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Talius is a new fungicide containing the new active substance proquinazid. Talius is a fungicide against cereal powdery mildew (Blumeria graminis) in cereals and grass seed. The risk assessment was finalized at a meeting Mai 29, 2012, by VKM’s Scientific Panel on plant protection products (VKM). VKM is in particular asked by the Norwegian Food Safety Authority to look at the following: The human health risk for operators related to the properties of the active substance and the product. o The effects seen in studies on dog and if these effects warrant a classification for chronic toxicity. o The oncogenic effects in liver and thyroid. o The establishment of reference values (ADI, AOEL and ARfD). o Dermal absorption. o The classification and labelling of the active substances and the product. • The fate and behaviour in the environment and the ecotoxical effects and risks with regard to the properties of Talius and proquinazid. VKM’s conclusion is as follows: Health: VKM concluded that a less serious effect (dose-related ocular discharge increase) was seen in dogs in both the 90 days study and 1 year-study. The opinion of the Panel is that cholangiocarcinomas is relevant for the classification of cancer and VKM is concerned about these effects. VKM proposes an NOAEL of 1.2 mg/kg bw/day for proquinazid based on the 2 year study with rats. VKM support: The proposed ADI value of 0.01 mg/kg bw/day. • The proposed AOEL value of 0.02 mg/kg bw/day. The proposed ARfD value of 0.2 mg/kg bw/day. • The proposed classification from The Norwegian Food Safety Authority. VKM supports The Norwegian Food and Safety Authority calculations for dermal absorption under Norwegian directions for use (2.6 times higher dilution). Environment: Proquinazid can be persistent under prevailing conditions in Norway and the Panel considers the results from the Finnish PEC calculator to be relevant for Norwegian conditions and expects that repeated annual applications may cause accumulation in soil up to an equilibrium level under Norwegian conditions.The potential for groundwater contamination from leaching of proquinazid and its metabolites are low. There are minimal risks for toxic effects of proquinazid to terrestrial organisms, sediment dwelling organisms, aquatic plants, and algae with the proposed application regime. For fish and invertebrates minimal risks are calculated provided that a 3 m buffer zone is used.

Movento 100 SC is a new insecticide containing the active substance spirotetramat. The intended use is in stone fruit, pome fruit, vegetables and ornamentals outdoors, and lettuce, tomatoes and cucumbers in greenhouses. VKM was requested by the Norwegian Food Safety Authority to consider possible health risk for operators related to the properties of Movento 100 SC; in particular the relevance of the effects of spirotetramat on thyroid hormones, brain, thymus and body weight observed in dogs, and the reproductive effects of spirotetramat observed in rats. VKM was also asked to consider the fate and behaviour of Movento 100 SC with the active ingredient spirotetramat in the environment, and the ecotoxicological effects and risks related to its use. The risk assessment was finalized in a meeting on May 24. 2013, by VKM’s Scientific Panel on Plant Protection Products. VKM’s conclusions are as follows: Health: VKM concludes that spirotetramat shows toxic effects in dogs and rats that could be relevant for humans. Thyroid and thymus glands are target organs in the oral subchronic toxicity studies of spirotetramat in dogs, and effects are observed from 19 mg/kg bw/day (600 ppm). Decreases in circulating thyroid hormone levels were detected in all three studies carried out with dogs (28-, 90-days and 1-year) and should be considered toxicologically relevant. The opinion of the Panel is that it cannot be excluded that the observed brain dilatation in dogs is treatment-related, and relevant to humans. Furthermore, VKM concludes that the reproductive effect observed in rats could be relevant for humans. VKM proposes a NOAEL of 5 mg/kg bw/day (200 ppm) for spirotetramat based on a 1- year toxicity study in dogs, and a NOAEL of 100 mg/kg bw/day based on the acute neurotoxicity study in rats. VKM supports/proposes: ADI: 0.05 mg/kg bw/day. AOEL: 0.05 mg/kg bw/day. ARfD: 1 mg/kg bw/day. Risk calculations show minimal risk if personal protective equipment is used. Environment: VKM concludes that spirotetramat and its metabolites are not expected to accumulate in soil. It is not expected that spirotetramat or any of its metabolites will reach concentrations in groundwater above the threshold level of 0.1 μg/L when the formulation Movento 100 SC is applied according to the intended use. VKM concludes that use of Movento 100 SC with the active substance spirotetramat according to the proposed application scheme in Norway represents a minimal risk of adverse effects on terrestrial mammals, birds, earthworms, and soil microorganisms. However, in-field effects on sensitive species of predatory mites in the crop cannot be excluded. The risk of adverse effects on bees is minimal providing that spirotetramat is not used on crops during flowering or when bees are actively foraging. For aquatic organisms in surface water, the risk is considered minimal, provided that a 5 m buffer zone to open water is used.

Luna Privilege is a new fungicide for use in apples, pears, outdoor and indoor strawberries, outdoor and indoor lettuces, peas, beans and indoor tomatoes, and contains the new active substance fluopyram. VKM was requested by the Norwegian Food Safety Authority to consider the possible health risk for operators related to the properties of Luna Privilege and fluopyram; in particular to evaluate the potential for bioaccumulation, reproductive and neurotoxic effects, and discuss possible mechanisms involved in liver and thyroid tumor formation observed in rats, and the establishment of NOAELs and reference values. VKM was also asked to evaluate the fate and behaviour of fluopyram in the environment, and the ecotoxicological effects and risks related to its use, in particular the potential for groundwater contamination, safety margins and possible effects on birds and aquatic organisms. The assessment was finalized in a meeting on December 13. 2013 by VKM’s Panel on Plant Protection Products. VKM’s conclusions are as follows: Health: It is the opinion of VKM that: The active ingredient fluopyram has low potential for bioaccumulation, and the data do not suggest a sex-specific excretion. An in vivo Comet Assay in rat liver could help to further elucidate the genotoxic potential of fluopyram. It can not be excluded that the reported incidence of “gall bladder absent” is treatment related. NOAEL for the 90-day feeding study in rats should be set to 3.06 mg/kg bw/day, resulting in an AOEL of 0.03 mg/kg bw/day. The “dumb-bell or incomplete ossification and/or bipartite/normal cartilage” should be considered as a malformation and regarded as treatment related. The time points used for neurotoxic measurements are not optimal to detect neurotoxic effects from acute exposure, since the time window between the first and second measurements is too long. The studies where effects of fluopyram and phenobarbital are compared can not be used to exclude human relevance of the tumor-inducing effect of fluopyram in the liver of female rats. The results from the mechanistic studies are insufficient to support the proposed mode of action for the induction of thyroid follicular cell tumors in male mice, and thus the induction of thyroid tumors in male mice could be relevant for humans. VKM proposes: NOAEL of 1.2 mg/kg bw/day based on a 2 year feeding study in rats. ADI: 0.012 mg/kg bw/day AOEL: 0.03 mg/kg bw/day ARfD: 0.25 mg/kg bw/day Risk calculations for field use of Luna Privilege show minimal risk if personal protective equipment is used. The AOEL of fluopyram for greenhouse use is not exceeded, even without protective equipment. Re-entry and bystander exposure is calculated to be well below the AOEL. Environment: It is the opinion of VKM that: Worst case degradation rates from laboratory studies should preferably be used to calculate PECsoil values for fluopyram. Both fluopyram and fluopyram-7-hydroxy have a high potential for groundwater contamination. The efficacy of buffer zones needs to be considered on a case-by-case basis, and further validation of values for efficacy from the model simulation is necessary. It is further the opinion of VKM that all the refinements used in the risk assessment for birds are relevant. Since the TER values estimated for all crops except for orchards are below the trigger following refinements, it is the view of VKM that the data indicate medium risk for strawberries and pulses, and high risk for lettuce in open field. The use of LC50 values for fluopyram in the TER calculations with a TER trigger value of 100 is overly conservative, and a reduction of the acute trigger for both invertebrates and fish from 100 to 10 for such calculations are suggested. The trigger for acute toxicity is not exceeded for any of the crops. For chronic toxicity, VKM concludes that there is a moderate risk for effects on fish when Luna Privilege is applied to beans/strawberries without the use of a vegetated mitigation buffer-strip.

Plenum is a new insecticide containing the new active substance pymetrozine. Plenum is an insecticide against different pests in ornamentals, lettuce, cucumber and tomato in greenhouse and against pollen beetles in oilseed- and turnip rapes. The risk assessment was finalized at a meeting Mai 29, 2012, by VKM’s Scientific Panel on plant protection products (VKM). The Panel is in particular asked by the Norwegian Food Safety Authority to look at the following: The human health risk for operators related to the properties of the active substance and the product. The Panel is in particular asked to look at the following: o The effects seen in studies on dog and if these effects warrant a classification for chronic toxicity. o The oncogenic effects in liver and lungs o The genotoxicity of metabolite CGA 300407. o The effects on reproduction and if the effects seen in teratology studies and developmental neurotoxicity study warrant a classification for developmental toxicity o The establishment of NOAELs and reference values (ADI, AOEL and ARfD). o The classification and labelling of the active substances and the product. VKM’s conclusion is as follows: The effects reported in the repeated dose toxicity studies with dogs should be considered as adverse. The increased incidence of liver and lung tumors should be considered as relevant for humans. It cannot be excluded that a genotoxic mechanism could be involved in the formation of the liver tumors, which would have implications for risk assessment. It should therefore be considered to test pymetrozine in more sensitive in vivo genotoxic endpoints in liver. The effects reported in the teratogenicity studies in rats and rabbits and in the developmental neurotoxicity study in rats should be considered for a classification of pymetrozine for developmental toxicity. Risk calculations with both the German model and the UK POEM show low risk if personal protection equipment is used. VKM propose: NOAEL of 0.6 mg/kg bw/day for pymetrozine based on the 1-year study in dogs. AOEL of 0.006 mg/kg bw/day for pymetrozine based on the NOAEL value at 0.6 mg/kg bw/day from the one year study in dogs and an UF of 100. ADI of 0.006 for pymetrozine based on the NOAEL value at 0.6 mg/kg bw/day from the one year study in dogs and an UF of 100. ARfD of 0.02 mg/kg bw/day for pymetrozine based on the LOAEL value at 8.1 mg/kg bw/day from the developmental neurotoxicity study and an UF of 500 (10 x interspecies difference, 10 x intraspecies difference, 3 x due to the use of a LOAEL value and 2 x due to the adversity of the neurodevelopmental effects). VKM supports the classification proposal from Norwegian Food Safety Authority.

Infinito is a new fungicide containing the two active substances fluopicolide and propamocarb-HCl intended to protect potatoes against the blight pathogen Phytophtora infestans. Fluopicolide is a new active substance in Norway, while propamocarb-HCl is already approved in several products. The risk assessment of Infinito was finalized in a meeting on 29. May 2012 by the Panel on Plant Protection Products of the Norwegian Scientific Committee for Food Safety (VKM). The main conclusions from the risk assessment of Infinito were: VKMs opinion is that both fluopicolide and its main metabolite M-01 (2,6- dichlorobenzamid (BAM)) are persistent in Norwegian soils and surface waters. Fluopicolide and its main metabolite M-01 may accumulate in Norwegian soils. The metabolite M-01 is highly mobile, while fluopicolide shows lower mobility in both studies and modelling. Based on the environmental properties pointed out in the risk assessment, The Norwegian Food Safety Authority (Mattilsynet) turned down the application for use of Infinito in Norway. The decision made by Mattilsynet has been appealed by the applicant, Bayer Crop Science. No new studies have been presented. Mattilsynet has asked VKM to consider the arguments in the appeal from Bayer Crop Science on the risk assessment of Infinito. The appeal and arguments were discussed in a meeting in VKMs Panel on Plant Protection Products on 13. December 2013. The conclusions were as follows: VKM maintains the previous conclusion on the risk assessment on persistence, accumulation and mobility of fluopicolide and one of the metabolites M-01. Our conclusion is further strengthened by additional documentation on: Persistence of M-01 as indicated by data from the National Monitoring program (JOVA) and international studies Accumulation as related to results from 4 year field studies and model calculations using the Finnish PEC calculator Mobility of M-01 as identified by FOCUS PELMO, FOCUS PEARL and MACRO simulations using Swedish and Norwegian scenarios in addition to monitoring results from groundwater in Norway Opinion from EFSA indicating that model simulations and risk of leaching to groundwater from row crops are under-estimated by a factor of six, and should be revised in the new FOCUS scenarios. Half-life calculations based on field studies of mobile substances should be avoided as some of the substances could have escaped to deeper layers and therefore showing artificially high degradation rates.

Aviator Xpro EC 225 is a new fungicide for use in cereals, containing the new active substance bixafen and the already approved active substance prothioconazole. Prothioconazole was assessed by the Norwegian Scientific Committee for Food Safety in 2006, and is therefore not included in this report. VKM was requested by the Norwegian Food Safety Authority to consider the possible health risk for operators related to the properties of bixafen used in Aviator Xpro EC 225; in particular to evaluate the relevance of the effects of bixafen on liver and coagulation parameters observed in rats and mice, thyroid tumours and reproductive effects observed in rats, and the establishment of NOAELs and reference values. VKM was also asked to evaluate the fate and behaviour of bixafen in the environment, and the ecotoxicological effects and risks related to its use. The risk assessment was finalized in a meeting on May 24. 2013 by VKM’s Scientific Panel on Plant Protection Products. VKM’s conclusions are as follows: Health: VKM concluded that the liver effects reported in sub-chronic and chronic studies in rats, dogs and mice exposed to bixafen, as well as the effects on coagulating parameters, should be considered adverse and of relevance to humans. It is the opinion of VKM that the thyroid follicular cell tumours reported in female rats exposed to bixafen is not sufficient to suggest that bixafen has a tumour inducing potential. It is further the opinion of VKM that it cannot be excluded that the reduced pup weight during lactation in the two-generation study is mediated by bixafen via the milk, and that bixafen has a direct effect on the number of stillborn pups in the F1 and F2 generation. Finally, the pup developmental variants and anomalies reported in the teratogenicity study are considered treatment related, and not as secondary effects related to maternal toxicity. VKM proposes a NOAEL of 2.0 mg/kg bw/day based on a 2 year feeding study in rats. VKM support/propose: ADI: 0.02 mg/kg bw/day AOEL: 0.13 mg/kg bw/day ARfD: 0.2 mg/kg bw/day Risk calculations show minimal risk if personal protective equipment is used. Environment: Bixafen is shown to be persistent in soil. VKM considers the results from a field study on a German site and the Finnish PEC calculator to be relevant and concludes that repeated annual applications can cause accumulation in soil under Norwegian conditions. There is also a potential for groundwater contamination from leaching of the metabolite M44. VKM has concluded that the use of bixafen as an ingredient in Aviator Xpro EC 225 with the proposed application regime in Norway will represent a minimal risk for toxic effects to terrestrial organisms. The toxicity of bixafen to aquatic organisms is high, and minimal risk of effects in surface waters can be achieved only if risk mitigation measures with bufferzones of 10 m are applied.

Milbeknock is a new insecticide/acaricide containing the new active substance milbemectin, which is a microbial fermentation product of Streptomyces. It is applied for control of mites and leafminers. The intended use is as a foliar spray in fruits (apples/pears), strawberries (only after harvest) and in ornamental plants growing in greenhouse and outdoors. The risk assessment was finalized at a meeting November 24, 2011, by VKM’s Scientific Panel on plant protection products (Panel 2). Panel 2 is in particular asked by the Norwegian Food Safety Authority to look at the following: 1) The human health risk for operators related to the properties of the active substance and the product. 2) The degree of oral absorption. 3) Acute toxicity. 4) The reproduction and developmental toxicity. 5) Establishment of reference values (ADI, AOEL and ARfD). 6) The fate and behaviour in the environment and environmental risk with regard to the properties of Milbeknock and milbemectin. 7) Bioavailability of milbemectin. 8) The microcosm study. VKM Panel 2’s conclusion is as follows: Health: Panel 2 proposes to set the absorption to 50%, which is in agreement with the EU DAR report (propose 47%). For acute toxicicity, Panel 2 concludes that milbemectin probably has a LD50 for dogs between 100 and 200 mg/kg body weight (bw)/day. Dogs seem to be the most sensitive experimental species and should be considered for hazard classification purposes. With respect to reproduction and developmental toxicity, Panel 2 concludes that the documentation of the role of the P-glycoprotein transporter is not convincing, and that the CF1 mice study cannot be used to support the argumentation of no developmental toxicity put forward by the applicant. The following reference values have been estimated: ADI of 0.03 mg/kg bw/day; AOEL of 0.015 mg/kg bw/day and ARfD of 0.067 mg/kg bw/day for milbemectin. Provided that personal protection equipment is used, the AOEL for operators is not exceeded. Environment: With respect to environmental fate, the opinion of the Panel is that the relatively rapid degradation in soil indicates a significant bioavailability in both soil and water-sediment systems. Further it concludes that 0.058µg/L should be regarded as NOECpopulation for the microcosm study. Panel 2 concludes that there is a medium risk of toxic effects on aquatic organisms due to exposure of milbemectin sprayed in fruits with the proposed application regime, provided that a buffer zone of 30 meters to surface water is applied. There are minimal risks of toxic effects on aquatic organisms with sufficient buffer zones in other applied crops. Panel 2 further considers the risk for foliage dwelling non-target predators and parasitoids to be high, and the risk to earthworms to be medium. The strong sorption to soil suggests that the bioavailability in soil may be limited. However, the opinion of the Panel is that the relatively rapid degradation in soil indicates a significant bioavailability. Also, for a substance with such a high log Kow the reported water solubility is relatively high, which will contribute to the availability for biological uptake. The toxicity observed in the microcosm study indicates high bioavailability also in a water/sediment system.

Bontima is a new fungicide containing the two active substances isopyrazam and cyprodinil. Bontima is a fungicide against the most important diseases in winter and spring barley. Isopyrazam is a new active ingredient with new mechanisms of action that may delay the development of fungicide resistance in treated crops. The risk assessment was finalized at a meeting May 29, 2012, by the Panel on plant protection products of the Norwegian Scientific Committee for Food Safety (VKM). The Norwegian Food Safety Authority would like, in this regard, an assessment of the following: The fate and behaviour in the environment and the ecotoxicological effects and risks with regard to the properties of Bontima and the active substances. The Panel is particularly asked to look at the following: o The persistence of isopyrazam and its metabolites. o The leaching potential of isopyrazam and its metabolites. VKM’s conclusion is as follows: Fate-related issues Isopyrazam is likely to be persistent in Norwegian soils with an associated risk of accumulation after repeated use. Isopyrazam exhibits low mobility in soil and is not expected to reach groundwater, however the two main metabolites are likely to exceed the EUs drinking water limit of 0.1 \(\mu\)g/L in groundwater. Since drainage and runoff, as well as drift, contributes to concentrations in surface water the risk assessment have to consider all these sources. In the EU DAR step 4 calculations, buffer zones of 20 m have been used to reduce runoff levels contributions by 80 %. VKM does not accept the use of these buffer zone modifications for Norwegian topographic conditions (with e.g. steeper agricultural areas). Risk to the environment There is minimal risk for toxic effects of isopyrazam to terrestrial organisms. For the aquatic compartment, there is a high risk for toxic effects of isopyrazam to aquatic organisms with the proposed application regime. This is based on calculations of runoff without buffer zone modifications, from which the resulting TER calculations show high risk of acute effects on fish and a medium risk of acute effects on invertebrates. A minimal risk for toxic effects was calculated for sediment dwelling organisms, aquatic plants, and algae.

Coragen 20 SC is a new product in Norway containing the active substance chlorantraniliprole. It is applied for use in apples against codling moth (Cydia pomonella), apple fruit moth (Argyresthia conjugella) and free leaf living larvae. The Norwegian Scientific Committee for Food Safety (VKM) was asked by the Norwegian Food Safety Authority to perform a risk assessment on human health, environmental fate and ecotoxicological of the active substance and the product. The risk assessment of the product was approved at a meeting 11th of May 2010 by VKMs Scientific Panel on Pesticides (Panel 2). VKMs Panel 2 concludes as following: The product and the active substance have low acute oral, dermal and inhalation toxicities. Both are non-irritating to the skin, and no allergenic potential by skin contact were shown. Coragen was non-irritating to the eyes, while chlorantraniliprole showed a weak irritating potential. Chlorantraniliprole is not shown to have any genotoxic or carcinogenic potential, or to be teratogenic or toxic to the reproduction of female animals. The potential for testicular toxicity of chlorantraniliprole is unclear because the study design and the limited number of young dogs (2/sex/group) do not provide basis for a firm conclusion. No particular target organ for toxicity in any species in the sub-chronic and chronic toxicity studies was seen. The observed dose- and time dependent increased degree of microvesiculation in the zona fasiculata of the adrenal cortex in male rats, is however of uncertain biological significance. All test species (rat, mice, dog) showed physiological adaption to chlorantraniliprole administration (increased liver metabolism with induction of cytochrome P450 enzymes) which was manifested as increased liver weight and hepatocellular hypertrophy. In the chronic toxicity study in mice, the increased liver weight and hepatocellular hypertophy was accompanied with eosinofilic foci, which was assessed as an adverse effect. The no observed effect level (NOAEL) derived from this study serves as basis for calculations of values for acceptable daily intake (ADI) and acceptable operator exposure level (AOEL). In Panel 2‘s opinion a sub-chronic study (90 days) with the technical material (E2Y45-282) including relevant concentration of the impurity IN-G2S78 should be performed. This would bring information on possible influence of the impurity on the toxicological profile of the technical material, and consequently on the assessment of the NOAELs in the various toxic studies. The estimated risk for operator and for bystanders or for workers re-entering treated crops is assessed as minimal. Chlorantraniliprole is persistent in soil with half live of about 1 year. The long half life indicates high potential for accumulation in soil after repeated use, which is confirmed by both model simulations and field studies. The Panel considers field data from the south of Europe not to be relevant for the Nordic conditions based on different climate conditions and soil properties contributing in different directions. The Panel considers that the existing documentation is not sufficient for a firm conclusion on the use of normalised field data for modelling purposes. However, the substance is persistent and expected to accumulate in soil. The Panel concludes that there is minimal risk for toxic effects on mammals, birds, bees, and microorganisms in soil due to chlorantraniliprole exposure with the proposed exposure regime. Panel concludes that there is “very high risk” for effects on in-field non target arthropods from chlorantraniliprole exposure. For soil living invertebrates, earthworms seem rather insensitive to chlorantraniliprole and the Panel considers the toxic effects to be minimal. Panel considers the risk for toxic effects on soil living arthropods to be high. Crustaceans and insects larvae are the aquatic organisms most sensitive to chlorantraniliprole. The Panel concludes that there is a minimal risk of toxic effects on aquatic organisms due to exposure to chlorantraniliprole with the proposed application regime provided that a buffer zone of 30 m to surface water is applied.

Centium is a new product in Norway containing the active substance clomazone. The product is applied for use as a herbicide in potato, carrot, cabbage, brussel sprout, swedes, oil seed rape, pea and bean. The Norwegian Institute for Agricultural and Environmental Research recommend approval in potato, carrot, swedes and pea. They do not recommend approval in oil seed rape, cabbage and bean because of lacking documentation on efficacy. The Norwegian Scientific Committee for Food Safety (VKM) was asked by the Norwegian Food Safety Authority to perform a risk assessment on human health and environmental fate of the active substance and the product. The risk assessment of the product was finalized at a meeting May 18, 2011, by VKM’s Scientific Panel on plant protection products (Panel 2). VKM Panel 2’s conclusion is as follows: The active ingredient clomazone is of moderate acute toxicity after oral and inhalation exposure and of low dermal toxicity. The product Centium is of low acute toxicity after oral exposure, by skin contact or by inhalation; however the product may contain a co-formulant, monomeric isocyanate, with sensitizing properties. Due to the lack of documentation on the monomer content, the Panel cannot evaluate the risk of sensitization. UK Poem model estimation of exposure show that exposure to operator is below AOEL and the health risk is therefore minimal. The effects observed in the dog study were considered due to the exposure of clomazone and should therefore be used to determine the NOAEL value. The fertility index used in the two-generation study in rats is not considered relevant as a measure of critical effect. The Panel suggests to use 4000 ppm (354 mg/kg bw/day) which is the highest dose tested in the study. Dose dependent responses were not always evident in the rat teratology study, but significant effects were found at the two highest dose levels (300 and 600 mg/kg bw/day). The observed effects were considered adverse. The proposed NOAEL of 100 mg/kg bw/day was therefore supported. The teratology study with rats is considered relevant for determination of acute reference dose (ARfD) since skeletal malformations can be induced after short exposure periods if the exposure is taking place during a sensitive period of foetal development. The Panel also concludes that based on the results from modeling with MACRO (4.4.2) using the Nordic groundwater scenarios and the input parameters agreed upon in the EU there is a potential for contamination of groundwater exceeding the trigger value of 0.1µg/L for the active ingredient following prescribed usage.

A total mixture of 29 persistent organic pollutants (POPs) modelled from Scandinavian blood concentrations was used to expose human A-498 kidney cells for 24 h over a concentration range spanning below to above blood level (1/10x, 1x, 50x, 100x, 500x). Its constituent submixtures (PFAA, Br, Cl) and co-mixtures (PFAA + Br, PFAA + Cl, Br + Cl) were also tested. Valinomycin (12 µM) was used as a cytotoxic comparative compound. Cell number (CN), nuclear area (NA), nuclear intensity (NI), mitochondrial membrane potential (MMP), and mitochondrial mass (MM) were assessed using high content analysis (HCA). Only the co-mixtures (PFAA + Cl, PFAA + Br) at 50x and 50x, 500x decreased CN, respectively. NI was increased by the total mixture at 500x and Cl mixture at all concentrations tested. MMP was increased by the total mixture at 100x and 500x, PFAA at 1x, Br + Cl and PFAA + Cl at 100x and 500x, respectively. MM was decreased by the total mixture at 500x. In contrast, valinomycin decreased CN and surviving cells showed a decrease in MMP and an increase in MM. In conclusion, POP exposure altered mitochondrial metabolism and induced cell death via an alternative mechanism to valinomycin. Only specific combinations of individual chemical classes, but not the total mixture, affected cell number.

Highlights • Mixtures of 29 halogenated POPs were reconstructed based on human blood levels. • In vitro assays permitted to evaluate DNT effects in human iPSC-derived NSCs. • POPs at human blood levels increased proliferating NSCs and decreased synapses. • Mathematical modelling showed that synaptogenesis was the most sensitive DNT endpoint. • Data support a possible link between POPs and neurodevelopmental disorders., Halogenated persistent organic pollutants (POPs) like perfluorinated alkylated substances (PFASs), brominated flame retardants (BFRs), organochlorine pesticides and polychlorinated biphenyls (PCBs) are known to cause cancer, immunotoxicity, neurotoxicity and interfere with reproduction and development. Concerns have been raised about the impact of POPs upon brain development and possibly neurodevelopmental disorders. The developing brain is a particularly vulnerable organ due to dynamic and complex neurodevelopmental processes occurring early in life. However, very few studies have reported on the effects of POP mixtures at human relevant exposures, and their impact on key neurodevelopmental processes using human in vitro test systems. Aiming to reduce this knowledge gap, we exposed mixed neuronal/glial cultures differentiated from neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) to reconstructed mixtures of 29 different POPs using concentrations comparable to Scandinavian human blood levels. Effects of the POP mixtures on neuronal proliferation, differentiation and synaptogenesis were evaluated using in vitro assays anchored to common key events identified in the existing developmental neurotoxicity (DNT) adverse outcome pathways (AOPs). The present study showed that mixtures of POPs (in particular brominated and chlorinated compounds) at human relevant concentrations increased proliferation of NSCs and decreased synapse number. Based on a mathematical modelling, synaptogenesis and neurite outgrowth seem to be the most sensitive DNT in vitro endpoints. Our results indicate that prenatal exposure to POPs may affect human brain development, potentially contributing to recently observed learning and memory deficits in children.

Telomere shortening can result in cellular senescence and in increased level of genome instability, which are key events in numerous of cancer types. Despite this, few studies have focused on the effect of nanomaterial exposure on telomere length as a possible mechanism involved in nanomaterial-induced carcinogenesis. In this study, effects of exposure to multiwalled carbon nanotubes (MWCNT) on telomere length were investigated in mice exposed by intrapleural injection, as well as in human lung epithelial and mesothelial cell lines. In addition, cell cycle, apoptosis, and regulation of genes involved in DNA damage repair were assessed. Exposure to MWCNT led to severe fibrosis, infiltration of inflammatory cells in pleura, and mesothelial cell hyperplasia. These histological alterations were accompanied by deregulation of genes involved in fibrosis and immune cell recruitment, as well as a significant shortening of telomeres in the pleura and the lung. Assessment of key carcinogenic mechanisms in vitro confirmed that long-term exposure to the long MWCNT led to a prominent telomere shortening in epithelial cells, which coincided with G1-phase arrest and enhanced apoptosis. Altogether, our data show that telomere shortening resulting in cell cycle arrest and apoptosis may be an important mechanism in long MWCNT-induced inflammation and fibrosis.

Arctic ecosystems are changing dramatically with warmer and wetter conditions resulting in complex interactions between herbivores and their forage. We investigated how Svalbard reindeer (Rangifer tarandus platyrhynchus) modify their late winter diets in response to long-term trends and interannual variation in forage availability and accessibility. By reconstructing their diets and foraging niches over a 17-year period (1995–2012) using serum δ13C and δ15N values, we found strong support for a temporal increase in the proportions of graminoids in the diets with a concurrent decline in the contributions of mosses. This dietary shift corresponds with graminoid abundance increases in the region and was associated with increases in population density, warmer summer temperatures and more frequent rain-on-snow (ROS) in winter. In addition, the variance in isotopic niche positions, breadths, and overlaps also supported a temporal shift in the foraging niche and a dietary response to extreme ROS events. Our long-term study highlights the mechanisms by which winter and summer climate changes cascade through vegetation shifts and herbivore population dynamics to alter the foraging niche of Svalbard reindeer. Although it has been anticipated that climate changes in the Svalbard region of the Arctic would be detrimental to this unique ungulate, our study suggests that environmental change is in a phase where conditions are improving for this subspecies at the northernmost edge of the Rangifer distribution.

Bonzi is a new plant growth regulator containing the active ingredient paclobutrazol of the triazole chemical class. Bonzi is for use on ornamental plants, such as Chrysanthemum, Perennial Plants, Bedding Plants, Foliage Plants, Geraniums, Hibiscus and Azaleas, in nurseries and greenhouses. VKM was requested by the Norwegian Food Safety Authority to consider the potential of Bonzi and paclobutrazol to induce reproductive effects in humans; in particular the relative sensitivity and relation between maternal effects and the effects on the offspring; and to what extent cleft palate observed in developmental studies in rat can be regarded as a specific teratogenic effect of relevance for humans. The risk assessment was finalized in a meeting in VKM’s Scientific Panel on Plant Protection Products on May 24. 2013. VKM’s conclusions are as follows: No adverse effect on fertility or reproductive performance was seen in rats. VKM concluded that the incidences of twisted snout observed in the F1 and F2 offspring of paclobutrazol treated rats are not likely to represent a developmental alteration, but rather an unspecific toxic effect. Marked maternal toxicity was seen in rats above 250 mg/kg bw/day, while partially ossified 7th cervical transverse processes and supernumerary 14th ribs were found in the offspring at 10 - 40 mg/kg/day. VKM concluded that the latter should be regarded as a direct teratogenic effect of paclobutrazol. Furthermore, VKM concluded that the data from the developmental toxicity studies are inconclusive regarding the effect on cleft palate development in rat foetuses. VKM proposes a NOAEL of 2.5 mg/kg bw/day for paclobutrazol based on a developmental toxicity study in rats.

Infinito is a new fungicide containing the two active substances fluopicolide and propamocarb-HCL. Infinito is a new generation fungicide to protect potatoes against the blight pathogen phytophtora infestans. The risk assessment was finalized at a meeting Mai 29, 2012, by the Panel on plant protection products of the Norwegian Scientific Committee for Food Safety (VKM). The Norwegian Food Safety Authority would like, in this regard, an assessment of the following: The fate and behaviour in the environment and the ecotoxicological effects and risks with regard to the properties of Infinito and the active substances. The Panel is particularly asked to look at the following: o The persistence of fluopicolide and its metabolites. o The leaching potential of fluopicolide and its metabolites. VKM considers both fluopicolide and its main metabolite M-01 (2,6-dichlorobenzamid (BAM)) to be persistent in Norwegian soils and surface waters. Other conclusions from VKM are as follows: Fluopicolide and its main metabolite may have a significant potential for soil accumulation after repeated use under Norwegian conditions. Fluopicolide shows low mobility in both studies and modelling. Metabolite M-01 (BAM) is however highly mobile. There is minimal risk for toxic effects of fluopicolide to terrestrial and aquatic organisms with the proposed application regime.

Purpose: Despite growing evidence that neuroinflammation and pro-inflammatory cytokines are involved in the pathogenesis of seizures and epilepsy, this knowledge has not been incorporated in the proposed mechanism of action of any of the current antiseizure medications (ASMs). Here, we tested the hypothesis by assessing inflammation markers in larval zebrafish (Danio rerio) exposed to lamotrigine (LTG). Methods: In order to establish the most appropriate LTG concentrations for the transcriptome analysis (RNAseq), we initially assessed for teratogenic (spinal cord deformation, heart oedema, failed inflation of the swim bladder) and behavioural effects (distance moved, time spent active, and average swimming speed during a light/dark test) in zebrafish larvae exposed to 0, 50, 100, 300, 500, 750, and 1000 μM LTG continuously between 5 and 120 h post fertilisation. Subsequently, we repeated the experiment with 0, 50, 100, or 300 μM LTG for transcriptomic analyses. Two databases (Kyoto Encyclopedia of Genes and Genomes; Gene Ontology) were used to interpret changes in gene expression between groups. Results: Major teratogenic effects were observed at concentrations of ≥ 500 μM LTG, whereas behavioural changes were observed at ≥ 300 μM LTG. Transcriptome analysis revealed a non-linear response to LTG. From the suite of differentially expressed genes (DEG), 85% (n = 80 DEGs) were upregulated following exposure to 50 μM LTG, whereas 58% (n = 12 DEGs) and 91% (n = 210 DEGs) were downregulated in response to 100 and 300 μM LTG. The metabolic pathways affected following exposure to 50 and 300 μM LTG were associated with responses to inflammation and pathogens as well development and regulation of the immune system in both groups. Notable genes within the lists of DEGs included component complement 3 (C3.a), which was significantly upregulated in response to 50 μM LTG, whereas interleukin 1β (IL-1β) was significantly downregulated in the 300 μM LTG group. The lowest exposure of 50 μM LTG is regarded as clinically relevant to therapeutic exposure. Conclusion: We demonstrated that LTG had an impact on the immune system, with a non-monotonic response curve. This dose-dependent relation could indicate that LTG can affect inflammatory responses and also at clinically relevant concentration. Further studies are needed to establish this method as a tool for screening the effects of ASMs on the immune system. This work was supported by Norwegian Epilepsy Federation (Norsk Epilespiforbund) and Norwegian Epilepsy Society (Norsk Epilepsiselskap).

Envidor is a new product in Norway containing the active substance spirodiclofen. The product is applied for use as an insecticide and acaricide in fruit, berries and ornamentals (field, glasshouses and tunnels). The Norwegian Scientific Committee for Food Safety (VKM) was asked by the Norwegian Food Safety Authority to perform a risk assessment on human health, environmental fate and ecotoxicological effects of the active substance and the product. The risk assessment of the product was finalized at a meeting November 25, 2010, by VKM’s Scientific Panel on plant protection products (Panel 2). VKM Panel 2’s conclusion is as follows: Both Envidor and the active substance spirodiclofen showed low acute oral -, dermal - and inhalation toxicities in animal studies, but showed skin sensitising properties. The estimated risk for operators is assessed as minimal provided adequate use of personal protective equipment. Spirodiclofen may have endocrine disrupting effects, but the in vivo data are not sufficient to make a firm conclusion at this point. Adrenals and other organs of the endocrine system, including the reproductive system, are target organs for chronic toxicity of spriodiclofen. Males seem to be more sensitive than females for adverse reproductive effects of spirodiclofen. Decreased testes and epididymides sizes, testes atrophy and decreased number of sperm were seen both in the parental and subsequent generation of rats. No teratogenic effect of spirodiclofen was seen. The disturbances in the endocrine system may account for the carcinogenic potential of spirodiclofen as evidenced by tumours in testicles, uterus and liver of animals. Spirodiclofen is not considered genotoxic. The Panel regards spirodiclofen to be carcinogenic and toxic to the reproduction in laboratory animals. Both spirodiclofen and its metabolites are rapidly degraded in soil, but while spirodiclofen has a low mobility due to high soil sorption, its metabolites are highly mobile. Use of Envidor with the proposed application regime implies a very high risk for adverse effects on bees and non-target arthropods due to exposure to the active substance spirodiclofen. The risk for adverse effects of spirodiclofen on other terrestrial organisms, and on aquatic organisms provided that a buffer zone of 30 m to surface water is applied, is considered to be minimal.

Simplex is a new herbicide in Norway containing the active substances aminopyralid and fluroxypyr. Aminopyralid is a new active substance in Norway, but fluroxypyr is registered in several authorized products. The intended use of the plant protection product is in established grassland for forage, established ley and pasture and in grass at the first year of sowing. During the spring of 2010, the Norwegian Scientific Committee for Food Safety (VKM) performed a human health risk assessment of the active substance aminopyralid and the product on request from the Norwegian Food Safety Authority. On further request from the Norwegian Food Safety Authority, VKM has performed a risk assessment on the fate and the behaviour in the environment and the environmental risk with regard to the properties of the active substance aminopyralid and the product Simplex, which was finalized at a meeting of VKM’s Scientific Panel on plant protection products (Panel 2) on November 25, 2010. VKM Panel 2’s conclusion is as follows: Aminopyralid is highly mobile in soil and the substance is very likely to reach ground water at concentrations above the threshold of 0.1 µg/L. Experimental data (watersediment studies) suggest that aminopyralid is persistent. However, aminopyralid concentrations in surface water are expected to decrease rapidly due to photolytic degradation. The overall risk for adverse effects on terrestrial and aquatic organisms following the proposed application of Simplex is considered to be minimal.

Simplex is a new herbicide in Norway containing the active substances aminopyralid and fluroxypyr. Aminopyralid is a new active substance in Norway, but fluroxypyr is found in several authorized products. The application concerns use in established grassland for forage, established ley and pasture and in grass at the first year of sowing. The Norwegian Scientific Committee for Food Safety (VKM) has on a request from the Norwegian Food Safety Authority performed a risk assessment on human health of the active substance and the product. The risk assessment of the product was approved at a meeting May 11 2010 by VKMs Scientific Panel on Pesticides (Panel 2). VKM’s Panel 2 concludes as following: Both Simplex and the active substance aminopyralid are characterized as extremely irritating to the eye based on persistent irritation to the eyes of rabbits. The product Simplex is also found irritating to the rabbit skin. Aminopyralid has low acute toxicity and is not shown to have genotoxic potential, or to be teratogenic or toxic to the reproduction in animals. There may however be a carcinogenic effect of aminopyralid based on an increased number of uterine sarcomas in mice. The main target organs for sub-chronic and chronic toxicity were the caecum (rats), the stomach (dogs, inflammation) and the liver (dogs, hyperthropy). No adverse effects for chronic toxicity were seen in mice. Rabbit was the most sensitive species for toxicity and the no observed effect levels (NOAELs) derived from studies in this species serve as base for calculations of values for acceptable daily intake (ADI) and acceptable operator exposure level (AOEL). The estimated risk for operator is assessed as minimal both by use of boom spraying (46% of AOEL) and knapsack sprayers (81% of AOEL). However, as a result of the hazard classification, a faceshield and gloves are necessary personal protective equipment (PPE) to be worn during mixing and loading operations, due to the risk of serious damage to the eyes and skin irritation.

1.The cost of reproduction on demographic rates is often assumed to operate through changing body condition. Several studies have found that reproduction depresses body mass more if the current conditions are severe, such as high population densities or adverse weather, than under benign environmental conditions. However, few studies have investigated the association between the fitness components and body mass costs of reproduction. 2.Using 25 years of individual-based capture–recapture data from Svalbard reindeer Rangifer tarandus platyrhynchus, we built a novel Bayesian state-space model that jointly estimated interannual change in mass, annual reproductive success and survival, while accounting for incomplete observations. The model allowed us to partition the differential effects of intrinsic and extrinsic factors on both non-reproductive mass change and the body mass cost of reproduction, and to quantify their consequences on demographic rates.3. Contrary to our expectation, the body mass cost of reproduction (mean = –5.8 kg) varied little between years (CV = 0.08), whereas the between-year variation in body mass changes, that were independent of the previous year's reproductive state, varied substantially (CV = 0.4) in relation to autumn temperature and the amount of rain-on-snow in winter. This body mass loss led to a cost of reproduction on the next reproduction, which was amplified by the same environmental covariates, from a 10% reduction in reproductive success in benign years, to a 50% reduction in harsh years. The reproductive mass loss also resulted in a small reduction in survival.4. Our results show how demographic costs of reproduction, driven by interannual fluctuations in individual body condition, result from the balance between body mass costs of reproduction and body mass changes that are independent of previous reproductive state. We illustrate how a strong context-dependent fitness cost of reproduction can occur, despite a relatively fixed body mass cost of reproduction. This suggests that female reindeer display a very conservative energy allocation strategy, either aborting their reproductive attempt at an early stage or weaning at a relatively constant cost. Such a strategy might be common in species living in a highly stochastic and food limited environment.

The presence of environmental pollutants in our ecosystem may impose harmful health effects to wildlife and humans. Several of these toxic chemicals have a potential to interfere with the endocrine system. The adrenal cortex has been identified as the main target organ affected by endocrine disrupting chemicals. The aim of this work was to assess exposure effects of defined and environmentally relevant mixtures of chlorinated, brominated and perfluorinated chemicals on steroidogenesis, using the H295R adrenocortical cell line model in combination with a newly developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method. By using this approach, we could simultaneously analyze 19 of the steroids in the steroid biosynthesis pathway, revealing a deeper insight into possible disruption of steroidogenesis. Our results showed a noticeable down-regulation in steroid production when cells were exposed to the highest concentration of a mixture of brominated and fluorinated compounds (10,000-times human blood values). In contrast, up-regulation was observed with estrone under the same experimental condition, as well as with some other steroids when cells were exposed to a perfluorinated mixture (1000-times human blood values), and the mixture of chlorinated and fluorinated compounds. Interestingly, the low concentration of the perfluorinated mixture alone produced a significant, albeit small, down-regulation of pregnenolone, and the total mixture a similar effect on 17-hydroxypregnenolone. Other mixtures resulted in only slight deviations from the control. Indication of synergistic effects were noted when we used a statistical model to improve data interpretation. A potential for adverse outcomes of human exposures is indicated, pointing to the need for further investigation into these mixtures.

Rabies is an important zoonotic disease with high fatality rates in animals and humans. In the Arctic, the Arctic fox (Vulpes lagopus) is regarded as the principal reservoir, but there is considerable debate about how the disease persists at the low population densities that are typical for this species. We describe an outbreak of rabies among Arctic foxes and Svalbard reindeer (Rangifer tarandus platyrhynchus) during 2011-12 on the remote Arctic archipelago of Svalbard, an area with a very low and relatively stable Arctic fox density. The aim of the research was to increase knowledge of Arctic rabies in this ecosystem and in the presumed spillover host, the Svalbard reindeer. Phylogenetic analysis of rabies virus (RABV) RNA isolates from Arctic fox and reindeer was performed, and clinical observations and histologic and immunohistochemical findings in reindeer were described. An ongoing capture-mark-recapture project allowed collection of serum samples from clinically healthy reindeer from the affected population for detection of rabies virus-neutralizing antibodies. The outbreak was caused by at least two different variants belonging to the RABV Arctic-2 and Arctic-3 clades, which suggests that rabies was introduced to Svalbard on at least two different occasions. The RABV variants found in Arctic fox and reindeer were similar within locations, suggesting that Arctic foxes and reindeer acquired the infection from the same source(s). The histopathologic and immunohistochemical findings in 10 reindeer were consistent with descriptions in other species infected with RABV of non-Arctic lineages. Evidence of RABV was detected in both brain and salivary gland samples. None of 158 examined serum samples from clinically healthy reindeer had virus-neutralizing antibodies against RABV.

The fasting endurance hypothesis (FEH) predicts strong selection for large body size in mammals living in environments where food supply is interrupted over prolonged periods of time. The Arctic is a highly seasonal and food restricted environment, but contrary to predictions from the FEH, empirical evidence shows that Arctic mammals are often smaller than their temperate conspecifics. Intraspecific studies integrating physiology and behaviour of different‐sized individuals, may shed light on this paradox. We tested the FEH in free‐living Svalbard reindeer (Rangifer tarandus platyrhynchus). We measured daily energy expenditure (DEE), subcutaneous body temperature (Tsc) and activity levels during the late winter in 14 adult females with body masses ranging from 46.3 to 57.8 kg. Winter energy expenditure (WEE) and fasting endurance (FE) were modelled dynamically by combining these data with body composition measurements of culled individuals at the onset of winter (14 years, n = 140) and variation in activity level throughout winter (10 years, n = 70). Mean DEE was 6.3±0.7 MJ day−1. Lean mass, Tsc and activity had significantly positive effects on DEE. Across all 140 individuals, mean FE was 85±17 days (range 48–137 days). In contrast to the predictions of the FEH, the dominant factor affecting FE was initial fat mass, while body mass and FE were not correlated. Furthermore, lean mass and fat mass were not correlated. FE was on average 80% (45 days) longer in fat than lean individuals of the same size. Reducing activity levels by ~16% or Tsc by ~5% increased FE by 7%, and 4%, respectively. Our results fail to support the FEH. Rather, we demonstrate that (i) the size of fat reserves can be independent of lean mass and body size within a species, (ii) ecological and environmental variation influence FE via their effects on body composition, and (iii) physiological and behavioural adjustments can improve FE within individuals. Altogether, our results suggest that there is a selection in Svalbard reindeer to accumulate body fat, rather than to grow structurally large. he Arctic, activity, biologging, daily energy expenditure, doubly labelled water, intraspecific scaling, subcutaneous body temperature, Svalbard reindeer to accumulate body fat, rather than to grow structurally large.

Background In recent decades, the incidence of metabolic disorders has increased internationally. This increase has been linked to exposure to persistent organic pollutants (POPs) but little is known about the metabolic effects of realistic human exposure mixtures at relevant concentrations. Objectives In this study we tested if POPs, representing real life exposure profiles and concentrations, were able to disrupt development and functions of adipose tissue in a direct way. Methods The lipogenic potency of a POP mixture modelled on levels found in human blood as detected in the Scandinavian population was assessed. The Total mixture comprises 29 compounds divided over three groups: chlorinated (Cl), brominated (Br), and perfluorinated compounds (PFAA). Individual PFAA chemicals, the Total mixture, and sub-mixtures (Cl, Br, PFAA, Cl + Br, Cl + PFAA, and Br + PFAA) at five (× 1/10, × 1, × 50, × 100, and × 500) human blood levels were tested in an optimised high content analysis (HCA) 3T3-L1 adipogenesis assay. Results Individual PFAAs; perfluorohexanesulfonic acid (PFHxS), perfluorooctanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), and perfluoroundecanoic acid (PFUnDA) promoted lipid accumulation in 3T3-L1 cells. The Total mixture, and the Cl, PFAA, Cl + Br, and Cl + PFAA sub-mixtures, promoted adipogenic differentiation and lipid accumulation. Increased lipid accumulation promoted adipose tissue expansion. Conclusions To the authors knowledge, this is the first in vitro bioassay study assessing the adipogenic effects of POP mixtures modelled on real-life human exposure levels. The findings highlight that such exposures may alter adipose tissue development and function, thus potentially playing a role in the globally increasing escalation of metabolic disorders.

Predator avoidance and food availability are both factors known to influence habitat selection and site fidelity around calving in caribou and reindeer. Here, we assess habitat selection and site fidelity during the calving period in the solitary, Arctic Svalbard reindeer Rangifer tarandus platyrhynchus, which is subject to limited predation risk and human disturbance. In this largely predator-free environment, we explore and discuss if habitat selection during the first week after calving is driven by food availability or remnants of anti-predatory behaviour. Based on GPS-collar data and ultrasound scanning (2009–2017; n = 134 individual-years) from two study areas, we estimated individual calving dates using recursive partitioning and first passage time and compared habitat selection and site fidelity of reproductive versus non-reproductive females. The K-select analysis suggested similar habitat selection during calving in reproductive and non-reproductive females. Female reindeer generally selected for lowland, flat habitats with high proportion of heath and moss tundra, i.e. habitats typically rich in terms of forage plants. Individuals producing a calf had significantly higher site fidelity in the calving period compared to the null model, and the mean distance between consecutive years' calving areas ranged between 1.5 and 3.9 km. Our study provides support for the prediction that in the absence of significant predation, ungulate calving site selection in the Arctic is mainly driven by the availability of spatially and temporally varying food resources.

Objectives - In extreme environments, such as the Arctic region, the anthropogenic influence is low and the presence of antimicrobial-resistant bacteria is unexpected. In this study, we screened wild reindeer (Rangifer tarandus platyrhynchus) from the Svalbard High Arctic Archipelago for antimicrobial-resistant Escherichia coli and performed in-depth strain characterisation. Methods - Using selective culturing of faecal samples from 55 animals, resistant E. coli were isolated and subjected to minimum inhibitory concentration (MIC) determination, conjugation experiments and whole-genome sequencing. Results - Twelve animals carried antimicrobial-resistant E. coli. Genomic analysis showed IncF plasmids as vectors both for resistance and virulence genes in most strains. Plasmid-associated genes encoding resistance to ampicillin, sulfonamides, streptomycin and trimethoprim were found in addition to virulence genes typical for colicin V (ColV)-producing plasmids. Comparison with previously reported IncF ColV plasmids from human and animal hosts showed high genetic similarity. The plasmids were detected in E. coli sequence types (STs) previously described as hosts for such plasmids, such as ST58, ST88 and ST131. Conclusion - Antimicrobial-resistant E. coli were detected from Svalbard reindeer. Our findings show that successful hybrid antimicrobial resistance–ColV plasmids and their host strains are widely distributed also occurring in extreme environmental niches such as arctic ecosystems. Possible introduction routes of resistant bacterial strains and plasmids into Svalbard ecosystems may be through migrating birds, marine fish or mammals, arctic fox (Vulpes lagopus) or via human anthropogenic activities such as tourism.

Primary cultures of cerebellar granule neurons (CGNs) derived from chicken embryos were used to explore the effects on developmental neurotoxicity by a complex defined mixture of persistent organic pollutants (POPs). Its chemical composition and concentrations were based on blood levels in the Norwegian/Scandinavian population. Perfluorooctane sulfonic acid (PFOS) alone, its most abundant compound was also evaluated. Different stages of CGNs maturation, between day in vitro (DIV) 1, 3, and 5 were exposed to the POP mixture, or PFOS alone. Their combination with glutamate, an excitatory endogenous neurotransmitter important in neurodevelopment, also known to cause excitotoxicity was evaluated. Outcomes with the mixture at 500x blood levels were compared to PFOS at its corresponding concentration of 20 µM. The POP mixture reduced tetrazolium salt (MTT) conversion at earlier stages of maturation, compared to PFOS alone. Glutamate-induced excitotoxicity was enhanced above the level of that induced by glutamate alone, especially in mature CGNs at DIV5. Glutathione (GSH) concentrations seemed to set the level of sensitivity for the toxic insults from exposures to the pollutants. The role of N-methyl-D-aspartate receptor (NMDA-R) mediated calcium influx in pollutant exposures was investigated using the non-competitive and competitive receptor antagonists MK-801 and CGP 39551. Observations indicate a calcium-independent, but still NMDA-R dependent mechanism in the absence of glutamate, and a calcium- and NMDA-R dependent one in the presence of glutamate. The outcomes for the POP mixture cannot be explained by PFOS alone, indicating that other chemicals in the mixture contribute its overall effect.

Exposure to chlorinated (Cl), brominated (Br) and perfluoroalkyl acid (PFAA) persistent organic pollutants (POPs) is associated with immunotoxicity and other adverse effects in humans and animals. Previous studies on POPs have mainly focused on single chemicals, while studies on complex mixtures are limited. Using DCF and luminol assays we examined effects on ROS generation in isolated human neutrophils, monocytes and lymphocytes, after in vitro exposure to a total mixture and sub-mixtures of 29 persistent compounds (Cl, Br, and PFAA). The mixtures were based on compounds prominent in blood, breast milk, and/or food. All mixture combinations induced ROS production in one or several of the cell models, and in some cases even at concentrations corresponding to human blood levels (compound range 1 pM – 16 nM). Whilst some interactions were detected (assessed using a mixed linear model), halogenated subgroups mainly acted additively. Mechanistic studies in neutrophils at 500× human levels (0.5 nM – 8 µM) indicated similar mechanisms of action for the Cl, PFAA, the combined PFAA + Cl and total (PFAA + Br + Cl) mixtures, and ROS responses appeared to involve β2-adrenergic receptor (β2AR) and Ca2+ signalling, as well as activation of NADPH oxidases. In line with this, the total mixture also increased cyclic AMP at levels comparable with the non-selective βAR agonist, isoproterenol. Although the detailed mechanisms involved in these responses remain to be elucidated, our data show that POP mixtures at concentrations found in human blood, may trigger stress responses in circulating immune cells. Mixtures of POPs, further seemed to interfere with adrenergic pathways, indicating a novel role of βARs in POP-induced effects.

Seasonal energetic challenges may constrain an animal's ability to respond to changing individual and environmental conditions. Here, we investigated variation in heart rate, a well-established proxy for metabolic rate, in Svalbard reindeer (Rangifer tarandus platyrhynchus), a species with strong seasonal changes in foraging and metabolic activity. In 19 adult females, we recorded heart rate, subcutaneous temperature and activity using biologgers. Mean heart rate more than doubled from winter to summer. Typical drivers of energy expenditure, such as reproduction and activity, explained a relatively limited amount of variation (2–6% in winter and 16–24% in summer) compared to seasonality, which explained 75% of annual variation in heart rate. The relationship between heart rate and subcutaneous temperature depended on individual state via body mass, age and reproductive status, and the results suggested that peripheral heterothermy is an important pathway of energy management in both winter and summer. While the seasonal plasticity in energetics makes Svalbard reindeer well-adapted to their highly seasonal environment, intraseasonal constraints on modulation of their heart rate may limit their ability to respond to severe environmental change. This study emphasizes the importance of encompassing individual state and seasonal context when studying energetics in free-living animals.This article is part of the theme issue ‘Measuring physiology in free-living animals (Part II)’.

Persistent organic pollutants (POPs) can reach the fetal brain and contribute to developmental neurotoxicity. To explore the distribution of POPs to the fetal brain, we exposed chicken embryos to a POP mixture, containing 29 different compounds with concentrations based on blood levels measured in the Scandinavian human population. The mixture was injected into the allantois at embryonic day 13 (E13), aiming at a theoretical concentration of 10 times human blood levels. POPs concentrations in the brain were measured at 0.5, 1, 2, 4, 6, 24, 48, and 72 h after administration. Twenty-seven of the individual compounds were detected during at least one of the time-points analyzed. Generally, the concentrations of most of the measured compounds were within the order of magnitude of those reported in human brain samples. Differences in the speed of distribution to the brain were observed between the per- and polyfluoroalkyl substances (PFASs), which have protein binding potential, and the lipophilic polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and brominated flame retardants (BFRs). Based on pharmacokinetic modeling, PFASs were best described by a one compartment model. PFASs displayed relatively slow elimination (Kel) and persisted at high levels in the brain. Lipophilic OCPs and PCBs could be fitted to a 2-compartment model. These showed high levels in the brain relative to the dose administrated as calculated by area under the curve (AUC)/Dose. Altogether, our study showed that chicken is a relevant model to explore the distribution of POPs into the developing brain at concentrations which are relevant for humans.

The ability of persistent organic pollutants (POPs) with endocrine disrupting properties to interfere with the developing reproductive system is of increasing concern. POPs are transferred from dams to offspring and the high sensitivity of neonates to endocrine disturbances may be caused by underdeveloped systems of metabolism and excretion. The present study aimed to characterize the effect of in utero and lactational exposure to a human relevant mixture of POPs on the female mammary gland, ovarian folliculogenesis and liver function in CD-1 offspring mice. Dams were exposed to the mixture through the diet at Control, Low or High doses (representing 0x, 5000x and 100 000x human estimated daily intake levels, respectively) from weaning and throughout mating, gestation, and lactation. Perinatally exposed female offspring exhibited altered mammary gland development and a suppressed ovarian follicle maturation. Increased hepatic cytochrome P450 enzymatic activities indirectly indicated activation of nuclear receptors and potential generation of reactive products. Hepatocellular hypertrophy was observed from weaning until 30 weeks of age and could potentially lead to hepatotoxicity. Further studies should investigate the effects of human relevant mixtures of POPs on several hormones combined with female reproductive ability and liver function.

Arctic ungulates are experiencing the most rapid climate warming on Earth. While concerns have been raised that more frequent icing events may cause die-offs, and earlier springs may generate a trophic mismatch in phenology, the effects of warming autumns have been largely neglected. We used 25 years of individual-based data from a growing population of wild Svalbard reindeer, to test how warmer autumns enhance population growth. Delayed plant senescence had no effect, but a six-week delay in snow-onset (the observed data range) was estimated to increase late winter body mass by 10%. Because average late winter body mass explains 90% of the variation in population growth rates, such a delay in winter-onset would enable a population growth of r = 0.20, sufficient to counteract all but the most extreme icing events. This study provides novel mechanistic insights into the consequences of climate change for Arctic herbivores, highlighting the positive impact of warming autumns on population viability, offsetting the impacts of harsher winters. Thus, the future for Arctic herbivores facing climate change may be brighter than the prevailing view. body mass, climate change, fitness, GPS, movement ecology, plant phenology, Rangifer, snow, space use, ungulates © 2020 The Authors. Global Change Biology published by John Wiley & Sons Ltd This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Perfluoroalkyl acids (PFAAs) are persistent man-made chemicals, ubiquitous in nature and present in human samples. Although restrictions are being introduced, they are still used in industrial processes as well as in consumer products. PFAAs cross the blood-brain-barrier and have been observed to induce adverse neurobehavioural effects in humans and animals as well as adverse effects in neuronal in vitro studies. The sulfonated PFAA perfluorooctane sulfonic acid (PFOS), has been shown to induce excitotoxicity via the N-methyl-D-aspartate receptor (NMDA-R) in cultures of rat cerebellar granule neurons (CGNs). In the present study the aim was to further characterise PFOS-induced toxicity (1–60 μM) in rat CGNs, by examining interactions between PFOS and elements of glutamatergic signalling and excitotoxicity. Effects of the carboxylated PFAA, perfluorooctanoic acid (PFOA, 300–500 μM) on the same endpoints were also examined. During experiments in immature cultures at days in vitro (DIV) 8, PFOS increased both the potency and efficacy of glutamate, whereas in mature cultures at DIV 14 only increased potency was observed. PFOA also increased potency at DIV 14. PFOS-enhanced glutamate toxicity was further antagonised by the competitive NMDA-R antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) at DIV 8. At DIV 8, PFOS also induced glutamate release (9–13 fold increase vs DMSO control) after 1−3 and 24 h exposure, whereas for PFOA a large (80 fold) increase was observed, but only after 24 h. PFOS and PFOA both also increased alanine and decreased serine levels after 24 h exposure. In conclusion, our results indicate that PFOS at concentrations relevant in an occupational setting, may be inducing excitotoxicity, and potentiation of glutamate signalling, via an allosteric action on the NMDA-R or by actions on other elements regulating glutamate release or NMDA-R function. Our results further support our previous findings that PFOS and PFOA at equipotent concentrations induce toxicity via different mechanisms of action.

Exposure to persistent organic pollutants (POPs), encompassing chlorinated (Cl), brominated (Br) and perfluoroalkyl acid (PFAA) compounds is associated with adverse neurobehaviour in humans and animals, and is observed to cause adverse effects in nerve cell cultures. Most studies focus on single POPs, whereas studies on effects of complex mixtures are limited. We examined the effects of a mixture of 29 persistent compounds (Cl + Br + PFAA, named Total mixture), as well as 6 sub-mixtures on in vitro exposed rat cerebellar granule neurons (CGNs). Protein expression studies of cerebella from in vivo exposed mice offspring were also conducted. The selection of chemicals for the POP mixture was based on compounds being prominent in food, breast milk or blood from the Scandinavian human population. The Total mixture and sub-mixtures containing PFAAs caused greater toxicity in rat CGNs than the single or combined Cl/Br sub-mixtures, with significant impact on viability from 500x human blood levels. The potencies for these mixtures based on LC50 values were Br + PFAA mixture > Total mixture > Cl + PFAA mixture > PFAA mixture. These mixtures also accelerated induced lipid peroxidation. Protection by the competitive N-methyl-D-aspartate (NMDA) receptor antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) indicated involvement of the NMDA receptor in PFAA and Total mixture-, but not Cl mixture-induced toxicity. Gene-expression studies in rat CGNs using a sub-toxic and marginally toxic concentration ((0.4 nM-5.5 µM) 333x and (1 nM-8.2 µM) 500x human blood levels) of the mixtures, revealed differential expression of genes involved in apoptosis, oxidative stress, neurotransmission and cerebellar development, with more genes affected at the marginally toxic concentration. The two important neurodevelopmental markers Pax6 and Grin2b were downregulated at 500x human blood levels, accompanied by decreases in PAX6 and GluN2B protein levels, in cerebellum of offspring mice from mothers exposed to the Total mixture throughout pregnancy and lactation. In rat CGNs, the glutathione peroxidase gene Prdx6 and the regulatory transmembrane glycoprotein gene Sirpa were highly upregulated at both concentrations. In conclusion, our results support that early-life exposure to mixtures of POPs can cause adverse neurodevelopmental effects.