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Hypoglycin A (HGA) and its homologue methylenecyclopropylglycine (MCPrG) are present in ackee and lychee as well as seeds, leaves, and seedlings of some maple (Acer) species. They are toxic to some animal species and humans. The determination of HGA, MCPrG, and their glycine and carnitine metabolites in blood and urine is a useful tool for screening for potential exposure to these toxins. In addition, HGA, MCPrG, and/or their metabolites have been detected in milk. In this work, simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) methods without derivatization were developed and validated for the quantification of HGA, MCPrG, and their metabolites in cow’s milk and urine. An extraction procedure from milk samples has been developed, whereas a dilute-and-shoot approach was implemented for urine samples. For quantification, the MS/MS analysis was performed in multiple reaction monitoring mode. The methods were validated according to the European Union guidelines using blank raw milk and urine as matrices. The limit of quantification presented here for HGA in milk (1.12 µg/L) is noticeably lower than the lowest published limit of detection (9 µg/L). Acceptable values for recovery (89–106% and 85–104% in milk and urine, respectively) and precision (≤ 20%) were obtained for all the quality control levels. The stability of HGA and MCPrG in frozen milk over a period of 40 weeks has been demonstrated. The method was applied to 68 milk samples from 35 commercial dairy farms and showed the absence of any quantifiable amounts of HGA, MCPrG, and their metabolites. Graphical Abstract

Phomopsins are mycotoxins mainly infesting lupines, with phomopsin A (PHOA) being the main mycotoxin. PHOA is produced by Diaporthe toxica, formerly assigned as toxigenic Phomopsis leptostromiformis, causing infections in lupine plants and harvested seeds. However, Diaporthe species may also grow on other grain legumes, similar to Aspergillus westerdijkiae as an especially potent ochratoxin A (OTA) producer. Formation of PHOA and OTA was investigated on whole field peas as model system to assess fungal growth and toxin production at adverse storage conditions. Field pea samples were inoculated with the two fungal strains at two water activity (aw) values of 0.94 and 0.98 and three different levels of 30, 50, and 80% relative air humidity.After 14 days at an aw value of 0.98, the fungi produced 4.49 to 34.3 mg/kg PHOA and 1.44 to 3.35 g/kg OTA, respectively. Strains of D. toxica also tested showed higher PHOA concentrations of 28.3 to 32.4 mg/kg.D. toxica strains did not grow or produce PHOA at an aw values of 0.94, while A. westerdijkiae still showed growth and OTA production.Elevated water activity has a major impact both on OTA and, even more pronouncedly, on PHOA formation and thus, proper drying and storage of lupins as well as other grain legumes is crucial for product safety.

Fungi of the genus Alternaria are ubiquitous in the environment. Their mycotoxins can leach out of contaminated plants or crop debris into the soil entering the plant via the roots. We aim to evaluate the importance of this entry pathway and its contribution to the overall content of Alternaria toxins (ATs) in wheat plants to better understand the soil–plant-phytopathogen system. A hydroponic cultivation system was established and wheat plants were cultivated for up to two weeks under optimal climate conditions. One half of the plants was treated with a nutrient solution spiked with alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA), whereas the other half of the plants was cultivated without mycotoxins. Plants were harvested after 1 and 2 weeks and analyzed using a QuEChERS-based extraction and an in-house validated LC–MS/MS method for quantification of the ATs in roots, crowns, and leaves separately. ATs were taken up by the roots and transported throughout the plant up to the leaves after 1 as well as 2 weeks of cultivation with the roots showing the highest ATs levels followed by the crowns and the leaves. In addition, numerous AOH and AME conjugates like glucosides, malonyl glucosides, sulfates, and di/trihexosides were detected in different plant compartments and identified by high-resolution mass spectrometry. This is the first study demonstrating the uptake of ATs in vivo using a hydroponic system and whole wheat plants examining both the distribution of ATs within the plant compartments and the modification of ATs by the wheat plants.

Background: The risk assessment of small particles (including nanoparticles) in products used in the food chain in the EU falls within the remit of the European Food Safety Authority (EFSA) and has been under thorough scientific considerations for over a decade. Now that more experience is gained with evaluating novel foods, food contact materials, food/feed additives and pesticides, the outlines for regulatory safety assessments and data requirements are established. Scope and approach: This paper reviews the principles underlying safety testing of small particles, referring to two recently published EFSA guidance documents. Examples and observations from assessing existing materials are provided to facilitate to a wider readership adequate implementation of the regulatory requirements. Main findings and conclusions: The starting point for safety testing is the physicochemical characterisation of the pristine material, being an engineered nanomaterial, a nanostructured material or a conventional material that contains a fraction of small particles that may retain properties at the nanoscale. Key parameters and threshold values for establishing the presence of small particles, the techniques and methods for characterisation in complex matrices, as well as approaches for dietary exposure assessment are outlined. Where there is the likelihood of small particles remaining after gastrointestinal digestion, hazard identification and hazard characterisation are required with special provisions. In particular, certain nano-specific considerations are highlighted that have to be considered during toxicological testing with the aim to demonstrate consumer safety of products to be used in the food chain in Europe.

Fungi of Aspergillus and Penicillium genus can infect peas (Pisum sativum), leading to a contamination with the nephrotoxic and carcinogenic ochratoxin A (OTA). Under unfavourable conditions, a fungus primarily found on lupines, Diapothe toxica, may also grow on peas and produce the hepatotoxic phomopsin A (PHOA). To study the effect of processing on OTA and PHOA content, two model products—wheat/rye-mixed bread with pea flour addition and pea pasta—were manufactured at small-business scale from artificially contaminated pea flour. The decrease of OTA and PHOA contents were monitored along the production process as indicators for toxin transformation. Pea bread dough was subjected to proofing for 30–40 min at 32 °C and baked at 250 °C to 230 °C for 40 min. OTA content (LODs < 0.1 μg/kg) showed a reduction in the bread crust (initially 17.0 μg/kg) to 88% and no reduction in the crumb (110%). For PHOA (LODs < 3.6 μg/kg), a decrease to approximately 21% occurred in the bread crust (initially 12.5 μg/kg), whilst for crumb, a less intense decrease to 91% was found. Pea pasta prepared with two toxin levels was extruded at room temperature, dried and cooked for 8 min in boiling water. In pea pasta, OTA was reduced from 29.8 to 13.9 μg/kg by 22% each after cooking, whilst 15% and 10% of the initial toxin amounts were found in the cooking water, respectively. For PHOA, 60% and 78% of initially 14.3 μg/kg and 7.21 μg/kg remained in the cooked pasta. As only the decrease of the initial content was measured and no specific degradation products could be detected, further research is needed to characterise potential transformation products. Heat treatment reduces the initial PHOA content stronger than the OTA content during pasta cooking and bread making. However, significant amounts of both toxins would remain in the final products.

Identifying nanomaterials (NMs) according to European Union legislation is challenging, as there is an enormous variety of materials, with different physico-chemical properties. The NanoDefiner Framework and its Decision Support Flow Scheme (DSFS) allow choosing the optimal method to measure the particle size distribution by matching the material properties and the performance of the particular measurement techniques. The DSFS leads to a reliable and economic decision whether a material is an NM or not based on scientific criteria and respecting regulatory requirements. The DSFS starts beyond regulatory requirements by identifying non-NMs by a proxy approach based on their volume-specific surface area. In a second step, it identifies NMs. The DSFS is tested on real-world materials and is implemented in an e-tool. The DSFS is compared with a decision flowchart of the European Commission’s (EC) Joint Research Centre (JRC), which rigorously follows the explicit criteria of the EC NM definition with the focus on identifying NMs, and non-NMs are identified by exclusion. The two approaches build on the same scientific basis and measurement methods, but start from opposite ends: the JRC Flowchart starts by identifying NMs, whereas the NanoDefiner Framework first identifies non-NMs.

The midbrain is a subcortical area involved in central functions such as integrating sensory modalities, movement initiation and bottom-up and top-down attention. In chicken, the midbrain roof is termed optic tectum (TeO) and consists of 15 layers with distinct in- and output regions. Visual input targets the superficial layers, while auditory input terminates in deeper layers. It has been shown that ablation of sensory epithelia leads to changes in the cellular patterning and structural organization of the sensory pathways. For the tectum, ablation of the eye anlagen was shown to affect retinorecipient neurons. While the gross morphology remained intact after enucleation, the shape of dendritic endings was changed presumably due to missing presynaptic input during synaptic pruning. We investigated the effect of deafferentation in a multisensory cell type, the Shepherd's crook neuron (SCN) in the TeO. SCNs have distinct dendritic branches in retinorecipient layers (superficial layers 1 to 5 and 7) and in layers where auditory input terminates. To assess whether removal of a single sensory input only affects the dendrites recipient for that input, we removed the eye anlagen and retrogradely labeled SCNs later in embryogenesis to visualize the morphology in lesioned and non-lesioned embryos. We found no changes in the gross morphology or in the basal dendrites, but an altered growth of the fine structures at the apical dendrite of SCNs in the retinorecipient layers. Our data indicate that the neuronal morphology of SCNs is mostly predefined before retinal innervation affect the fine structure.

The midbrain is an important subcortical area involved in distinct functions such as multimodal integration, movement initiation, bottom-up, and top-down attention. Our group is particularly interested in cellular computation of multisensory integration. We focus on the visual part of the avian midbrain, the optic tectum (TeO, counterpart to mammalian superior colliculus). This area has a layered structure with the great advantage of distinct input and output regions. In chicken, the TeO is organized in 15 layers where visual input targets the superficial layers while auditory input terminates in deeper layers. One specific cell type, the Shepherd's crook neuron (SCN), extends dendrites in both input regions. The characteristic feature of these neurons is the axon origin at the apical dendrite. The molecular identity of this characteristic region and thus, the site of action potential generation are of particular importance to understand signal flow and cellular computation in this neuron. We present immunohistochemical data of structural proteins (NF200, Ankyrin G, and Myelin) and ion channels (Pan-Nav , Nav 1.6, and Kv 3.1b). NF200 is strongly expressed in the axon. Ankyrin G is mainly expressed at the axon initial segment (AIS). Myelination starts after the AIS as well as the distribution of Nav channels on the axon. The subtype Nav 1.6 has a high density in this region. Kv 3.1b is restricted to the soma, the primary neurite and the axon branch. The distribution of functional molecules in SCNs provides insight into the information flow and the integration of sensory modalities in the TeO of the avian midbrain.

Single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS) promises fast and selective determination of nanoparticle size and number concentrations. While several studies on practical applications have been published, data on formal, especially interlaboratory validation of sp-ICP-MS, is sparse. An international interlaboratory study was organized to determine repeatability and reproducibility of the determination of the median particle size and particle number concentration of Ag nanoparticles (AgNPs) in chicken meat. Ten laboratories from the European Union, the USA, and Canada determined particle size and particle number concentration of two chicken meat homogenates spiked with polyvinylpyrrolidone (PVP)-stabilized AgNPs. For the determination of the median particle diameter, repeatability standard deviations of 2 and 5% were determined, and reproducibility standard deviations were 15 and 25%, respectively. The equivalent median diameter itself was approximately 60% larger than the diameter of the particles in the spiking solution. Determination of the particle number concentration was significantly less precise, with repeatability standard deviations of 7 and 18% and reproducibility standard deviations of 70 and 90%. Electronic supplementary material The online version of this article (doi:10.1007/s00216-017-0427-2) contains supplementary material, which is available to authorized users.

Estimating consumer exposure to nanomaterials (NMs) in food products and predicting their toxicological properties are necessary steps in the assessment of the risks of this technology. To this end, analytical methods have to be available to detect, characterize and quantify NMs in food and materials related to food, e.g. food packaging and biological samples following metabolization of food. The challenge for the analytical sciences is that the characterization of NMs requires chemical as well as physical information. This article offers a comprehensive analysis of methods available for the detection and characterization of NMs in food and related products. Special attention was paid to the crucial role of sample preparation methods since these have been partially neglected in the scientific literature so far. The currently available instrumental methods are grouped as fractionation, counting and ensemble methods, and their advantages and limitations are discussed. We conclude that much progress has been made over the last 5 years but that many challenges still exist. Future perspectives and priority research needs are pointed out. Graphical Abstract Two possible analytical strategies for the sizing and quantification of Nanoparticles: Asymmetric Flow Field-Flow Fractionation with multiple detectors (allows the determination of true size and mass-based particle size distribution); Single Particle Inductively Coupled Plasma Mass Spectrometry (allows the determination of a spherical equivalent diameter of the particle and a number-based particle size distribution).

Die Authentizitatsprufung von Lebens- und Futtermitteln umfasst eine grose Bandbreite an Fragestellungen, Matrizes, analytischen Techniken und Forschungsansatzen, weshalb die Vernetzung von offentlichen Instituten eine immer grosere Rolle spielt. Um den Austausch zwischen Experten auf eine breitere Basis zu stellen, organisierte das Bundesinstitut fur Risikobewertung (BfR) erstmals eine bundesweite Veranstaltung fur offentliche Institute mit dem Titel „Status quo der Authentizitatsprufung von Lebens- und Futtermitteln - Analytische Routineverfahren und Forschungsansatze“. Auf Einladung des BfR berichteten Expertinnen und Experten diverser Einrichtungen des Bundes, der Landesuberwachungsbehorden und Universitaten am 13. und 14. Juni 2016 in den Tagungsraumen des BfR uber ihre bisherigen Erfahrungen und Aktivitaten in der Echtheitsuberprufung – Authentizitatsprufung – von Lebens- und Futtermitteln. Das Ziel des BfR, mit dieser Veranstaltung den wissenschaftlichen Austausch in diesem aktuellen Forschungsgebiet durch den Aufbau eines Expertennetzwerkes zu fordern, wurde erreicht.

cited By 0; The European Commission's recommendation on the definition of nanomaterial (2011/696/EU) established an applicable standard for material categorization. However, manufacturers face regulatory challenges during registration of their products. Reliable categorization is difficult and requires considerable expertise in existing measurement techniques (MTs). Additionally, organizational complexity is increased as different authorities' registration processes require distinct reporting. The NanoDefine project tackled these obstacles by providing the NanoDefiner e-tool: A decision support expert system for nanomaterial identification in a regulatory context. It providesMT recommendations for categorization of specific materials using a tiered approach (screening/confirmatory), and was constructed with experts from academia and industry to be extensible, interoperable, and adaptable for forthcoming revisions of the nanomaterial definition. An implemented MT-driven material categorization scheme allows detailed description. Its guided workflow is suitable for a variety of user groups. Direct feedback and explanation enable transparent decisions. Expert knowledge is held in a knowledge base for representation ofMT performance criteria and physicochemical particle type properties. Continuous revision ensured data quality and validity. Recommendations were validated by independent case studies on industry-relevant particulate materials. Besides supporting material identification and registration, the free and open-source e-tool may serve as template for other expert systems within the nanoscience domain. © 2019 by the authors.

cited By 1; Worldwide there is a variety of regulatory provisions addressing nanomaterials. The identification as nanomaterial in a regulatory context often has the consequence that specific legal rules apply. In identifying nanomaterials, and to find out whether nanomaterial-specific provisions apply, the external size of particles is globally used as a criterion. For legal certainty, its assessment for regulatory purposes should be based on measurements and methods that are robust, fit for the purpose and ready to be accepted by different stakeholders and authorities. This should help to assure the safety of nanomaterials and at the same time facilitate their international trading. Therefore, we propose a categorisation scheme which is driven by the capabilities of common characterisation techniques for particle size measurement. Categorising materials according to this scheme takes into account the particle properties that are most important for a determination of their size. The categorisation is exemplified for the specific particle number based size metric of the European Commission's recommendation on the definition of nanomaterial, but it is applicable to other metrics as well. Matching the performance profiles of the measurement techniques with the material property profiles (i) allows selecting the most appropriate size determination technique for every type of material considered, (ii) enables proper identification of nanomaterials, and (iii) has the potential to be accepted by regulators, industry and consumers alike. Having such a scheme in place would facilitate the regulatory assessment of nanomaterials in regional legislation as well as in international relations between different regulatory regions assuring the safe trade of nanomaterials. © 2019 The Royal Society of Chemistry.

The European Food Safety Authority has produced this Guidance on human and animal health aspects (Part 1) of the risk assessment of nanoscience and nanotechnology applications in the food and feed chain. It covers the application areas within EFSA's remit, e.g. novel foods, food contact materials, food/feed additives and pesticides. The Guidance takes account of the new developments that have taken place since publication of the previous Guidance in 2011. Potential future developments are suggested in the scientific literature for nanoencapsulated delivery systems and nanocomposites in applications such as novel foods, food/feed additives, biocides, pesticides and food contact materials. Therefore, the Guidance has taken account of relevant new scientific studies that provide more insights to physicochemical properties, exposure assessment and hazard characterisation of nanomaterials. It specifically elaborates on physicochemical characterisation of nanomaterials in terms of how to establish whether a material is a nanomaterial, the key parameters that should be measured, the methods and techniques that can be used for characterisation of nanomaterials and their determination in complex matrices. It also details the aspects relating to exposure assessment and hazard identification and characterisation. In particular, nanospecific considerations relating to in vivo/in vitro toxicological studies are discussed and a tiered framework for toxicological testing is outlined. It describes in vitro degradation, toxicokinetics, genotoxicity as well as general issues relating to testing of nanomaterials. Depending on the initial tier results, studies may be needed to investigate reproductive and developmental toxicity, immunotoxicity, allergenicity, neurotoxicity, effects on gut microbiome and endocrine activity. The possible use of read‐across to fill data gaps as well as the potential use of integrated testing strategies and the knowledge of modes/mechanisms of action are also discussed. The Guidance proposes approaches to risk characterisation and uncertainty analysis, and provides recommendations for further research in this area., This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2018.EN-1430/full

The reliable quantification of nanomaterials (NMs) in complex matrices such as food, cosmetics and biological and environmental compartments can be challenging due to interactions with matrix components and analytical equipment (vials and tubing). The resulting losses along the analytical process (sampling, extraction, clean-up, separation and detection) hamper the quantification of the target NMs in these matrices as well as the compatibility of results and meaningful interpretations in safety assessments. These issues can be overcome by the addition of known amounts of internal/recovery standards to the sample prior to analysis. These standards need to replicate the behaviour of target analytes in the analytical process, which is mainly defined by the surface properties. Moreover, they need to carry a tag that can be quantified independently of the target analyte. As inductively coupled plasma mass spectrometry is used for the identification and quantification of NMs, doping with isotopes, target analytes or with chemically related rare elements is a promising approach. We present the synthesis of a library of TiO 2 NMs doped with hafnium (Hf) and zirconium (Zr) (both low in environmental abundance). Zirconia NMs doped with Hf were also synthesized to complement the library. NMs were synthesized with morphological and size properties similar to commercially available TiO 2 . Characterization included: transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy, X-ray diffraction spectroscopy, Brunauer–Emmett–Teller total specific surface area analysis, cryofixation scanning electron microscopy, inductively coupled plasma optical emission spectroscopy and UV–visible spectrometry. The Ti : Hf and Ti : Zr ratios were verified and calculated using Rietveld refinement. The labelled NMs can serve as internal standards to track the extraction efficiency from complex matrices, and increase method robustness and traceability of characterization/quantification.

Nanotechnology has the potential to innovate the agricultural, feed and food sectors (hereinafter referred to as agri/feed/food). Applications that are marketed already include nano-encapsulated agrochemicals or nutrients, antimicrobial nanoparticles and active and intelligent food packaging. Many nano-enabled products are currently under research and development, and may enter the market in the near future. As for any other regulated product, applicants applying for market approval have to demonstrate the safe use of such new products without posing undue safety risks to the consumer and the environment. Several countries all over the world have been active in examining the appropriateness of their regulatory frameworks for dealing with nanotechnologies. As a consequence of this, different approaches have been taken in regulating nano-based products in agri/feed/food. The EU, along with Switzerland, were identified to be the only world region where nano-specific provisions have been incorporated in existing legislation, while in other regions nanomaterials are regulated more implicitly by mainly building on guidance for industry. This paper presents an overview and discusses the state of the art of different regulatory measures for nanomaterials in agri/feed/food, including legislation and guidance for safety assessment in EU and non-EU countries.

Determining composition, shape, and size of nanoparticles dispersed in a complex matrix is necessary in the assessment of toxicity, for regulatory actions, and environmental monitoring. Many types of nanoparticles that are currently used in consumer products contain more than one metal which are often not uniformly distributed (e.g., core-shell nanoparticles). This compositional and structural complexity makes their characterization difficult. In this study, we investigate the capability of single particle inductively coupled plasma mass spectrometry (spICP-MS) using time-of-flight (TOF) and quadrupole (Q) mass analyzers to determine the composition, size distribution, and concentration of a series of nanoparticles that are used in a variety of industrial applications: BiVO4, (Bi0.5Na0.5)TiO3 and steel (which contains Fe, Cr, Ni, Mo) nanoparticles. In addition, we tested both types of mass analyzers with Au-core/Ag-shell nanoparticles, which are well-characterized and have already been used for assessment of multi-element capabilities of spICP-MS. The results confirm that both types of mass analyzers produce accurate estimations of the size of Au-core/Ag-shell particles. For other multi-element nanoparticles, spICP-MS provided the size of aggregates and/or agglomerates in the prepared suspensions. In general, particle size detection limits (dLOD) of spICP-TOFMS instruments with values of 29 nm for Ti, 14 nm for Mo, and 7 nm for Au, are smaller than those obtained for the quadrupole instruments. This study finds that only spICP-TOFMS can accurately assess the elemental composition of nano-steel particles. By contrast, spICP-QMS is limited to the detection of 2 elements in an individual particle and the elemental composition of nano-steel particles is less accurate. In general, spICP-TOFMS was able to quantify multiple elements with high precision and that currently makes it the first choice for multi-element detection of unknown nanoparticles.

In this paper the NanoDefiner e-tool for classification of potential nanomaterial, according to the European Commission's definition, is introduced. Its knowledge base enables rule-based multi-criteria decision making on recommendation of suitable measurement techniques for a specific material. Its rule base was established with help of experts from industry and academia via literature analysis and lab experiments. Beside its potential to support material classification, its capability to facilitate suitability testing of measurement techniques for materials by manufacturers is outlined.

Nanomaterials are developed for and applied in food, food additives, supplements and food contact materials. In an inventory of internet databases 140 products in the food and food-related sectors were identified that claim to contain nanomaterials. A great diversity of nanomaterials is applied, ranging from inorganic metal and metal oxides to organic nanomaterials that carry bioactive ingredients. Here we present an overview of nanomaterial applications that are currently available, discuss state of the art analytical chemical characterization and toxicological assessment methods, as well as categorization methods to support the safety evaluation of the application of nanomaterials throughout food production.

The potential impact of nanomaterials on the environment and on human health has already triggered legislation requiring labelling of products containing nanoparticles. However, so far, no validated analytical methods for the implementation of this legislation exist. This paper outlines a generic approach for the validation of methods for detection and quantification of nanoparticles in food samples. It proposes validation of identity, selectivity, precision, working range, limit of detection and robustness, bearing in mind that each “result” must include information about the chemical identity, particle size and mass or particle number concentration. This has an impact on testing for selectivity and trueness, which also must take these aspects into consideration. Selectivity must not only be tested against matrix constituents and other nanoparticles, but it shall also be tested whether the methods apply equally well to particles of different suppliers. In trueness testing, information whether the particle size distribution has changed during analysis is required. Results are largely expected to follow normal distributions due to the expected high number of particles. An approach of estimating measurement uncertainties from the validation data is given.

Silver nanoparticles (AgNPs) are of interest due to their antimicrobial activity and are seen as potential candidates to replace antibiotics in animal husbandry. A few studies have focused on this new application, but they lack any considerations about residual accumulation of AgNPs in edible animal tissues and animal products. In this research, a 22 day in vivo study was carried out by oral administration of 20 nm spherical PVP coated AgNPs to hens. Six doses of approximately 1 mg kg-1 of AgNPs-PVP each were administered to animals throughout the experimentation. Atomic absorption spectroscopy (AAS) was used for quantitative determination of residual total Ag in different organs and matrices. The analyses showed that Ag accumulates in livers (concentration ranging from 141 μg kg-1 to 269 μg kg-1) and yolks (concentration ranging from 20 μg kg-1 to 49 μg kg-1) but not in muscles, kidneys, and albumen belonging to hens of the treated group (tG2). Ag was not detected in animals of the control group (uG1) (i.e., total Ag < LOD = 10 μg kg-1). Single particle inductively coupled plasma mass spectrometry (spICP-MS) and scanning electron microscopy with energy dispersive X-ray detection (SEM-EDX) were employed to elucidate the presence of AgNPs in livers and yolks belonging to tG2 animals. spICP-MS highlighted that part of residual Ag found in livers (about 5-20%) is in NP form with an average dimension of approximately 20 nm. SEM-EDX technique confirmed the presence of AgNPs only in livers of treated animals. The results show that feeding AgNPs to hens may become a source of consumer exposure to AgNPs. As far as we know this is the first study showing transfer of AgNPs or reaction products thereof from animal feed to animal products. Chemicals/CAS: silver, 7440-22-4; Anti-Bacterial Agents; Silver

Currently established and projected regulatory frameworks require the classification of materials (whether nano or non-nano) as specified by respective definitions, most of which are based on the size of the constituent particles. This brings up the question if currently available techniques for particle size determination are capable of reliably classifying materials that potentially fall under these definitions. In this study, a wide variety of characterisation techniques, including counting, fractionating, and spectroscopic techniques, has been applied to the same set of materials under harmonised conditions. The selected materials comprised well-defined quality control materials (spherical, monodisperse) as well as industrial materials of complex shapes and considerable polydispersity. As a result, each technique could be evaluated with respect to the determination of the number-weighted median size. Recommendations on the most appropriate and efficient use of techniques for different types of material are given. Graphical Abstract Electronic supplementary material The online version of this article (doi:10.1007/s11051-016-3461-7) contains supplementary material, which is available to authorized users.

Processing multimodal sensory information is vital for behaving animals in many contexts. The barn owl, an auditory specialist, is a classic model for studying multisensory integration. In the barn owl, spatial auditory information is conveyed to the optic tectum (TeO) by a direct projection from the external nucleus of the inferior colliculus (ICX). In contrast, evidence of an integration of visual and auditory information in auditory generalist avian species is completely lacking. In particular, it is not known whether in auditory generalist species the ICX projects to the TeO at all. Here we use various retrograde and anterograde tracing techniques both in vivo and in vitro, intracellular fillings of neurons in vitro, and whole-cell patch recordings to characterize the connectivity between ICX and TeO in the chicken. We found that there is a direct projection from ICX to the TeO in the chicken, although this is small and only to the deeper layers (layers 13-15) of the TeO. However, we found a relay area interposed among the IC, the TeO, and the isthmic complex that receives strong synaptic input from the ICX and projects broadly upon the intermediate and deep layers of the TeO. This area is an external portion of the formatio reticularis lateralis (FRLx). In addition to the projection to the TeO, cells in FRLx send, via collaterals, descending projections through tectopontine-tectoreticular pathways. This newly described connection from the inferior colliculus to the TeO provides a solid basis for visual-auditory integration in an auditory generalist bird. J. Comp. Neurol. 525:513-534, 2017. © 2016 Wiley Periodicals, Inc.

Seven commercial titanium dioxide pigments and two other well-defined TiO2 materials (TiMs) were physicochemically characterised using asymmetric flow field flow fractionation (aF4) for separation, various techniques to determine size distribution and inductively coupled plasma mass spectrometry (ICPMS) for chemical characterization. The aF4-ICPMS conditions were optimised and validated for linearity, limit of detection, recovery, repeatability and reproducibility, all indicating good performance. Multi-element detection with aF4-ICPMS showed that some commercial pigments contained zirconium co-eluting with titanium in aF4. The other two TiMs, NM103 and NM104, contained aluminium as integral part of the titanium peak eluting in aF4. The materials were characterised using various size determination techniques: retention time in aF4, aF4 hyphenated with multi-angle laser light spectrometry (MALS), single particle ICPMS (spICPMS), scanning electron microscopy (SEM) and particle tracking analysis (PTA). PTA appeared inappropriate. For the other techniques, size distribution patterns were quite similar, i.e. high polydispersity with diameters from 20 to >700 nm, a modal peak between 200 and 500 nm and a shoulder at 600 nm. Number-based size distribution techniques as spICPMS and SEM showed smaller modal diameters than aF4-UV, from which mass-based diameters are calculated. With aF4-MALS calculated, light-scattering-based “diameters of gyration” (Øg) are similar to hydrodynamic diameters (Øh) from aF4-UV analyses and diameters observed with SEM, but much larger than with spICPMS. A Øg/Øh ratio of about 1 indicates that the TiMs are oblate spheres or fractal aggregates. SEM observations confirm the latter structure. The rationale for differences in modal peak diameter is discussed.

We have designed a cell culture system for thoracic neurons of adult Locusta migratoria that enables the establishment of functional synapses in vitro. Patch-clamp recordings revealed three different neuron classes. About half of the neurons (47%) had unexcitable somata with outward and no inward conductance. The other half generated either single (37%) or multiple action potentials (18%) and differed mainly in lower outward conductance. Selectively stained motor neurons were analyzed to demonstrate varied physiological properties due to culture conditions. Using paired patch clamp recordings we demonstrate directly synaptic transmission in morphologically connected neurons in vitro. Presynaptic stimulation resulted in postsynaptic potentials in 42 pairs of neurons tested, independent of the type of neuron. According to pharmacological experiments most of these synapses were either glutamatergic or GABAergic. In addition to these chemical synapses, electrical synapses were found. With the demonstration of synapse formation in cell culture of adult locust neurons, this study provides the basis for the future analysis of more defined insect neuronal circuits in culture.

The presence, dissolution, agglomeration state, and release of materials in the nano-size range from food containing engineered nanoparticles during human digestion is a key question for the safety assessment of these materials. We used an in vitro model to mimic the human digestion. Food products subjected to in vitro digestion included (i) hot water, (ii) coffee with powdered creamer, (iii) instant soup, and (iv) pancake which either contained silica as the food additive E551, or to which a form of synthetic amorphous silica or 32 nm SiO2 particles were added. The results showed that, in the mouth stage of the digestion, nano-sized silica particles with a size range of 5-50 and 50-500 nm were present in food products containing E551 or added synthetic amorphous silica. However, during the successive gastric digestion stage, this nano-sized silica was no longer present for the food matrices coffee and instant soup, while low amounts were found for pancakes. Additional experiments showed that the absence of nano-sized silica in the gastric stage can be contributed to an effect of low pH combined with high electrolyte concentrations in the gastric digestion stage. Large silica agglomerates are formed under these conditions as determined by DLS and SEM experiments and explained theoretically by the extended DLVO theory. Importantly, in the subsequent intestinal digestion stage, the nano-sized silica particles reappeared again, even in amounts higher than in the saliva (mouth) digestion stage. These findings suggest that, upon consumption of foods containing E551, the gut epithelium is most likely exposed to nano-sized silica. © 2012 American Chemical Society.

Silver nanoparticles are recognized as effective antimicrobial agents and have been implemented in various consumer products including washing machines, refrigerators, clothing, medical devices, and food packaging. Alongside the silver nanoparticles benefits, their novel properties have raised concerns about possible adverse effects on biological systems. To protect consumer’s health and the environment, efficient monitoring of silver nanoparticles needs to be established. Here, we present the development of human metallothionein (MT) based surface plasmon resonance (SPR) sensor for rapid detection of nanosilver. Incorporation of human metallothionein 1A to the sensor surface enables screening for potentially biologically active silver nanoparticles at parts per billion sensitivity. Other protein ligands were also tested for binding capacity of the nanosilver and were found to be inferior to the metallothionein. The biosensor has been characterized in terms of selectivity and sensitivity towards different types of silver nanoparticles and applied in measurements of real-life samples—such as fresh vegetables and river water. Our findings suggest that human MT1-based SPR sensor has the potential to be utilized as a routine screening method for silver nanoparticles, that can provide rapid and automated analysis dedicated to environmental and food safety monitoring. Figure Surface plasmon resonance biosensor for rapid detection of silver nanoparticles was developed. Human metallothionein 1A (hMT1A) was immobilized on the sensor chip surface and showed dose dependent binding to the silver nanoparticles with part per billion sensitivity. Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-5920-z) contains supplementary material, which is available to authorized users.

A liquid chromatography–tandem mass spectrometry method for the analysis of seven antiviral drugs, zanamivir, ribavirin, oseltamivir, oseltamivir carboxylate, amantadine, rimantadine and arbidol, in poultry muscle is reported. The antiviral drugs were extracted from the homogenized poultry muscle sample using methanol. The extract was purified using tandem solid-phase extraction combining a cation exchange cartridge and a phenylboronic acid cartridge. To prevent excessive matrix effects, the analytes were separated from the matrix constituents using a column-switch liquid chromatography system combining a reversed-phase and a Hypercarb analytical column. Detection was carried out using tandem mass spectrometry. The method was fully validated according to 2002/657/EC [1] and proved to be adequate for quantification and confirmation of zanamivir and ribavirin at 10 μg kg−1, oseltamivir, oseltamivir carboxylate, amantadine and rimantadine at levels below 1.0 μg kg−1 and for qualitative confirmatory analysis of arbidol at levels below 1 μg kg−1. Electronic supplementary material The online version of this article (doi:10.1007/s00216-011-5581-3) contains supplementary material, which is available to authorized users.

Peking duck is one of the most well-known Chinese dishes. GC–MS analysis revealed that, either benzo(a)pyrene alone, or the total amount of major PAHs with carcinogenic potential for humans, reached levels of 8.7 and 54.7 μg/kg, respectively. These levels greatly exceeded those found in grilled poultry in European countries. However, the intake of benzo(a)pyrene from a Peking duck meal (about 0.65 μg) was not higher than that from the daily intake (for residents in the same area) from vegetables alone (about 2 μg/day). Since Peking duck is still not a plebeian meat in this country, people would not consume it very often.

This article presents research based on a case study of Bern University Hospital and the Greater Bern Area that analyses the hospital's influence on innovation in the regional medical device industry. The study draws upon the concept of sectoral innovation systems enlarged with a space-oriented model of agents' innovation-related behaviour. Its main data stem from semi-structured interviews with representatives from regional companies and the hospital. The findings confirm the systemic character of innovation in medical technology and the role of university hospitals as the main functional source of medical device innovation. At the inception of the medical device industry, innovations were implemented in a regional setting, but with the maturation of the industry, this setting has yielded to an almost global setting for innovation. This international setting became prevalent largely due to the marketing considerations of the industry, showing that from an industrial management point of view, product inno...

Interest in nanoparticles (NPs) has increased explosively over the past two decades. Using NPs, high loadings of vitamins and health-benefit actives can be achieved in food, and stable flavors as well as natural food-coloring dispersions can be developed. Detection and characterization of NPs are essential in understanding the benefits as well as the potential risks of the application of such materials in food. While many such applications are described in the literature, methods for detection and characterization of such particles are lacking. Organic NPs suitable for application in food are lipid-, protein- or polysaccharide-based particles, and this review describes current analytical techniques that are used, or could be used, for identification and characterization of such particles in food products. We divide the analytical approaches into four sections: sample preparation; separation; imaging; and, characterization. We discuss techniques and reported applications for NPs or otherwise related particle compounds. The results of this investigation show that, for a successful characterization of NPs in food, at least some kind of sample preparation will be required. While a simple sample preparation may be satisfactory for imaging techniques for known analytes, for other techniques, a further separation using chromatography, field-flow fractionation or ion-mobility separation is necessary. Subsequently, photon-correlation spectroscopy and especially mass spectrometry techniques as matrix-assisted laser desorption/ionization combined with time-of-flight mass spectrometry, seem suitable techniques for characterizing a wide variety of organic NPs.

Carbon nanotubes (CNTs) are among the most promising novel nanomaterials and their unique chemical and physical properties suggest an enormous potential for many areas of research and applications. As a consequence, the production of CNT-based material and thus the occupational and public exposure to CNTs will increase steadily. Although there is evidence that nanoparticles (NPs) can enter the nervous system via the blood stream, olfactory nerves or sensory nerves in the skin, there is still only little knowledge about possible toxic effects of CNTs on cells of the nervous system. The goal of the present study was to analyse the influences of single-walled CNTs (SWCNTs) with different degrees of agglomeration on primary cultures derived from chicken embryonic spinal cord (SPC) or dorsal root ganglia (DRG). As measured by the Hoechst assay treatment of mixed neuro-glial cultures with up to 30mug/mL SWCNTs significantly decreased the overall DNA content. This effect was more pronounced if cells were exposed to highly agglomerated SWCNTs as compared to better dispersed SWCNT-bundles. Using a cell-based ELISA we found that SWCNTs reduce the amount of glial cells in both peripheral nervous system (PNS) and central nervous system (CNS) derived cultures. Neurons were only affected in DRG derived cultures, where SWCNT treatment resulted in a decreased number of sensory neurons, as measured by ELISA. Additionally, whole-cell patch recordings revealed a diminished inward conductivity and a more positive resting membrane potential of SWCNT treated DRG derived neurons compared to control samples. The SWCNT suspensions used in this study induced acute toxic effects in primary cultures from both, the central and peripheral nervous system of chicken embryos. The level of toxicity is at least partially dependent on the agglomeration state of the tubes. Thus if SWCNTs can enter the nervous system at sufficiently high concentrations, it is likely that adverse effects on glial cells and neurons might occur.

The performance of a previously developed immunochemical biosensor screening method for fluoroquinolone (FQ) antibiotics in poultry muscle, fish and egg was compared with established methods. Blank sample material of the target matrices was individually spiked with the FQs at half maximum residue levels. Homogeneity of the test materials was confirmed by liquid chromatography-mass spectrometry (LC-MS/MS). Identical sets of spiked samples as well as incurred samples from a previous feeding experiment were sent to three independent laboratories and analysed by LC-MS/MS, a microbiological assay and the new biosensor assay. The new method correctly identified all contaminated samples and demonstrated advantages in sensitivity and analysis time compared to the microbiological screening assay.

A surface plasmon resonance biosensor immunoassay has been developed for multi-residue determination of 13 (fluoro)quinolone antibiotics in poultry meat, eggs and fish. The following performance characteristics were determined according to the guidelines laid down for screening assay validation in European Decision 2002/657/EC: detection capability, specificity/selectivity, decision limit, repeatability, ruggedness and stability. The detection capability estimated for norfloxacin, the reference fluoroquinolone, was below 0.5, 1 and 1.5 ng g⁻¹ for poultry meat, egg and fish, respectively. The screening assay proved specific and showed satisfactory sensitivity below the MRL levels even though flumequine and oxolinic acid had lower cross-reactivities. A wide range of non-MRL substances were also detected at concentrations below 10 ng g⁻¹. Repeatability was good with both intra- and inter-assay coefficients of variation 56%; ruggedness was also demonstrated.

Analysis of honey for the antibiotic streptomycin, which is not approved for use in apiculture within the European Union, by immunochemical biosensor screening was found to be disturbed by matrix influence related to the color of the honeys, as also known from enzyme-immunoassays. Effect of interfering compounds most likely being glycosides was eliminated by meticulous pH-control followed by ultrafiltration of the dissolved honey samples prior to analysis resulting in a rapid and simple analytical method with a detection limit

The aim of this study was to develop an optical biosensor inhibition immunoassay, based on the surface plasmon resonance (SPR) principle, for use as a screening test for 13 (fluoro)quinolones, including flumequine, used as veterinary drugs in food-producing animals. For this, we immobilised various quinolone derivatives on the sensor chip and tested binding of a range of different antibodies (polyclonal and one engineered antibody) in the presence and absence of free (fluoro)quinolones. The main challenge was to detect flumequine in an assay giving good results for the other compounds. One antigen-antibody combination proved satisfactory: polyclonal antibodies raised against a dual immunogen and, on the sensor chip, a fluoroquinolone derivative. It was the first time that this concept of the bi-active antibody was described in the literature. The assay, optimised for detection in three matrices (poultry muscle, fish, and egg), was tested on incurred samples prepared by liquid extraction followed by two washing steps. This rapid, simple method proved adequate for detecting at least 13 (fluoro)quinolones at concentrations below established maximum residue levels (MRLs). The reference molecule norfloxacin could be detected in the range of 0.1-10 microg kg(-1) in extracts of egg and poultry meat and in the range of 0.1-100 microg kg(-1) in extracts of fish. The determined midpoints of these calibration curves were about 1, 1.5 and 3 microg kg(-1) in poultry meat, egg and fish, respectively.

Solid-phase extraction of 20 L seawater samples enabled the enrichment and determination of a wide array of organic substances, including compounds of medium to high polarity, in the pg/L-range. A number of contaminants was detected and quantified throughout the North Sea, among them the pesticides dichlobenil (2,6-dichlorobenzonitrile), metolachlor and terbuthylazine as well as the industrial chemicals dichloropyridines (DCPy, 4 isomers) and nitrobenzene. Concentrations attained values up to 1.4 ng/L for dichlobenil, 0.83 ng/L for terbuthylazine, 0.61 ng/L for metolachlor, 0.13 ng/L for 2,6-DCPy, 4.37 ng/L for nitrobenzene and 1-8 ng/L for tris(chloropropyl)phosphates (TCPP). A number of North Sea water samples was screened for non-target compounds, revealing the presence of further contaminants, e.g., lindane and TCPP, as well as several biogenic and/or anthropogenic organobromine substances, among which bromoindols, -phenols and -anisoles were identified.

Immunochemical screening assays using surface plasmon resonance have been developed for chloramphenicol and chloramphenicol glucuronide residues in poultry muscle, honey, prawn and cows' milk using a sensor chip coated with a chloramphenicol derivative and an antibody. The antibody cross-reacted with chloramphenicol glucuronide 73.8% (poultry), 69.2% (honey), 75.7% (prawn) and 84.8% (milk). There was no cross-reaction with similar drugs or other commonly used antibiotics. The assay allowed the direct analysis of bovine milk (fat content ∼3.5%). Poultry, honey and prawn samples were extracted with ethyl acetate followed by analysis on the biosensor. The decision limits (CCa) for each assay were determined as: poultry (0.005 μg kg -1 ), honey (0.02 μg kg -1 ), prawn (0.04 μg kg -1 ) and milk (0.04 μg kg -1 ) and the detection capabilities (CCβ) were 0.02, 0.02, 0.07 and 0.05 μg kg -1 , respectively. Poultry muscle, honey and milk were spiked at 0.1 μg kg -1 and prawn at 0.15 μg kg -1 and the intra-assay precision (n = 10) calculated as 10.5, 5.0, 4.6 and 8.8%, respectively. Between run precision (n = 3) performed at the same levels yielded the following results: 3.0% (poultry), 4.7% (honey), 7.6% (milk) and 5.5% (prawn).

Seven polymeric solid-phase extraction (SPE) sorbents were evaluated with regard to their ability to extract acidic, neutral and basic pharmaceuticals and estrogens simultaneously from water at neutral pH. Highest recoveries (70-100%) for the majority of the analytes were obtained with styrene-methacrylate and styrene-N-vinylpyrrolidone co-polymers. The latter one (Oasis HLB) was chosen for further refinement of an extraction method for the quantitative determination of acidic and neutral drugs in surface water samples at detection limits below 1 ng/l. A sequential elution protocol was applied for clean-up and separation of the extracted analytes into fractions suitable for further compound specific processing. The neutral analytes as well as the acidic compounds after derivatisation were quantified by GC-MS. Caffeine, ibuprofen, its metabolites and diclofenac were detected in river water samples in the 1-100 ng/l range.

Detection and characterization of nanoparticles (NPs) in complex media as consumer products, food and toxicological test media is an essential part of understanding the potential benefits and risks of the application of nanoparticles. Single particle ICP-MS (spICP-MS) was studied as a screening tool for the detection and characterization of nanoparticles in complex matrices such as food and biological tissues. A data evaluation tool was created for the calculation of particle size, concentration and size distribution from the raw data. spICP-MS measurements were carried out on a standard quadrupole instrument as well as on a sector-field instrument. Performance characteristics were determined for four types of NPs. For the quadrupole instrument the size detection limits were 20 nm (Au and Ag), 50 (TiO2) and 200 nm (SiO2). For the sector-field instrument size detection limits are lower, 10 nm (Au). Concentration detection limits ranged from 1 ng L−1 for 60 nm Au NPs to 0.1 μg L−1 for 500 nm SiO2 particles. The dynamic range of spICP-MS is limited to two orders of magnitude and as a consequence sample dilution is often required. The precision of the method was found to be

A bioassay-directed fractionation and identification (toxicity identification evaluation procedure) was performed on extracts of 10 l River Elbe water samples. The experimental method included a SDB-1 solid phase extraction followed by RP-HPLC fractionation and subfractionation. Chemical analysis by GC–MS as well as acute toxicity testing using a luminescent bacteria assay were conducted in the respective fractions. Many substances were identified, among which were pesticides and pharmaceuticals, but many compounds remained unknown.