Multi-isotopic composition of brown crab (Cancer pagurus) and seafloor sediment from a mine tailing sea disposal impacted fjord ecosystem.

Sea disposal of mine tailings in fjord ecosystems is an important coastal management issue in Norway and occurs at the land-sea interface. Here we studied accumulation of heavy metals in brown crab (Cancer pagurus) and seafloor sediment from Jøssingfjord, Norway during 2018 to evaluate long-term, legacy pollution effects of coastal mine tailing sea disposal activities. Nickel and copper sediment pollution in the mine tailing sea disposal area was classified as moderate and severe, respectively, under Norwegian environmental quality standards, and highlights the persistent hazard and legacy impacts of heavy metals in these impacted fjord ecosystems. Mercury, zinc, and arsenic had stronger affinities to brown crab muscle likely due to the presence of thiols, and availability of metal binding sites. Our multi-isotopic composition data showed that lead isotopes were the most useful source apportionment tool for this fjord. Overall, our study highlights the importance and value of measuring several different heavy metals and multiple isotopic signatures in different crab organs and seafloor sediment to comprehensively evaluate fjord pollution and kinetic uptake dynamics. Brown crabs were suitable eco-indicators of benthic ecosystem heavy metal pollution in a fjord ecosystem still experiencing short- and long-term physical and chemical impacts from coastal mining sea disposal activities.

Mixture toxicity of 6PPD-quinone and polystyrene nanoplastics in zebrafish.

Plastic pollution, including micro- and nanoplastics, is a growing concern. Tyre-wear particles (TWPs) are the second largest source of microplastics in the ocean following abrasion of synthetic fibres. In addition to the particles themselves, TWPs contain many harmful chemicals, including 6PPD. This chemical reacts with atmospheric ozone and forms the toxic compound 6PPD-quinone (6PPDq), which poses a danger to aquatic life. There is a knowledge gap in understanding risks associated with the combined toxicity of nanoplastics (NPs) and 6PPDq. The present study aimed to investigate the toxicity of NPs and 6PPDq on adult zebrafish using phenotypic (behaviour, histology) and transcriptomic endpoints. Zebrafish were exposed to four treatments: control (contaminant-free), 50 µg/L 6PPDq, 3 mg/L polystyrene (PS)-NPs, and a combination of 50 µg/L 6PPDq and 3 mg/L PS-NPs. We did not observe locomotory dysregulation in zebrafish exposed to NPs. However, we found significant hyperlocomotion in zebrafish exposed to 6PPDq and this effect was even more substantial after co-exposure with PS-NPs. This study explores the molecular mechanisms behind these effects, identifying genes associated with neurotransmitters and fatty acid metabolism that were dysregulated by the co-exposure. Transcriptomic analysis further showed that both 6PPDq and PS-NPs impacted cellular processes associated with sterol biosynthesis, cholesterol metabolism, and muscle tissue development. The effects on these mechanisms were stronger in co-exposed zebrafish, indicating a heightened risk to cellular integrity and mitochondrial dysfunction. These results highlight the significance of mixture toxicity when studying the effects of NPs and associated chemicals like 6PPDq.

Quick and efficient microplastic isolation from fatty fish tissues by surfactant-enhanced alkaline digestion.

For monitoring microplastic contamination in fish tissues, tissue digestion into filterable components prior to microplastic identification and quantification should be quick and efficient, providing satisfying microplastic recoveries of relevant particle sizes. Filtration with a small pore size, necessary to target small particles, is a challenge. Some proposed protocols take several days. To improve this, a combination of surfactants (Tween®-20 and Triton™ X-100) with potassium hydroxide (KOH) and pH neutralization was used. Fish bones were removed in tissue preparation prior to digestion. Recovery down to ca. 60-80 µm worked well for PA-66, PE, PET, PP, PS and PVC. In conclusion, we developed a comparatively swift digestion protocol, enabling filtration of 100 g samples with a pore size of 10 µm, for fish fillets with high (mackerel), intermediate (salmon, plaice) and low (cod) fat contents, fish liver, head kidney and oil samples, within 16-24 h.

Efficient extraction of small microplastic particles from rat feed and feces for quantification.

To date, microplastic is ubiquitously encountered in the environment. Studies analyzing microplastic in terrestrial ecosystems, including animal feces and feed, are few. Microplastic quantification method validation and harmonization are not yet far developed. For the analysis of small microplastic, approximately <0.5 mm, extraction from organic and inorganic materials is fundamental prior to quantitative and qualitative analysis. Method validation, including recovery studies, are necessary throughout the analytical chain. In this study, we developed an optimized, efficient protocol with a duration of 72 h for the digestion of laboratory rat feed and feces. A combination of a mild acidic (H2O2 15%; HNO3 5%) and an alkaline treatment (10% KOH) dissolving the previous filter, followed by enzymatic digestion (Viscozyme®L) proved to be efficient for the extraction and identification of spiked polyamide (15-20 µm) and polyethylene (40-48 µm) from feed and feces samples from rats, showing high recovery rates. Extracted rat feces samples from an in vivo study in which Wistar rats were fed with feed containing microplastic were analyzed with pyrolysis-gas chromatography-Orbitrap™ mass spectrometry, quantifying recovered microplastic in rat feces in environmentally relevant concentrations. The presented three-step protocol provides a suitable, time and cost-effective method to extract microplastic from rat feed and feces.

Polystyrene nanoplastics enhance the toxicological effects of DDE in zebrafish (Danio rerio) larvae.

Anthropogenic releases of plastics, persistent organic pollutants (POPs), and heavy metals can impact the environment, including aquatic ecosystems. Nanoplastics (NPs) have recently emerged as pervasive environmental pollutants that have the ability to adsorb POPs and can cause stress in organisms. Among POPs, DDT and its metabolites are ubiquitous environmental pollutants due to their long persistence. Despite the discontinued use of DDT in Europe, DDT and its metabolites (primarily p,p'-DDE) are still found at detectable levels in fish feed used in salmon aquaculture. Our study aimed to look at the individual and combined toxicity of NPs (50 mg/L polystyrene) and DDE (100 µg/L) using zebrafish larvae as a model. We found no significant morphological, cardiac, respiratory, or behavioural changes in zebrafish larvae exposed to NPs alone. Conversely, morphological, cardiac and respiratory alterations were observed in zebrafish larvae exposed to DDE and NPs + DDE. Interestingly, behavioural changes were only observed in zebrafish larvae exposed to NPs + DDE. These findings were supported by RNA-seq results, which showed that some cardiac, vascular, and immunogenic pathways were downregulated only in zebrafish larvae exposed to NPs + DDE. In summary, we found an enhanced toxicological impact of DDE when combined with NPs.

Intestinal permeability and gene expression after polyethylene and polyamide microplastic ingestion in Wistar rats.

Microplastic particles are ubiquitous in the environment. However, little is known about their uptake and effects in humans or mammalian model organisms. Here, we studied the effects of pristine polyamide (15-20 µm) and polyethylene (40-48 µm) particles after oral ingestion in rats. The animals received feed containing microplastic particles (0.1% polyamide or polyethylene, or a mixture of both polymers) or a control diet without microplastic particles, for 5 weeks. The permeability of the duodenum was investigated in an Ussing chamber, whereas gene expression and concentration of tight junction proteins were measured in gut tissue and plasma. Microplastic particles were quantified by pyrolysis-gas chromatography/mass spectrometry in rats' feces. Rats fed with microplastic particles had higher duodenal permeability. Expression of gene coding for the tight junction protein occludin (OCLN) was higher in PE treated animals compared to control or the PA group. No changes in the expression of the gene coding for zonula occludens protein 1 were detected. Occludin protein concentrations were below the limit of detection of the applied method in both gut and plasma. Zonula occludens protein 1 concentrations in the gut were significantly higher in groups exposed to PA and PE as compared to control, while zonula occludens protein 1 concentrations in plasma did not show significant changes. These results demonstrated that short-term exposure to a dose of 0.1% (w/w) microplastic particles in feed had limited effects on duodenal permeability, expression of pro-inflammatory protein genes and tight junction protein genes in the duodenum.

Distinct polymer-dependent sorption of persistent pollutants associated with Atlantic salmon farming to microplastics.

Interactions of microplastics and persistent organic pollutants (POPs) associated with Atlantic salmon farming were studied to assess the potential role of microplastics in relation to the environmental impact of aquaculture. HDPE, PP, PET and PVC microplastics placed for 3 months near fish farms sorbed POPs from aquafeeds. PET and PVC sorbed significantly higher levels of dioxins and PCBs compared to HDPE, while the levels sorbed to PP were intermediate and did not differ statistically from PET, PVC or HDPE. In addition, the composition of dioxins accumulated in caged blue mussels did not reflect the patterns observed on the microplastics, probably due to polymer-specific affinity of POPs. In conclusion, the results of this study show that microplastics occurring near fish farms can sorb aquafeed-associated POPs and, therefore, microplastics could potentially be vectors of such chemicals in the marine environment and increase the environmental impact of fish farming.

Co-occurrence of contaminants in marine fish from the North East Atlantic Ocean: Implications for human risk assessment.

Marine fish from the North East Atlantic Ocean (NEAO) are nutrient rich and considered a valuable economic resource. However, marine fish are also a major dietary source of several contaminants, including persistent organic pollutants (POPs) and heavy metals. Using one of the world's largest seafood datasets (n > 25,000 individuals), comprising 12 commercially important fish species collected during 2006-2019 in the NEAO, we assessed the co-occurrence of elements and POPs, and evaluated potential risks to human consumers. Several positive correlations between concentrations of mercury (Hg), dioxins, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) were observed. Concentrations of Hg, dioxins, PCBs and PBDEs increased from North to South and associations between marine sediment contamination, sea temperature, and fish Hg and POPs concentrations were identified using multi-linear regression (MLR) models. In general, Hg concentrations in fillet and liver of fish were positively associated with increases in both sediment contamination and sea temperature. POPs concentrations in both fillet and liver were positively associated with increases in sediment contamination, and only POPs concentrations in the liver of benthopelagic and demersal species were found to be positively correlated with sea temperature. Using a probabilistic approach to estimate human contaminant exposure from seafood, we showed that intake of pelagic species posed the highest risk of dioxins and dioxin-like PCBs (DL-PCBs) exposure, while intake of benthopelagic and demersal species posed the highest risk of Hg exposure. This study can serve as a model to further understand the distribution, co-occurrence, and trends of contaminants in seafood harvested from the NEAO and their potential risks to human consumers.

Moving forward in microplastic research: A Norwegian perspective.

Given the increasing attention on the occurrence of microplastics in the environment, and the potential environmental threats they pose, there is a need for researchers to move quickly from basic understanding to applied science that supports decision makers in finding feasible mitigation measures and solutions. At the same time, they must provide sufficient, accurate and clear information to the media, public and other relevant groups (e.g., NGOs). Key requirements include systematic and coordinated research efforts to enable evidence-based decision making and to develop efficient policy measures on all scales (national, regional and global). To achieve this, collaboration between key actors is essential and should include researchers from multiple disciplines, policymakers, authorities, civil and industry organizations, and the public. This further requires clear and informative communication processes, and open and continuous dialogues between all actors. Cross-discipline dialogues between researchers should focus on scientific quality and harmonization, defining and accurately communicating the state of knowledge, and prioritization of topics that are critical for both research and policy, with the common goal to establish and update action plans for holistic benefit. In Norway, cross-sectoral collaboration has been fundamental in supporting the national strategy to address plastic pollution. Researchers, stakeholders and the environmental authorities have come together to exchange knowledge, identify knowledge gaps, and set targeted and feasible measures to tackle one of the most challenging aspects of plastic pollution: microplastic. In this article, we present a Norwegian perspective on the state of knowledge on microplastic research efforts. Norway's involvement in international efforts to combat plastic pollution aims at serving as an example of how key actors can collaborate synergistically to share knowledge, address shortcomings, and outline ways forward to address environmental challenges.

Corrigendum to ''Sampling protocol for the determination of nutrients and contaminants in fish and other seafood - The EAF-Nansen programme" [MethodsX 7 (2020) 101063].

[This corrects the article DOI: 10.1016/j.mex.2020.101063.].

Sampling protocol for the determination of nutrients and contaminants in fish and other seafood - The EAF-Nansen Programme.

Seafood plays a central role in global food and nutrition security. However, there is a lack of data on the concentration of nutrients and contaminants in fish and other seafood, especially in low- and middle-income countries. In order to assess the potential risks and benefits associated with seafood intake, reliable and up-to-date food composition data is crucial. The quality of food composition data is affected by several factors, such as sampling protocols and the suitability and quality of the methods applied for sample preparation and analysis. In this paper, we describe the sampling methodology and protocols related to the sampling of fish and other seafood and the corresponding analytical methods used to analyse the nutrient and contaminant content of such species. For nutrients, the determination of protein, fat, ash, energy, fatty acids, cholesterol, and amino acids is described, in addition to analyses for determination of the vitamin and mineral content in fish and other seafood. For contaminants, analyses for the determination of organic pollutants and microplastics are described. The methodology described in this paper is used for sampling data through scientific surveys in low- and middle-income countries with research vessel Dr. Fridtjof Nansen under the EAF-Nansen Programme. The Programme aims to improve knowledge on the nutritional composition of fish and ensure the fish is safe to consume.•In this paper, we describe the sampling protocols used for sampling fish and other seafood during scientific surveys under the EAF-Nansen Programme.•This paper describes the methodology and quality control for analysing nutrients and contaminants in fish and other seafood.

Micro- and nanoplastic toxicity on aquatic life: Determining factors.

Plastic pollution has become a major environmental concern due to its omnipresence and degradation to smaller particles. The potential toxicological effects of micro- and nanoplastic on biota have been investigated in a growing number of exposure studies. We have performed a comprehensive review of the main determining factors for plastic particle toxicity in the relevant exposure systems, from publications until including the year 2018. For a focused scope, effects of additives or other pollutants accumulated by the plastic particles are not included. In summary, current literature suggests that plastic particle toxicity depends on concentration, particle size, exposure time, particle condition, shape and polymer type. Furthermore, contaminant background, food availability, species, developmental stage and sex have major influence on the outcome of plastic particles exposures. Frequently reported effects were on body and population growth, energy metabolism, feeding, movement activity, physiological stress, oxidative stress, inflammation, the immune system, hormonal regulation, aberrant development, cell death, general toxicity and altered lipid metabolism. Several times reported were increased growth and food consumption, neuro-, liver- or kidney pathology and intestinal damage. Photosynthesis disruption was reported in studies investigating effects on phytoplankton. For the currently unquantified plastic particles below 10 µm, more toxic effects were reported in all aquatic life, as compared to plastic particles of larger size.

Iodine content of six fish species, Norwegian dairy products and hen's egg.

Iodine is a trace element required for the production of thyroid hormones, essential for metabolism, growth and brain development, particularly in the first trimester of pregnancy. Milk and lean fish are the main dietary sources of iodine in the Norwegian diet. Thus, the aim of the present study was to provide updated analysed values of iodine concentration in six fish species, 27 selected Norwegian iodine-rich dairy foods and Norwegian hen's eggs. The iodine concentrations in the wild fish species varied between 18 µg/100 g (Atlantic halibut) and 1,210 µg/100 g (pollack). The iodine concentration of cow milk varied between 12 and 19 µg/100 g and the iodine concentration of the eggs varied between 23 and 43 µg/100 g. The results in this study deviate somewhat from the current iodine concentrations in the Norwegian Food Composition Table. This deviation may have a large impact on the assessment of the iodine intake. Hence, updated knowledge about the variation in iodine level of fish, milk, dairy products and hen's egg are of great importance when estimating the iodine intake in the population. These data will contribute substantially to future estimations of dietary iodine intake and will be made available for the public Norwegian Food Composition Table.

Novel microscopy-based screening method reveals regulators of contact-dependent intercellular transfer.

Contact-dependent intercellular transfer (codeIT) of cellular constituents can have functional consequences for recipient cells, such as enhanced survival and drug resistance. Pathogenic viruses, prions and bacteria can also utilize this mechanism to spread to adjacent cells and potentially evade immune detection. However, little is known about the molecular mechanism underlying this intercellular transfer process. Here, we present a novel microscopy-based screening method to identify regulators and cargo of codeIT. Single donor cells, carrying fluorescently labelled endocytic organelles or proteins, are co-cultured with excess acceptor cells. CodeIT is quantified by confocal microscopy and image analysis in 3D, preserving spatial information. An siRNA-based screening using this method revealed the involvement of several myosins and small GTPases as codeIT regulators. Our data indicates that cellular protrusions and tubular recycling endosomes are important for codeIT. We automated image acquisition and analysis to facilitate large-scale chemical and genetic screening efforts to identify key regulators of codeIT.

Intercellular transfer of transferrin receptor by a contact-, Rab8-dependent mechanism involving tunneling nanotubes.

Intercellular communication between cancer cells, especially between cancer and stromal cells, plays an important role in disease progression. We examined the intercellular transfer of organelles and proteins in vitro and in vivo and the role of tunneling nanotubes (TNTs) in this process. TNTs are membrane bridges that facilitate intercellular transfer of organelles of unclear origin. Using 3-dimensional quantitative and qualitative confocal microscopy, we showed that TNTs contain green fluorescent protein (GFP)-early endosome antigen (EEA) 1, GFP Rab5, GFP Rab11, GFP Rab8, transferrin (Tf), and Tf receptor (Tf-R) fused to mCherry (Tf-RmCherry). Tf-RmCherry was transferred between cancer cells by a contact-dependent but secretion-independent mechanism. Live cell imaging showed TNT formation preceding the transfer of Tf-RmCherry and involving the function of the small guanosine triphosphatase (GTPase) Rab8, which colocalized with Tf-RmCherry in the TNTs and was cotransferred to acceptor cells. Tf-RmCherry was transferred from cancer cells to fibroblasts, a noteworthy finding that suggests that this process occurs between tumor and stromal cells in vivo. We strengthened this hypothesis in a xenograft model of breast cancer using enhanced (e)GFP-expressing mice. Tf-RmCherry transferred from tumor to stromal cells and this process correlated with an increased opposite transfer of eGFP from stromal to tumor cells, together pointing toward complex intercellular communication at the tumor site.

CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation.

: The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening.

Rab3D is critical for secretory granule maturation in PC12 cells.

Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs.

Lung endothelial ADAM17 regulates the acute inflammatory response to lipopolysaccharide.

Acute lung injury (ALI) is associated with increased vascular permeability, leukocyte recruitment, and pro-inflammatory mediator release. We investigated the role of the metalloproteinase ADAM17 in endotoxin-induced ALI with focus on endothelial ADAM17. In vitro, endotoxin-mediated induction of endothelial permeability and IL-8-induced transmigration of neutrophils through human microvascular endothelial cells required ADAM17 as shown by inhibition with GW280264X or shRNA-mediated knockdown. In vivo, ALI was induced by intranasal endotoxin-challenge combined with GW280264X treatment or endothelial adam17-knockout. Endotoxin-triggered upregulation of ADAM17 mRNA in the lung was abrogated in knockout mice and associated with reduced ectodomain shedding of the junctional adhesion molecule JAM-A and the transmembrane chemokine CX3CL1. Induced vascular permeability, oedema formation, release of TNF-α and IL-6 and pulmonary leukocyte recruitment were all markedly reduced by GW280264X or endothelial adam17-knockout. Intranasal application of TNF-α could not restore leukocyte recruitment and oedema formation in endothelial adam17-knockout animals. Thus, activation of endothelial ADAM17 promotes acute pulmonary inflammation in response to endotoxin by multiple endothelial shedding events most likely independently of endothelial TNF-α release leading to enhanced vascular permeability and leukocyte recruitment.

Roles of myosin Va and Rab3D in membrane remodeling of immature secretory granules.

Neuroendocrine secretory granules (SGs) are formed at the trans-Golgi network (TGN) as immature intermediates. In PC12 cells, these immature SGs (ISGs) are transported within seconds to the cell cortex, where they move along actin filaments and complete maturation. This maturation process comprises acidification-dependent processing of cargo proteins, condensation of the SG matrix, and removal of membrane and proteins not destined to mature SGs (MSGs) into ISG-derived vesicles (IDVs). We investigated the roles of myosin Va and Rab3 isoforms in the maturation of ISGs in neuroendocrine PC12 cells. The expression of dominant-negative mutants of myosin Va or Rab3D blocked the removal of the endoprotease furin from ISGs. Furthermore, expression of mutant Rab3D, but not of mutant myosin Va, impaired cargo processing of SGs. In conclusion, our data suggest an implication of myosin Va and Rab3D in the maturation of SGs where they participate in overlapping but not identical tasks.