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The prevalent application of engineered nanomaterials (ENMs) has led to an extensive effort to develop tools facilitating their risk assessment and management. This interlaboratory trial (encompassing two laboratories) sought to investigate the genotoxic impact of ENMs using the hypoxanthine phosphoribosyltransferase (HPRT) forward mutation assay and in vitro cytokinesis-blocked micronucleus (CBMN) assay, utilising harmonised protocols. In the CBMN assay, human lymphoblast (TK6) cells were exposed to zinc oxide (ZnO), titanium dioxide (TiO2) and tungsten carbide-cobalt (WC/Co) for 24-hours after which 1000 binucleated cells were scored for micronuclei (n=2). For the HPRT assay, TK6 and mouse fibroblast (V79) cells were exposed to ZnO, TiO2, WC/Co, CuO and Nanocyl-CNTs for 24-hours after which 600 wells were scored for point mutations (n=2). Significant cytotoxicity and micronuclei frequency was reported following ZnO exposure (20µg/ml). Significant micronuclei induction was reported following WC/Co exposures (2-fold over control, 100µg/ml). No significant mutagenicity was detected with ZnO or TiO2; both laboratories observed significant cytotoxicity following exposure to ZnO (20µg/ml; 37% reduction in cell viability). In the second interlaboratory trial CuO induced significant cytotoxicity (36% reduction in viability: 0.5µg/ml), and a 32-fold increase in mutagenicity. The Nanocyl induced a 6-fold increase in mutagenicity. The data generated here has shown good statistical concordance between laboratories and has highlighted a promising route forward in supporting risk decision making for ENMs. The authors would like to acknowledge this research has received funding from the European Union’s Horizon 2020 research and innovation program for the RiskGONE project, grant agreement #814425.

Air pollutions are shown to increase the occurrence of cardiovascular diseases and ischemic stroke. These effects imply an indirect exposure of secondary organs downstream of the respiratory system as the primary target of exposure. Mechanistic studies of air pollution on secondary organs have traditionally been performed on organ specific monocultures at submerged conditions. Direct exposure to organ specific cells secondary exposed to air pollution have the drawback that it does not imply realistic exposure conditions. In this study we have indirectly exposed organ specific cells to particulate matter via exposure of a bronchial lung model at the air liquid interphase (ALI). The lung model was exposed to a model diesel particle (DEP) and an equivalent dose of an acetone extract of the DEP. The DEP induced induction of IL-6 formation of which the DEP was more potent than the DEP extract. The DEP extract inhibited CXCL8 formation, which appeared contradictory since both the DEP and DEP-extract increased expression of CXCL8 mRNA. Our results showed a substantial increased expression of CyP1A1 and CyP1B1 in both epithelial and endothelial cells in the lung model of which no differences were observed between the DEP and DEP-extract. However, the solvent extract induced the CYPs with a higher potency in the secondary exposed HepG2 liver cells, probably due to faster permeation of dissolved substances in the DEP extract through the lung barrier. Thus, organ specific cell models can be combined to achieve a more realistic so-called body-specific model to study secondary effects.

Determination of acute toxicity to vertebrates in aquatic environments is mainly performed following OECD test guideline 203, requiring the use of a large number of fish and with mortality as endpoint. This test is also used to determine toxicity of nanomaterials in aquatic environments. Since a replacement method for animal testing in nanotoxicity studies is desirable, the feasibility of fish primary cultures or cell lines as a model for nanotoxicity screenings is investigated here. Dicentrarchus labrax primary cultures and RTgill-W1 cell line were exposed to several concentrations (0.1 to 200 ug/mL) of different nanoparticles (TiO2, polystyrene and silver), and cytotoxicity, metabolic activity and reactive oxygen species formation were investigated after 24 and 48 h of exposure. Protein corona as amount of protein bound, as well as the influence of surface modification (-COOH, -NH2), exposure media (Leibovitz’s L15 or seawater), weathering and cell type were the experimental variables included to test their influence on the results of the assays. Data from all scenarios was split based on the significance each experimental variable had in the result of the cytotoxicity tests, in an exploratory approach that allows for better understanding of the determining factors affecting toxicity. Data shows that more variables significantly influenced the outcome of toxicity tests when the primary cultures were exposed to the different nanoparticles. Toxicity tests performed in RTgill-W1 were influenced only by exposure time and nanoparticle concentration. The whole data set was integrated in a biological response index to show the overall impact of nanoparticle exposures. This study was supported by a postdoctoral grant to AJ-R (Basque Government; POSDOC program 2017–2019), Basque Country, Spain; and Federal Ministry of Education and Research, BMBF (NanoUmwelt, grant agreement Nº 030150B), Germany.

13 Pág. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, 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., The work leading to this article has received funding from the European Union’s Horizon 2020 Research and Innovation programme, Grant Agreements no. 646221 (NanoReg2, 2015–2019), no. 814401 (Gov4Nano, 2019–2022) and no. 814425 (RiskGONE 2019–2023). In addition, the European Union’s 7th Framework Programme projects NANoREG (2013–2017, Grant Agreement no. 310584), NanoTEST (2008–2012, no. 201335) and ENPRA (2009–2012, no. 228789), the European Union’s Health Programme Joint Action project NANOGENOTOX (2010–2013, no. 2009 21 01) and the US NIH NCI caNanoLab portal are acknowledged for providing data

BACKGROUND. Methodology for estimation of cerebrospinal fluid (CSF) tracer clearance could have wide clinical application in predicting excretion of intrathecal drugs and metabolic solutes from brain metabolism and for diagnostic workup of CSF disturbances. METHODS. The MRI contrast agent gadobutrol (Gadovist) was used as a CSF tracer and injected into the lumbar CSF. Gadobutrol is contained outside blood vessels of the CNS and is eliminated along extravascular pathways, analogous to many CNS metabolites and intrathecal drugs. Tracer enrichment was verified and assessed in CSF by MRI at the level of the cisterna magna in parallel with obtaining blood samples through 48 hours. RESULTS. In a reference patient cohort (n = 29), both enrichment within CSF and blood coincided in time. Blood concentration profiles of gadobutrol through 48 hours varied between patients diagnosed with CSF leakage (n = 4), idiopathic normal pressure hydrocephalus dementia (n = 7), pineal cysts (n = 8), and idiopathic intracranial hypertension (n = 4). CONCLUSION. Assessment of CSF tracer clearance is clinically feasible and may provide a way to predict extravascular clearance of intrathecal drugs and endogenous metabolites from the CNS. The peak concentration in blood (at about 10 hours) was preceded by far peak tracer enhancement at MRI in extracranial lymphatic structures (at about 24 hours), as shown in previous studies, indicating a major role of the spinal canal in CSF clearance capacity. FUNDING. The work was supported by the Department of Neurosurgery, Oslo University Hospital; the Norwegian Institute for Air Research; and the University of Oslo.

Microfluidic technology is a valuable tool for realizing more in vitro models capturingcellular and organ level responses for rapid and animal-free risk assessment ofnew chemicals and drugs. Microfluidic cell-based devices allow high-throughputscreening and flexible automation while lowering costs and reagent consumption dueto their miniaturization. There is a growing need for faster and animal-free approachesfor drug development and safety assessment of chemicals (Registration, Evaluation,Authorisation and Restriction of Chemical Substances, REACH). The work presenteddescribes a microfluidic platform for in vivo-like in vitro cell cultivation. It is equippedwith a wafer-based silicon chip including integrated electrodes and a microcavity. Aproof-of-concept using different relevant cell models shows its suitability for labelfreeassessment of cytotoxic effects. A miniaturized microscope within each modulemonitors cell morphology and proliferation. Electrodes integrated in the microfluidicchannels allow the noninvasive monitoring of barrier integrity followed by a label-freeassessment of cytotoxic effects. Each microfluidic cell cultivation module can beoperated individually or be interconnected in a flexible way. The interconnection ofthe different modules aims at simulation of the whole-body exposure and responseand can contribute to the replacement of animal testing in risk assessment studies incompliance with the 3Rs to replace, reduce, and refine animal experiments.

Intrinsically fluorescent carbon dots may form the basis for a safer and more accurate sensor technology for digital counting in bioanalytical assays. This work presents a simple and inexpensive synthesis method for producing fluorescent carbon dots embedded in hollow silica particles. Hydrothermal treatment at low temperature (160 °C) of microporous silica particles in presence of urea and citric acid results in fluorescent, microporous and hollow nanocomposites with a surface area of 12 m2 /g. High absolute zeta potential (-44 mV) at neutral pH demonstrates the high electrosteric stability of the nanocomposites in aqueous solution. Their fluorescence emission at 445 nm is remarkably stable in aqueous dispersion under a wide pH range (3-12) and in the dried state. The biocompatibility of the composite particles is excellent, as the particles were found to show low genotoxicity at exposures up to 10 μg/cm2 .

Microfluidic technology is a valuable tool for realizing more in vitro models capturing cellular and organ level responses for rapid and animal-free risk assessment of new chemicals and drugs. Microfluidic cell-based devices allow high-throughput screening and flexible automation while lowering costs and reagent consumption due to their miniaturization. There is a growing need for faster and animal-free approaches for drug development and safety assessment of chemicals (Registration, Evaluation, Authorisation and Restriction of Chemical Substances, REACH). The work presented describes a microfluidic platform for in vivo-like in vitro cell cultivation. It is equipped with a wafer-based silicon chip including integrated electrodes and a microcavity. A proof-of-concept using different relevant cell models shows its suitability for label-free assessment of cytotoxic effects. A miniaturized microscope within each module monitors cell morphology and proliferation. Electrodes integrated in the microfluidic channels allow the noninvasive monitoring of barrier integrity followed by a label-free assessment of cytotoxic effects. Each microfluidic cell cultivation module can be operated individually or be interconnected in a flexible way. The interconnection of the different modules aims at simulation of the whole-body exposure and response and can contribute to the replacement of animal testing in risk assessment studies in compliance with the 3Rs to replace, reduce, and refine animal experiments., T.K. and Y.K. contributed equally to this work. The authors thank Frank Bauerfeld for system assembly and electrical testing, Werner Haberer for assembly of the microfluidic cartridges, Axel Brenner for the fabrication of the silicon micro cavity chips, Hoffman La Roche for providing the Ro 19-8022, NorGenoTech for providing the Fpg enzyme, and Karen Steenson for proofreading. This research was funded by the European Commission under the Horizon2020 programme (HISENTS, Grant Agreement No. 685817 and VISION, Grant Agreement No. 857381); and by the Norwegian Research Council Norway (RCN) via the European Research Area Network (ERA-NET) EuroNanoMed II project INNOCENT (RCN 271075) and the ERA-NET EuroNanoMed III project Graphene-encapsulated magnetic nanoparticles (RCN 246672/O70). K.K. received Short Term Scientific Mission Grant (ID 42926) under COST Action CA 17140 “Cancer Nanomedicine from the Bench to the Bedside” supported by COST (European Cooperation in Science and Technology). E.E. received funding from the Norwegian Research Council (272412/F40). O.H.M. received funding from the Spanish Ministry of Economy and Competitiveness (SEV-2013-0295-17-3).

The implementation of Safe(r) by Design (SbD) in industrial innovations requires an integrated approach where the human, environmental and economic impact of the SbD measures is evaluated across and throughout the nanomaterial (NM) life cycle. SbD was implemented in six industrial companies where SbD measures were applied to NMs, nano-enabled products (NEPs) and NM/NEP manufacturing processes. The approach considers human and environmental risks, functionality of the NM/NEP and costs as early as possible in the innovation process, continuing throughout the innovation progresses. Based on the results of the evaluation, a decision has to be made on whether to continue, stop or re-design the NM/NEP/process or to carry out further tests/obtain further data in cases where the uncertainty of the human and environmental risks is too large. However, SbD can also be implemented at later stages when there is already a prototype product or process available, as demonstrated in some of the cases. The SbD measures implemented in some of the case studies did not result in a viable solution. For example the coating of silicon nanoparticles with amorphous carbon increased the conductivity, the stability and reduced the dustiness of the particles and therefore the risk of explosion and the exposure to workers. However the socioeconomic assessment for their use in lithium-ion batteries for cars, when compared to the use of graphite, showed that the increase in performance did not overcome the higher production costs. This work illustrates the complexities of selecting the most appropriate SbD measures and highlights that SbD cannot be solely based on a hazard and exposure assessment but must include other impacts that any SbD measures may have on sustainability including energy consumption and waste generation as well as all associated monetary costs.

Concerns have been raised as to whether gunshot fumes induce prolonged reduced lung capacity or even cancer due to inhalation. Gunshot fumes from three different types of ammunition calibre 5.56 mm × 45 NATO were investigated. SS109 has a soft lead (Pb) core, while NM255 and NM229 have a harder steel core. Emissions from ammunitions were characterized with respect to particle number- and mass-size, and mass distribution, heavy metal content, and different gases. Lung epithelial cells were exposed to the fumes at the air liquid interface to elucidate cytotoxicity and genotoxicity. Irrespectively of ammunition type, the largest mass fraction of generated particulate matter (PM) had a size between 1 and 3 μm. The highest number of particles generated was in the size range of 30 nm. Fumes from NM255 and NM229 induced cytotoxic effects of which the emission from NM229 induced the highest effect. Fumes from NM229 induced a dose-related increase in DNA-damage. Significant effects were only achieved at the highest exposure level, which led to approximately 40% reduced cell viability after 24 h. The effect probably relates to the mass of emitted particles where the size may be of importance, in addition to emission of Cu and Zn. A complex mixture of chemical substances and PM may increase the toxicity of the fumes and should encourage measures to reduce exposure.

Nanotechnologies have reached maturity and market penetration that require nano‐specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)‐by‐Design (SbD) concepts. This paper provides an overview of the state‐of‐the‐art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re‐thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science‐based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally., This study received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 814425 (RiskGONE) and No 814572 (NanoSolveIT).

Understanding nanomaterial (NM)-protein interactions is a key issue in defining the bioreactivity of NMs with great impact for nanosafety. In the present work, the complex phenomena occurring at the bio/nano interface were evaluated in a simple case study focusing on NM-protein binding thermodynamics and protein stability for three representative metal oxide NMs, namely, zinc oxide (ZnO; NM-110), titanium dioxide (TiO

(1) In compliance with the 3Rs policy to reduce, refine and replace animal experiments, the development of advanced in vitro models is needed for nanotoxicity assessment. Cells cultivated in 3D resemble organ structures better than 2D cultures. This study aims to compare cytotoxic and genotoxic responses induced by titanium dioxide (TiO2), silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) in 2D monolayer and 3D spheroid cultures of HepG2 human liver cells. (2) NPs were characterized by electron microscopy, dynamic light scattering, laser Doppler anemometry, UV-vis spectroscopy and mass spectrometry. Cytotoxicity was investigated by the alamarBlue assay and confocal microscopy in HepG2 monolayer and spheroid cultures after 24 h of NP exposure. DNA damage (strand breaks and oxidized base lesions) was measured by the comet assay. (3) Ag-NPs were aggregated at 24 h, and a substantial part of the ZnO-NPs was dissolved in culture medium. Ag-NPs induced stronger cytotoxicity in 2D cultures (EC50 3.8 &micro, g/cm2) than in 3D cultures (EC50 &gt, 30 &micro, g/cm2), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC50 10.1&ndash, 16.2 &micro, g/cm2). Ag- and ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO2-NPs showed no toxicity (EC50 &gt, 75 &micro, g/cm2). (4) This study shows that the HepG2 spheroid model is a promising advanced in vitro model for toxicity assessment of NPs.

Changes in the genetic material can lead to serious human health defects, as mutationsin somatic cells may cause cancer and can contribute to other chronic diseases. Genotoxic eventscan appear at both the DNA, chromosomal or (during mitosis) whole genome level. The study ofmechanisms leading to genotoxicity is crucially important, as well as the detection of potentiallygenotoxic compounds. We consider the current state of the art and describe here the main endpointsapplied in standard human in vitro models as well as new advanced 3D models that are closer tothe in vivo situation. We performed a literature review of in vitro studies published from 2000–2020(August) dedicated to the genotoxicity of nanomaterials (NMs) in new models. Methods suitable for detection of genotoxicity of NMs will be presented with a focus on advances in miniaturization,organ-on-a-chip and high throughput methods.

Nerve agent-induced seizures can cause varying degrees of neuropathology depending on level of poisoning and duration of seizing. The intention of this review was to validate a novel approach for establishing effective treatment regimens against soman poisoning. Identification of seizure controlling sites in the forebrain of rats poisoned by soman was made by means of lesions, and the anticonvulsive properties of a number of relevant drugs were tested by microinfusions into the identified areas. By using these procedures, procyclidine emerged as the most potent drug. Its potency was confirmed in systemic studies and is further enhanced when combined with levetiracetam. Acute treatment with a combination of HI-6, levetiracetam and procyclidine (procyclidine regimen) can effectively manage supralethal poisoning by any of the classical nerve agents. Extended treatment with the procyclidine regimen is able to terminate residual "silent", local epileptiform activity in the severely poisoned rats. Evident advantages are seen when the same regimen exerts both powerful anticonvulsant and neuroprotectant efficacies. According to the results presented, the new strategy for establishing therapies against soman-induced seizures appears to be valid.

Soil contamination of ammunition residues at shooting ranges for small arms may be followed by leaching of lead (Pb), copper (Cu), zinc (Zn), and antimony (Sb). Mixing stabilizing agents into the soil may reduce the mobility of the contaminants. To avoid risk of unexpected effects of a stabilizing agent in large-scale measures, the effect of an amendment should be tested on a small scale in advance. Two different amendments, ferric oxyhydroxide powder (CFH-12 from a commercial provider) and zerovalent iron (powder or grit), were mixed into different soil types in order to test their generic effects as stabilizing agents in contaminated soil from shooting ranges. Factors that were considered for their effects were soil water pH, limestone addition, soil chemical composition, and content of organic matter. The stabilizing agents (2–4% weight basis) were mixed into four different soil types contaminated with ammunition residues. The effects of the amendments were elucidated in two column experiments, one small-scale and one larger-scale experiment. Leaching of Pb, Cu, Zn, and Sb from the soil mixed with stabilizing agents was compared with reference soil with no amendments added. Best performance was achieved on leaching of Sb irrespective of the type of iron amendment and soil type. The Sb concentrations in the soil leachates were 55–94% less than in the leachates from the reference soils. Both amendments mixed into an acidic soil reduced the Pb, Cu, and Zn concentrations in the soil leachates in the range of 79–99%. The ability of the amendment to reduce leaching of Pb, Cu, and Zn from the other soil types was highly dependent on soil pH. CFH-12 was acidic and pH had to be balanced with limestone. The general trend was that the iron amendments reduced leaching of the elements in the order Sb>>Cu > Pb ≥ Zn. Iron amendment may be suitable as stabilizing agents for Pb, Cu, Zn, and Sb in soil. The soil pH appeared to be the most important factor governing the mobility of the ammunition residues in the soils. Overall, best effect was achieved with zerovalent iron, which can be purchased at low cost and appeared to have minor influence on the properties of the soils.

Genotoxicity is associated with serious health effects and includes different types of DNA lesions, gene mutations, structural chromosome aberrations involving breakage and/or rearrangements of chromosomes (referred to as clastogenicity) and numerical chromosome aberrations (referred to as aneuploidy). Assessing the potential genotoxic properties of chemicals, including nanomaterials (NMs), is a key element in regulatory safety assessment. State-of-the-art genotoxicity testing includes a battery of assays covering gene mutations, structural and numerical chromosome aberrations. Typically various in vitro assays are performed in the first tier. It is not very likely that NMs may induce as yet unknown types of genotoxic damage beyond what is already known for chemicals. Thus, principles of genotoxicity testing as established for chemicals should be applicable to NMs as well. However, established test guidelines (i.e., OECD TG) may require adaptations for NM testing, as currently under discussion at the OECD. This chapter gives an overview of genotoxicity testing of NMs in vitro based on experiences from various research projects. We recommend a combination of a mammalian gene mutation assay (at either Tk or HPRT locus), the in vitro comet assay, and the cytokinesis-block micronucleus assay, which are discussed in detail here. In addition we also include the Cell Transformation Assay (CTA) as a promising novel test for predicting NM-induced cell transformation in vitro.

Delirium is an acute state of confusion and a fluctuating level of consciousness. It is precipitated by physical illness or trauma, such as pneumonia, heart infarction, or hip fracture. Delirium is common among elderly hospitalized patients, and as many as 50% of hip fracture patients may develop delirium. Delirium may precipitate dementia, but recent studies indicate that delirium is caused by unknown neurotoxic mechanisms that are different from those that are associated with dementia. Experimental studies have shown that high extracellular levels of sodium are neurotoxic. We sampled lumbar cerebrospinal fluid (CSF) from hip fracture patients during hip surgery and analyzed metal ions that influence neuronal function. Eight patients who developed delirium after surgery had 21% higher CSF sodium than 17 patients who did not develop delirium (median value 175 mmol/L; range 154-188, vs. 145 mmol/L (112-204; p 0.008) or 39 patients who underwent elective surgery under spinal anesthesia without developing delirium (145 mmol/L; 140-149; p = 0.0004). Seven patients who had developed delirium before CSF sampling had a median CSF sodium of 150 mmol/L (144-185; p = 0.3). CSF potassium was also 21% higher in patients who developed delirium (p = 0.024), but remained within the physiological range. Serum sodium and potassium were normal in all patient groups. This study, on a small sample of patients, confirms the neurotoxic potential and clinical importance of high extracellular levels of sodium in the brain. High CSF sodium would likely affect cerebral function and could precipitate delirium; further, it could interact with dementia-specific mechanisms to precipitate dementia development.

Organ specific uptake and depuration, and biological effects in Atlantic salmon (Salmo salar) exposed to 2, 4, 6-trinitrotoluene (TNT) were studied. Two experiments were conducted, the first using radiolabeled TNT (14C-TNT, 0.16mg/L) to study uptake (48h) and depuration (48h), while the second experiment focused on physiological effects in fish exposed to increasing concentrations of unlabeled TNT (1μg-1mg/L) for 48h. The uptake of 14C-TNT in the gills and most of the organs increased rapidly during the first 6h of exposure (12h in the brain) followed by a rapid decrease even though the fish were still exposed to TNT in the water. The radioactivity in the gall bladder reached a maximum after 55h, 7h after the transfer to the clean water. A high concentration of 14C-TNT in the gall bladder indicates that TNT is excreted through the gall bladder. Mortality (2 out of 14) was observed at a concentration of 1mg/L, and the surviving fish had hemorrhages in the dorsal muscle tissue near the spine. Analysis of the physiological parameters in blood from the high exposure group revealed severe effects, with an increase in the levels of glucose, urea and HCO3, and a decrease in hematocrit and the levels of Cl and hemoglobin. No effects on blood physiology were observed in fish exposed to the lower concentrations of TNT (1-100μg/L). TNT and the metabolites 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT) were found in the muscle tissue, whereas only 2-ADNT and 4-ADNT were found in the bile. The rapid excretion and estimated bioconcentration factors (range of 2-18 after 48h in gills, blood, liver, kidney, muscle and brain) indicated a low potential for bioaccumulation of TNT.

The Norwegian Armed Forces’ shooting ranges contain contamination by metals such as lead (Pb) and copper (Cu) and are often used as grazing pastures for livestock. To determine whether the sheep were at risk from grazing at a shooting range in Nord-Trondelag (the Leksdalen shooting field), a study was conducted wherein the aim was to determine the amount of soil the sheep were eating, the accumulation of Cu and Pb in the livers of lambs grazing on the shooting ranges, and the accumulation of Pb and Cu in the grass. The grazing behavior of the sheep was mapped using GPS tracking and wildlife cameras. Soil, grass, feces, and liver samples were collected. All the samples were analyzed for Pb, Cu, and molybdenum (Mo), and soil and feces were also analyzed for titanium (Ti). Mean concentrations in grass, soil, feces, and liver was 41–7189, 1.3–29, 4–5, and 0.3 mg/kg Pb, respectively, and 42–580, 4.2–11.9, 19–23, and 273 mg/kg Cu, respectively. The soil ingestion rate was calculated using Ti in feces and soil. From these results, the theoretical dose of Cu and Pb ingested by grazing sheep was calculated. The soil ingestion rate was found to be 0.1–0.4%, significantly lower than the soil ingestion rate of 5–30% usually used for sheep. Little or no accumulation of Cu and Pb in the grass was found. There was no difference between the metal concentrations in the washed and unwashed grass. According to the calculated dose, the sheep were at little or no risk of acute or chronic Pb and Cu poisoning from grazing on the Leksdalen shooting range. The analysis of liver samples showed that lambs grazing on the shooting range did not have higher levels of Cu or Pb than lambs grazing elsewhere. None of the lambs had concentrations of Cu or Pb in their livers indicating poisoning.

Formation of a bacterial brain abscess entails loss of brain cells and formation of pus. The mechanisms behind the cell loss are not fully understood. Staphylococcus aureus, a common cause of brain abscesses, produces various exotoxins, including α-hemolysin, which is an important factor in brain abscess formation. α-Hemolysin may cause cytolysis by forming pores in the plasma membrane of various eukaryotic cells. However, whether α-hemolysin causes lysis of brain cells is not known. Nor is it known whether α-hemolysin in the brain causes cell death through pore formation or by acting as a chemoattractant, recruiting leukocytes and causing inflammation. Here we show that α-hemolysin injected into rat brain causes cell damage and edema formation within 30 min. Cell damage was accompanied by an increase in extracellular concentrations of zinc, GABA, glutamate, and other amino acids, indicating plasma membrane damage, but leukocytic infiltration was not seen 0.5-12h after α-hemolysin injection. This was in contrast to injection of S. aureus, which triggered extensive infiltration with neutrophils within 8h. In vitro, α-hemolysin caused concentration-dependent lysis of isolated nerve endings and cultured astrocytes. We conclude that α-hemolysin contributes to the cell death inherent in staphylococcal brain abscess formation as a pore-forming neurotoxin.

Metals and metalloids from ammunition residues at small arms shooting ranges leach into the soil and surrounding watercourses and may pose a threat to exposed wildlife and humans. To reduce the potential impact of heavy metal on the environment a field study was performed with different sorbents in order to reduce the metal concentration in polluted water from a shooting range. Two sorbents were tested in situ for their ability to reduce the concentration of Cu, Sb and Pb: Brimac(®) charcoal and Kemira(®) iron hydroxide. The mean sorption of Cu, Sb and Pb was 85%, 65%, and 88% respectively when using the charcoal and 60%, 85% and 92% respectively with the iron hydroxide. Even better sorption of the elements was achieved when the two sorbents were combined in order to increase their selectivity. The best results were achieved in the filter in which the water percolated the charcoal first and the iron hydroxide last, with a mean sorption of Cu, Sb and Pb of 89%, 90% and 93% respectively. This preparation gave a significant better sorption of Cu compared to the filter in which the water percolated the iron hydroxide first and the charcoal last. The different effect between the two filters may be due to pH, since charcoal has alkaline properties and iron hydroxide has acidic properties. For large scale experiments or in filter devices we therefore recommend use of a combination of different reactive sorbents.

Brain abscesses frequently cause symptoms such as seizures, delirium, paresis and sensory deficits that could reflect brain edema, increased intracranial pressure, or tissue destruction. However, it is also possible that pus constituents could disturb neuronal function in the surrounding brain tissue. In pus from 16 human brain abscesses, extracellular potassium ([K(+)]o) was 10.6 ± 4.8 mmol/L (mean ± SD; maximum value 22.0 mmol/L). In cerebrospinal fluid (CSF), [K(+)]o was 2.7 ± 0.6 mmol/L (N = 14; difference from pus p 0.001), which is similar to previous control values for [K(+)]o in CSF and brain parenchyma. Zinc and iron were40-fold higher in pus than in CSF; calcium, copper, manganese, and chromium were also higher, whereas sodium and magnesium were similar. Pus from 10 extracerebral abscesses (empyemas) also had higher [K(+)]o, zinc, iron, calcium, copper, manganese, and chromium than did CSF. Brain abscess [K(+)]o was significantly higher than serum potassium (3.8 ± 0.5 mmol/L; p = 0.0001), indicating that the elevated abscess [K(+)]o originated from damaged cells (e.g. brain cells and leukocytes), not from serum. High [K(+)]o could depolarize neurons, high levels of zinc could inhibit glutamate and GABA receptors, and high levels of iron and copper could cause oxidative damage, all of which could contribute to neuronal dysfunction in brain abscess patients.

An environmental survey was performed in Lake Kyrtjønn, a small lake within an abandoned shooting range in the south of Norway. In Lake Kyrtjønn the total water concentrations of Pb (14µg/L), Cu (6.1µg/L) and Sb (1.3µg/L) were elevated compared to the nearby reference Lake Stitjønn, where the total concentrations of Pb, Cu and Sb were 0.76, 1.8 and 0.12µg/L, respectively. Brown trout (Salmo trutta) from Lake Kyrtjønn had very high levels of Pb in bone (104mg/kg w.w.), kidney (161mg/kg w.w.) and the gills (137mg/kg d.w), and a strong inhibition of the ALA-D enzyme activity were observed in the blood (24% of control). Dry fertilized brown trout eggs were placed in the small outlet streams from Lake Kyrtjønn and the reference lake for 6 months, and the concentrations of Pb and Cu in eggs from the Lake Kyrtjønn stream were significantly higher than in eggs from the reference. More than 90% of Pb accumulated in the egg shell, whereas more than 80% of the Cu and Zn accumulated in the egg interior. Pb in the lake sediments was elevated in the upper 2-5cm layer (410-2700mg/kg d.w), and was predominantly associated with redox sensitive fractions (e.g., organic materials, hydroxides) indicating low potential mobility and bioavailability of the deposited Pb. Only minor amounts of Cu and Sb were deposited in the sediments. The present work showed that the adult brown trout, as well as fertilized eggs and alevins, may be subjected to increased stress due to chronic exposure to Pb, whereas exposure to Cu, Zn and Sb were of less importance.

Hassel, Bjornar; Dahlberg, Daniel; Mariussen, Espen; Goverud, Ingeborg Lostegaard; Antal, Ellen-Ann; Tonjum, Tone; Maehlen, Jan. Brain infection with Staphylococcus aureus leads to high extracellular levels of glutamate, aspartate, γ-aminobutyric acid, and zinc. Journal of Neuroscience Research 2014 ;Volum 92.(12) s. 1792-1800

A treatment regimen consisting of HI-6, scopolamine, and physostigmine (termed the physostigmine regimen) has been based on the serendipitous discovery that it exerts powerful antidotal effects against high levels of soman poisoning if it is administered 1 min after exposure. A medical therapy with corresponding efficacy, but without the time limitation of the latter regimen, has been developed through studies of microinfusions of anticonvulsants into seizure controlling sites in the forebrain of rats. From these studies procyclidine emerged as the most potent anticonvulsant, and its potency was further enhanced when being combined with the antiepileptic levetiracetam during systemic administration. In the present study, the capacity of HI-6, levetiracetam, and procyclidine (termed the procyclidine regimen) was tested against that of the physostigmine regimen. The results showed that both regimens were very effective against supralethal doses of soman (3, 4, 5 × LD₅₀) when given 1 and 5 min after intoxication. When the treatments were administered 10 and 14 or 20 and 24 min after soman exposure, only the procyclidine regimen was able to terminate seizures and preserve lives. When used as prophylactic therapies, both regimens protected equally well against seizures, but only the procyclidine regimen provided neuroprotection. The procyclidine regimen has apparently capacities to serve as a universal therapy against soman intoxication in rats.

Current treatment of nerve agent poisoning with ionotropic drugs proves inadequate, and alternative treatment strategies are searched for. Based on positive findings with metabotropic glutamate modulators in microinfusion studies, the present study was initiated to examine anticonvulsant effects of MPEP (2-Methyl-6-(phenylethynyl)pyridine hydrochloride), a metabotropic glutamate receptor 5 antagonist, and DCG-IV ((2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine), a metabotropic glutamate receptor 2/3 agonist, when administered systemically in combinations with HI-6 (1-[([4-(aminocarbonyl)pyridino]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium) and procyclidine or HI-6 and levetiracetam relative to the combination of HI-6, procyclidine, and levetiracetam. The results showed that MPEP or DCG-IV combined with HI-6 and procyclidine resulted in substantial antidotal efficacy when administered 20 min after onset of seizures elicited by soman. MPEP or DCG-IV combined with HI-6 and levetiracetam did not terminate seizures and preserve lives. When given 20 min before challenge with soman, DCG-IV in combination with HI-6 and procyclidine provided protection, whereas MPEP combined with HI-6 and procyclidine did not. Combinations with metabotropic glutamate receptor modulators did not achieve the same high level of antidotal efficacy as the combination of HI-6, procyclidine, and levetiracetam. MPEP alone inhibited pseudocholinesterase activity in the brain markedly. A positive correlation was found between latency to seizure onset or full protection and level of pseudocholinesterase activity in brain. MPEP and DCG-IV can serve as effective anticonvulsants against nerve agent poisoning when combined with HI-6 and procyclidine. Metabotropic glutamate receptor modulators may represent an alternative or supplement to treatment with ionotropic drugs.

An environmental survey was performed on shooting ranges for small arms located on minerotrophic mires. The highest mean concentrations of Pb (13 g/kg), Cu (5.2 g/kg), Zn (1.1 g/kg), and Sb (0.83 g/kg) in the top soil were from a range located on a poor minerotrophic and acidic mire. This range had also the highest concentrations of Pb, Cu, Zn, and Sb in discharge water (0.18 mg/L Pb, 0.42 mg/L Cu, 0.63 mg/L Zn, and 65 μg/L Sb) and subsurface soil water (2.5 mg/L Pb, 0.9 mg/L Cu, 1.6 mg/L Zn, and 0.15 mg/L Sb). No clear differences in the discharge of ammunition residues between the mires were observed based on the characteristics of the mires. In surface water with high pH (pH ~7), there was a trend with high concentrations of Sb and lower relative concentrations of Cu and Pb. The relatively low concentrations of ammunition residues both in the soil and soil water, 20 cm below the top soil, indicates limited vertical migration in the soil. Channels in the mires, made by plant roots or soil layer of less decomposed materials, may increase the rate of transport of contaminated surface water into deeper soil layers and ground water. A large portion of both Cu and Sb were associated to the oxidizable components in the peat, which may imply that these elements form inner-sphere complexes with organic matter. The largest portion of Pb and Zn were associated with the exchangeable and pH-sensitive components in the peat, which may imply that these elements form outer-sphere complexes with the peat.

The world is facing large environmental threats such as climate changes and spread of contaminants. Environmental awareness has become a part of our daily lives. In order to engage youth in environmental issues and ignite their interest for natural sciences, a scientifically based project was run where pupils participated in environmental research on dioxins in fish. Dioxins and dioxin-like PCBs were screened by the use of BDS DR CALUX® bioassay in 203 fish samples from 13 countries: Australia, Croatia, the Czech Republic, Denmark, Estonia, Finland, France, Iceland, Latvia, Liechtenstein, Norway, Poland and Sweden. The project was part of the Norwegian education and outreach initiative of the International Polar Year (IPY), and schools were invited to participate. Pupils from 54 schools followed a scientific protocol for fish sampling; they recorded and published important field and fish data through a Web interface and labelled, packed and shipped the samples for CALUX analysis. We conclude that collaboration between schools and research institutions was beneficial for both partners. The results showed that the majority of the fish samples had dioxin levels below the maximum limit set by the EU commission. These results would be difficult to obtain without the effort of the involved schools.

The present study reports diastereomer-specific accumulation of HBCD from a point source in five marine species representing a typical food web in a Norwegian coastal area. Samples of mussels, polychaetes, crabs and seabird eggs were analyzed for the diastereomers α-, β- and γ-HBCD, as well as lipid content and stable isotopes of nitrogen (15N/14N) to estimate trophic level. Accumulated HBCD did not correlate well with lipid content for most of the species, thus wet-weight based concentrations were included in an assessment of biomagnification. In contrast to β- and γ-HBCD, the α-diastereomer increased significantly with trophic level, resulting in magnification factors > 1 in this coastal marine ecosystem. Data for poikilotherms did not show the same positive correlation between the α-diastereomer and trophic position as homeotherms. The apparent biomagnification of the α-HBCD could be due to bioisomerization or diastereomer-specific elimination that differed between poikilotherms and homeotherms.

To optimize clean-up procedures for the analysis of alpha-, beta-, and gamma-hexabromocyclododecanes (HBCD) in environmental and biological extracts, their retention behavior on silica gel and florisil was investigated using diverse mobile-phase solvents and accounting for matrix effects. The beta-diastereomer, relative to the alpha- and gamma-diastereomers, is substantially retained on both florisil and silica gel regardless of the solvent used. The beta-diastereomer is therefore prone to undergo selective loss during clean-up. This sequence is counterintuitive to sequences based on reverse-phase chromatography with a C18-column, in which the alpha- (and not the beta-) isomer is eluted first when using a polar solvent. There has been some discrepancy regarding the structures of these diastereomers in the literature, with structures based on X-ray crystallography only becoming recently available. Based on these X-ray crystal structures, physical-chemical properties (the octanol-water partitioning constant, the Henry's law constant, subcooled liquid vapour pressures and subcooled liquid water solubilities) of the HBCD diastereomers were estimated using the quantum-chemistry based software COSMOtherm, and were found to differ from previously calculated values using different structures (e.g. log K(aw) for alpha-, beta-, and gamma-HBCD are here estimated to be -8.3, -9.3 and -8.2 respectively). Hypothesis relating differences in structure to physical-chemical properties and retention sequences are presented. The extra retention of the beta-diastereomer on silica gel and florisil is likely because it can form both greater specific (i.e. polar) and non-specific (i.e. non-polar) interactions with surfaces than the other diastereomers. Non-specific interactions can also account for the counter-intuitive elution orders with C(18)-reverse-phase chromatography. These results indicate that care should be taken when isolating HBCDs and other molecular diastereomers from environmental and biological samples, and that reported concentrations of beta-HBCD in the literature may be negatively biased.

Many toxic substances have been distributed to the environment, some of which have properties that promote accumulation and biomagnification in living organisms. Approximately 1.2 million metric tons of polychlorinated biphenyls (PCBs) have been produced and about 30% have been discharged to the environment. Approximately 200 000 metric tons of brominated flame retardants (BFRs) are produced annually, of which considerable amounts have been spread globally, even to the Polar Regions. Behavioral testing of animals has shown that these compounds may affect learning, memory and fine motor functions. Animals are most sensitive during early development. Several epidemiological studies have shown that PCBs and BFRs may be responsible for similar effects in humans. Of especially concern are possible effects of PCBs and BFRs in mixtures containing the highly neurotoxic methyl mercury. The compounds affect several targets in the nervous system that seem to be interconnected, and may be responsible for the observed behavioral deficits. It was shown early that PCBs affect dopamine and serotonin levels in the brain. Later studies showed that transport mechanisms of these neurotransmitters appear to be particularly sensitive to PCBs. Furthermore, PCBs affect intracellular calcium levels and induce formation of reactive oxygen species both in vivo and in vitro, and reduce cell viability in vitro. Neuroendocrine functions, particularly the thyroid hormone system, are also sensitive to disruption by PCBs and BFRs. Their metabolites, such as hydroxy-metabolites, appear to be particularly potent. We conclude that PCBs are particularly toxic during early development and that the toxic effects are a combination of several factors, including disturbance of calcium homeostasis, oxidative stress, and influence on neurotransmitter transport. Monoaminergic cells appear to be particularly vulnerable.

The brominated flame retardant hexabromocyclododecane (HBCD) has been reported in environmental samples worldwide. The three diastereomers, alpha-, beta- and gamma-HBCD, behave differently in aquatic food webs; likely depending on different factors influencing assimilation efficiency and metabolism. In the present study, two oral exposure experiments with rainbow trout were performed to assess the role of selective uptake on diastereomer-specific accumulation and disposition of HBCD to liver, brain and muscle. In both experiments, juvenile rainbow trout (Oncorhynchus mykiss) were administrated a technical HBCD-mixture in commercial feed (10mgkg(-1)), followed by up to 21 days of food deprivation. Already 6h after exposure, the HBCD accumulation was significant, and the concentrations peaked 4-8 days after the exposure. The relative change in HBCD pattern during the accumulation process (0-8 days), suggested that there was a diastereomer-selective uptake of alpha- and beta-HBCD in the rainbow trout. During the initial 48h, considerable amounts of all three diastereomers were distributed to liver, brain and muscle. A 70% reduction in SigmaHBCD levels after 21 days, indicated elimination of HBCD from brain and liver, but no clear elimination from the muscle was observed. Differences in HBCD pattern between organs at the end of the experiment support a proposal of an organ-specific diastereomer accumulation.

Regulatory limit values for toxicants are in general determined by the toxicology of the single compounds. However, little is known about their combined effects. Methyl mercury (MeHg), polychlorinated biphenyls (PCBs), and brominated flame retardants (BFRs) are dominant contaminants in the environment and food. MeHg is a well known neurotoxicant, especially affecting the developing brain. There is increasing evidence that PCB and BFRs also have neurotoxic effects. An enhanced effect of these toxicants, due to either synergistic or additive effects, would be considered as a risk for the fetal development. Here we studied the combinatorial effects of MeHg in combination with PCB or BFR on the reuptake of glutamate in synaptosomes. To provide the optimal conclusion regarding type of interaction, we have analyzed the data using two mathematical models, the Löewe model of additivity and Bliss' model of independent action. Binary and ternary mixtures in different proportions were made. The toxicants had primarily additive effects, as shown with both models, although tendencies towards synergism were observed. MeHg was by far the most potent inhibitor of uptake with an EC(50) value of 0.33 microM. A reconstituted mixture from a relevant fish sample was made in order to elucidate which chemical was responsible for the observed effect. Some interaction was experienced between PCB and MeHg, but in general MeHg seemed to explain the observed effect. We also show that mixture effects should not be assessed by effect addition.

Polychlorinated biphenyls (PCBs) are reported to induce the formation of reactive oxygen species (ROS) in human neutrophil granulocytes through the activation of the NADPH oxidase. The purpose of the present study is to elucidate the cellular mechanisms responsible for the activation of the NADPH oxidase after exposure to PCB. We have previously shown that PCB activates human neutrophil granulocytes through a calcium dependent activation of phospholipase D and/or phospholipase C, followed by the activation of protein kinase C. In the present study, pharmacological characterization of Aroclor (A) 1242-induced respiratory burst in human neutrophils was conducted by the use of enzymatic inhibitors. Pre-incubation with U0126, SB203580, SP600125, cyclosporin A and FK506 attenuated the A 1242-induced respiratory burst, measured by DCF-fluorescence, and luminol-amplified chemiluminescence. Our results show that the Erk1/2 kinases and p38MAPK/JNK are involved in ROS formation in neutrophils exposed to A 1242.

Small-arm shooting ranges often receive a significant input of lead (Pb), copper (Cu) and antimony (Sb) from ammunition. The goal of the present study was to investigate the mobility, distribution and speciation of Pb and Sb pollution under field conditions in both untreated and sorbent-amended shooting range soil. Elevated Sb (19-349μgL(-1)) and Pb (7-1495μgPbL(-1)) concentrations in the porewater of untreated soil over the four-year test period indicated a long-term Sb and Pb source to the adjacent environment in the absence of remedial measures. Mixing ferric oxyhydroxide powder (CFH-12) (2%) together with limestone (1%) into the soil resulted in an average decrease of Sb and Pb porewater concentrations of 66% and 97%, respectively. A similar reduction was achieved by adding 2% zerovalent iron (Fe°) to the soil. The remediation effect was stable over the four-year experimental period indicating no remobilization. Water- and 1M NH4NO3-extractable levels of Sb and Pb in field soil samples indicated significant immobilization by both treatments (89-90% for Sb and 89-99% for Pb). Results from sequential extraction analysis indicate fixation of Sb and Pb in less accessible fractions like amorphous iron oxides or even more crystalline and residual mineral phases, respectively. This work shows that amendment with Fe-based sorbents can be an effective method to reduce the mobility of metals both in cationic and anionic form in polluted shooting range soil.