zebrafish fed with Se-ENNs supplemented diets (60 and 120 nm) showed normal swimming behavior compared to the control. No significant alteration was determined in the AChE activity of the fish fed with the Se-ENNs supplemented diet. In contrast, feeding the zebrafish with a diet containing 240 mg kg(-1)Se led to lethal effects. These observations clearly depict the potential benefits of usi, Funding Agencies:European Union (EU) 652831ERDF "Multidisciplinary research to increase application potential of nanomaterials in agricultural practice" CZ.02.1.01/0.0/0.0/16_025/0007314Grant Agency of the Czech Republic 17-15451S