Lufenuron treatment temporarily represses gene expression and affects the SUMO pathway in liver of Atlantic salmon.

Lufenuron is a benzoylurea insecticide currently in use to combat sea lice infestation in salmon aquaculture in Chile. With pending approval in Norway, the aim of this work was to study the uptake and toxicity of lufenuron in liver tissue of Atlantic salmon. Juvenile salmon weighing 40 g were given a standard 7-day oral dose, and bioaccumulation and transcriptional responses in the liver were examined 1 day after the end-of-treatment (day 8) and after 1 week of elimination (day 14). Bioaccumulation levels of lufenuron were 29 ± 3 mg/kg at day 8 and 14 ± 1 mg/kg at day 14, indicating relatively rapid clearance. However, residues of lufenuron were still present in the liver after 513 days of depuration. The exposure gave a transient inhibition of transcription in the liver at day 8 (2437 significant DEGs, p-adj < .05), followed by a weaker compensatory response at day 14 (169 significant DEGs). Pathways associated with RNA metabolism such as the sumoylation pathway were most strongly affected at day 8, while the apelin pathway was most profoundly affected at day 14. In conclusion, this study shows that lufenuron easily bioaccumulates and that a standard 7-day oral dose induces a transient inhibition of transcription in liver of salmon.

Ecotoxicological assessment of Cu-rich acid mine drainage of Sulitjelma mine using zebrafish larvae as an animal model.

Acid mine drainage (AMD) is widely acknowledged as a substantial threat to the biodiversity of aquatic ecosystems. The present study aimed to study the toxicological effects of Cu-rich AMD from the Sulitjelma mine in zebrafish larvae. The AMD from this mine was found to contain elevated levels of dissolved metals including Mg (46.7 mg/L), Al (20.2 mg/L), Cu (18.3 mg/L), Fe (19.8 mg/L) and Zn (10.6 mg/L). To investigate the toxicological effects, the study commenced by exposing zebrafish embryos to various concentrations of AMD (ranging from 0.75% to 9%) to determine the median lethal concentration (LC50). Results showed that 96 h LC50 for zebrafish larvae following AMD exposure was 2.86% (95% CI: 2.32-3.52%). Based on acute toxicity results, zebrafish embryos (<2 hpf) were exposed to 0.1% AMD (Cu: 21.7 µg/L) and 0.45% AMD (Cu: 85.7 µg/L) for 96 h to assess development, swimming behaviour, heart rate, respiration and transcriptional responses at 116 hpf. Light microscopy results showed that both 0.1% and 0.45% AMD reduced the body length, eye size and swim bladder area of zebrafish larvae and caused phenotypic abnormalities. Swimming behaviour results showed that 0.45% AMD significantly decreased the locomotion of zebrafish larvae. Heart rate was not affected by AMD exposure. Furthermore, exposure caused a significant increase in oxygen consumption indicating vascular stress in developing larvae. Taken altogether, the study shows that even heavily diluted AMD with environmentally relevant levels of Cu caused toxicity in zebrafish larvae.

Effects of mine tailing exposure on the development of early life stages of the marine copepod Calanus finmarchicus.

The demand for mineral resources is increasing mining activities worldwide. In Norway, marine tailing disposal (MTD) is practiced, introducing mineral particles into fjord ecosystems. We investigated the effects of two concentrations (high and low) of fine tailings from a CaCO3 processing plant on early life stages of the marine copepod Calanus finmarchicus. Results show that the exposure did not significantly impact hatching success or development in non- and early feeding life stages. However, feeding stage nauplii ingested tailings, which caused a significantly slower development in later nauplii stages in high exposure groups, with most individuals being two stages behind the control group. Further, high mortality occurred in late nauplii and early copepodite stages in low exposure groups, which could be caused by insufficient energy accumulation and depleted energy reserves during development. Individuals exposed to high exposure concentrations seemed to survive by arresting development and potentially by reduced activity, thereby conserving energy reserves. In nature, slower development could affect lipid storage buildup and reproduction.

Environmentally realistic concentrations of chlorinated, brominated, and fluorinated persistent organic pollutants induce the unfolded protein response as a shared stress pathway in the liver of Atlantic cod (Gadus morhua).

In the North Sea and North Atlantic coastal areas, fish experience relatively high background levels of persistent organic pollutants. This study aimed to compare the mode of action of environmentally relevant concentrations of mixtures of halogenated compounds in Atlantic cod. Juvenile male cod with mean weight of 840 g were exposed by gavage to dietary mixtures of chlorinated (PCBs, DDT analogs, chlordane, lindane, and toxaphene), brominated (PBDEs), and fluorinated (PFOS) compounds for 4 weeks. One group received a combined mixture of all three compound groups. The results showed that the accumulated levels of chemicals in cod liver after 4 weeks of exposure reflected concentrations found in wild fish in this region. Pathway analysis revealed that the treatment effects by each of the three groups of chemicals (chlorinated, brominated, and fluorinated) converged on activation of the unfolded protein response (UPR). Upstream regulator analysis predicted that almost all the key transcription factors (XBP1, ERN1, ATF4, EIF2AK3, and NFE2L2) regulating the UPR were significantly activated. No additive effect was observed in cod co-treated with all three compound groups. In conclusion, the genome-wide transcriptomic study suggests that the UPR pathway is a sensitive common target of halogenated organic environmental pollutants in fish.

Dietary chlorpyrifos-methyl exposure impair transcription of immune-, detoxification- and redox signaling genes in leukocytes isolated from cod (Gadus morhua).

Inclusion of new environmental toxicants increase with the amount of plant ingredients substituting marine proteins and oils in feed for farmed Atlantic salmon (Salma salar). Agricultural pesticides like chlorpyrifos-methyl, present in commercial salmon feeds, may affect salmon immune and detoxification responses. Atlantic cod (Gadus morhua), surrounding the net pens, grazing on feces and uneaten pellets may be affected accordingly. The aim of this study was to analyze transcription responses in Atlantic cod head kidney tissue and isolated leukocytes following dietary chlorpyrifos-methyl inclusions and possible interactions with proinflammatory signals. Head kidney tissues and leukocytes were isolated from cod fed diets contaminated with chlorpyrifos-methyl (0.5 mg/kg, 2.4 mg/kg, 23.2 mg/kg) for 30 days. The isolated leukocytes were further challenged with bacteria (lipopolysaccharide (LPS), virus (polyinosinic acid:polycytidylic acid (PIC) mimic and l-arginine, an immuno-modulating amino acid, in vitro. The LPS-induced transcription of the interleukin genes il-1ß, il-6, il-8 increased in leukocytes isolated from cod fed chlorpyrifos-methyl 23.2 mg/kg, compared to cod fed the control diet, indicating increased inflammation. Transcriptional levels of carnitine palmitoyl transferase (cpt1a), aryl hydrogen receptor (ahr) and catalase (cat) were all reduced by dietary inclusions of chlorpyrifos-methyl in the leukocytes. The findings suggests that dietary chlorpyrifos-methyl exposure impair inflammation, detoxification and redox signaling in cod leukocytes.

Effects of mine tailing exposure on early life stages of cod (Gadus morhua) and haddock (Melanogrammus aeglefinus).

Mining and processing of minerals produce large quantities of tailings as waste. Some countries, including Norway, allow disposal of mine tailings in the sea. In this study we investigated the impacts of tailings from a calcium carbonate (CaCO3) processing plant on early live stages of haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua). Fish eggs (3 days post fertilisation; dpf) were exposed for 48 h to three concentrations of tailings, nominally 1 mg L-1 (low, L); 10 mg L-1 (medium, M) and 100 mg L-1 (high, H); with L and M representing concentrations occurring at tailing release points. Results show that tailings rapidly adhered to eggs of both species, causing negative buoyancy (sinking of eggs) in M and H exposures. While tailings remained on egg surfaces in both species also after exposure termination, adhesion seemed more pronounced in cod, leading to larger impacts on buoyancy even after exposure. Tailing exposure further induced early hatching and significantly reduced survival in M and H exposed embryos in both fish species, and in cod from the L exposure group. Moreover, tailing exposure caused reduced survival and malformations in larvae, potentially related to premature hatching. This study shows that mineral particles adhere to haddock and cod eggs, affecting egg buoyancy, survival and development.

A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts.

To avoid negative environmental impacts of escapees and potential inter-breeding with wild populations, the Atlantic salmon farming industry has and continues to extensively test triploid fish that are sterile. However, they often show differences in performance, physiology, behavior and morphology compared to diploid fish, with increased prevalence of vertebral deformities and ocular cataracts as two of the most severe disorders. Here, we investigated the mechanisms behind the higher prevalence of cataracts in triploid salmon, by comparing the transcriptional patterns in lenses of diploid and triploid Atlantic salmon, with and without cataracts. We assembled and characterized the Atlantic salmon lens transcriptome and used RNA-seq to search for the molecular basis for cataract development in triploid fish. Transcriptional screening showed only modest differences in lens mRNA levels in diploid and triploid fish, with few uniquely expressed genes. In total, there were 165 differentially expressed genes (DEGs) between the cataractous diploid and triploid lens. Of these, most were expressed at lower levels in triploid fish. Differential expression was observed for genes encoding proteins with known function in the retina (phototransduction) and proteins associated with repair and compensation mechanisms. The results suggest a higher susceptibility to oxidative stress in triploid lenses, and that mechanisms connected to the ability to handle damaged proteins are differentially affected in cataractous lenses from diploid and triploid salmon.

Whole-animal accumulation, oxidative stress, transcriptomic and metabolomic responses in the pink shrimp (Pandalus montagui) exposed to teflubenzuron.

The benzoylurea chitin synthesis inhibitor teflubenzuron, widely used against sea lice in North Atlantic aquaculture, may pose an environmental threat to non-targeted crustaceans. In this experiment, laboratory acclimated pink shrimp (Pandalus montagui), a species found in fjords with Atlantic salmon farming, were exposed to dietary teflubenzuron for 46 days (control; low dose: 0.01 µg/g; high dose: 0.1 µg/g). The exposure doses represent 0.1% and 1% of a standard treatment dose for Atlantic salmon. Mortality and prevalence of deformities, pharmacokinetics, oxidative stress and transcriptomic and metabolomic profiling were used to assess the response to teflubenzuron exposure. Mortality in the high-dose group was 25% (five of 20 individuals). No control or low-dose group shrimps died. Phenotypic responses,i.e., leg deformities (0 control, 6 low, 8 high) and cloudy eyes (0 control, 3 low, 7 high), were observed in some surviving shrimps (control n = 15, low n = 17, high n = 15). Accumulated levels of teflubenzuron in shrimps from the high-dose group ranged from 4.7 to 369 ng/g wet weight. Transcriptomic profiling showed very few significantly altered genes in the exposed shrimps. Teflubenzuron-induced changes to the metabolome pointed to well-known effects of benzoylurea agents, with reduced levels of N-acetylglucosamine indicating an effect on chitin synthesis. The metabolomic profiling showed that teflubenzuron exposure was associated with reduced energy metabolism. Some metabolites pointed to increased necrosis and/or bacterial overgrowth in the teflubenzuron-exposed shrimps. In conclusion, this study shows that teflubenzuron causes phenotypic effects in P. montagui exposed to 0.1% of the treatment dose given to Atlantic salmon.

Metabolic effects of p,p'-DDE on Atlantic salmon hepatocytes.

Decades after being banned in many countries, DDT and its metabolites are still considered major environmental hazards. The p,p'-DDE isomer, the DDT metabolite found in highest concentration in aquaculture feeds, is an endocrine disruptor with demonstrated ability to induce epigenetic effects. This study aimed at examining the impact of p,p'-DDE on Atlantic salmon. Primary hepatocytes were exposed to four concentrations of p,p'-DDE (0.1, 1, 10, 100 µm) for 48 hours, and endpoints included cytotoxicity, global DNA methylation, targeted transcription and metabolomics profiling (100 µm). p,p'-DDE was moderately cytotoxic at 100 µm. No impact was seen on global DNA methylation. Vtg1 and esr1 transcription, markers of endocrine disruption, was most strongly induced at 10 µm p,p'-DDE, while ar showed strongest response at 100 µm. Metabolomics profiling showed that p,p'-DDE at 100 µm most strongly affected carbohydrate metabolism, primary bile acid metabolism, leucine, isoleucine and valine metabolism, diacylglycerol and sphingolipid metabolism. Observed changes in lipid levels suggest that p,p'-DDE interferes with phospholipid membrane biosynthesis. Elevation of bile acid levels in p,p'-DDE-exposed hepatocytes indicates upregulation of synthesis of bile acids after cytochrome P450 activation. Pathway analysis showed that the superpathway of methionine degradation was the most significantly affected pathway by p,p'-DDE exposure, while endocrine system disorder topped the diseases and disorder ranking. In conclusion, this work predicts an endocrine response to p,p'-DDE exposure, and demonstrates how this legacy pesticide might interfere with mechanisms linked to DNA methylation in Atlantic salmon hepatocytes.

ß-naphthoflavone interferes with cyp1c1, cox2 and IL-8 gene transcription and leukotriene B4 secretion in Atlantic cod (Gadus morhua) head kidney cells during inflammation.

The objective of this study was to evaluate how ß-naphthoflavone interacts with lipopolysaccharide (LPS) and polyinosinic acid: polycytidylic acid (poly I: C) induced innate immune parameters as well as phase I and phase II detoxification enzymes in head kidney cells isolated from Atlantic cod. ß-naphthoflavone is a pure agonist of aryl hydrocarbon receptor (AhR) while LPS and poly I: C are not. ß-naphthoflavone was added to head kidney leukocytes alone or together with LPS or poly I: C and the responses were evaluated in terms of protein and gene expression. The results showed that ß-naphthoflavone (25 nM), with and without LPS, significantly induced cytochrome P450 (cyp1c) transcription in cod head kidney cells. ß-naphthoflavone (100 nM) in the presence of the virus mimic, poly I: C, also increased cyp1c1transcription. LPS induced cyp1c1, cyclooxygenase 2 (cox2), interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and interleukin 8 (IL-8) transcription, genes that were not affected by the tested ß-naphthoflavone concentrations alone. However, ß-naphthoflavone (25 and 50 nM) strengthened LPS induced cox2 and IL-8 transcription. Cod head kidney cells exposed to ß-naphthoflavone concentrations ranging from 25 to 100 nM, with and without LPS or poly I: C, expressed AhR protein. LPS or ß-naphthoflavone (5-50 nM) significantly induced leukotriene B4 (LTB4) secretion compared to control. In conclusion, this study suggests that ß-naphthoflavone could interfere with LPS induced immune cell signaling in cod head kidney cells.

Exposure of first-feeding cod larvae to dispersed crude oil results in similar transcriptional and metabolic responses as food deprivation.

Exposure of first-feeding cod larvae (Gadus morhua) to dispersed oil results in reduced feeding during an important transition period. First-feeding cod larvae were subjected to a 4-d treatment of food deprivation and sampled for microarray analyses. These microarray data were combined with data from cod larvae treated with mechanically and chemically dispersed oil in an attempt to understand to what extent starvation might explain some of the effects observed in first-feeding cod larvae during oil exposure. Transcriptional profiling of cod larvae suggested that the influence of oil exposure was almost as dramatic as being completely deprived of food. Protein and cellular degradation and loss of amino acids and glucose appear to be concomitant responses to both oil exposure and starvation. Fluorescence imaging of gut content indicated low uptake of food, and reduced growth (decrease in dry weight and in carbon and nitrogen content) was also noted in oil-exposed larvae, providing phenotypic anchoring of microarray data. The study displays the importance in combining use of high-throughput molecular tools with assessment of fitness-related endpoints in order to provide a greater understanding of toxicant-induced responses. This combined-approach investigation suggests that reduction of food uptake is an important process to be included when predicting effects of accidental oil spills. Finally, when comparing data from two oil treatments, exposure to chemically dispersed oil did not appear to result in greater toxicity than exposure to mechanically dispersed oil.

Transcriptional effects of metal-rich acid drainage water from the abandoned Løkken Mine on Atlantic salmon (Salmo salar) smolt.

Runoff of metals represents one of the major environmental challenges related to historic and ongoing mining activity. In this study, transcriptomics (direct RNA sequencing [RNA-seq] and reverse-transcription quantitative polymerase chain reaction [RT-qPCR]) was used to predict toxicity of metal-rich acid mine drainage (AMD) water collected in the abandoned copper (Cu) mine called Løkken Mine on Atlantic salmon liver and kidney, the main target organs of Cu-induced toxicity in fish. Smolts were exposed to control and diluted AMD water, which contains a mixture of metals but is especially enriched with Cu, at 4 concentrations in freshwater (FW) for 96 h, and then were transferred to and kept in seawater (SW) for another 24 h. Significant accumulation of Cu was observed in the gills, but not liver and kidney tissues, after 96 h of exposure. Short-term exposure to metal-rich ADM (high exposure group) significantly upregulated 3201 transcripts and downregulated 3782 transcripts in liver. The strongest effect attributed to exposure was observed on the KEGG pathway "protein processing in endoplasmic reticulum," followed by "steroid biosynthesis." Gene ontology (GO) analysis suggested that exposure predominantly affected "protein folding," possibly by disrupting disulfide bonds as a result of endoplasmic-reticulum-generated stress, and "sterol biosynthetic processes." Transfer to uncontaminated SW for 24 h amended the transcription of several genes, suggesting a transient effect of treatment on some mechanisms. In conclusion, the data show that trace metals in AMD from abandoned pyrite mines might disturb molecular mechanisms linked to protein folding in Atlantic salmon smolt endoplasmic reticulum.

Endocrine effects of real-life mixtures of persistent organic pollutants (POP) in experimental models and wild fish.

A series of studies have assessed the occurrence, levels, and potential adverse effects of persistent organic pollutants (POP) in fish from Lake Mjøsa. In this lake, high levels of various POP were detected in biota. Fish from the nearby Lake Losna contain background levels of POP and served as reference (controls) in these studies. Significantly higher prevalence of mycobacteriosis and pathological changes were documented in burbot (Lota lota) from Mjøsa compared to burbot from Losna. Further, transcriptional profiling identified changes in gene expression in burbot from Mjøsa compared to burbot from Losna associated with drug metabolism enzymes and oxidative stress. POP extracted from burbot liver oil from the two lakes was used to expose zebrafish (Danio rerio) during two consecutive generations. During both generations, POP mixtures from both lakes increased the rate of mortality, induced earlier onset of puberty, and skewed sex ratio toward males. However, opposite effects on weight gain were found in exposure groups compared to controls during the two generations. Exposure to POP from both lakes was associated with suppression of ovarian follicle development. Analyses of genome-wide transcription profiling identified functional networks of genes associated with weight homeostasis, steroid hormone functions, and insulin signaling. In human cell studies using adrenocortical H295R and primary porcine theca and granulosa cells, exposure to lake extracts from both populations modulated steroid hormone production with significant difference from controls. The results suggest that POP from both lakes may possess the potential to induce endocrine disruption and may adversely affect health in wild fish.

Fish pre-acclimation temperature only modestly affects cadmium toxicity in Atlantic salmon hepatocytes.

An emerging focus in environmental toxicology is how climate change will alter bioavailability and uptake of contaminants in organisms. Ectothermic animals unable to adjust their temperature by local migration, such as farmed fish kept in net pens, may become more vulnerable to contaminants in warmer seas. The aim of this work was to study cadmium (Cd) toxicity in cells obtained from fish acclimated to sub-optimal growth temperature. Atlantic salmon hepatocytes, harvested from fish pre-acclimated either at 15°C (optimal growth temperature) or 20°C (heat-stressed), were exposed in vitro to two concentrations of Cd (control, 1 and 100µM Cd) for 48h. Cd-induced cytotoxicity, determined with the xCELLigence system, was more pronounced in cells from fish pre-acclimated to a high temperature than in cells from fish grown at optimal temperature. A feed spiked with antioxidants could not ameliorate the Cd-induced cytotoxicity in cells from temperature-stressed fish. At the transcriptional level, Cd exposure affected 11 out of 20 examined genes, of which most are linked to oxidative stress. The transcriptional levels of a majority of the altered genes were changed in cells harvested from fish grown at sub-optimal temperature. Interaction effects between Cd exposure and fish pre-acclimation temperature were seen for four transcripts, hmox1, mapk1, fth1 and mmp13. Overall, this study shows that cells from temperature-stressed fish are modestly more vulnerable to Cd stress, and indicate that mechanisms linked to oxidative stress may be differentially affected in temperature-stressed cells.

Role of the GH-IGF-1 system in Atlantic salmon and rainbow trout postsmolts at elevated water temperature.

A comparative experiment with Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) postsmolts was conducted over 35 days to provide insight into how growth, respiration, energy metabolism and the growth hormone (GH) and insulin-like growth factor 1 (IGF-1) system are regulated at elevated sea temperatures. Rainbow trout grew better than Atlantic salmon, and did not show reduced growth at 19 °C. Rainbow trout kept at 19 °C had increased blood hemoglobin concentration compared to rainbow trout kept at 13 °C, while salmon did not show the same hemoglobin response due to increased temperature. Both species showed reduced length growth and decreased muscle glycogen stores at 19 °C. Circulating IGF-1 concentration was higher in rainbow trout than in Atlantic salmon, but was not affected by temperature in either species. Plasma IGF-binding protein 1b (IGFBP-1b) concentration was reduced in Atlantic salmon reared at 19 °C after 15 days but increased in rainbow trout at 19 °C after 35 days. The igfbp1b mRNA level in liver showed a positive correlation to plasma concentrations of glucose and IGFBP-1b, suggesting involvement of this binding protein in carbohydrate metabolism at 19 °C. At this temperature muscle igfbp1a mRNA was down-regulated in both species. The muscle expression of this binding protein correlated negatively with muscle igf1 and length growth. The plasma IGFBP-1b concentration and igfbp1b and igfbp1a expression suggests reduced muscle igf1 signaling at elevated temperature leading to glucose allostasis, and that time course is species specific due to higher thermal tolerance in rainbow trout.

Thermal stress alters expression of genes involved in one carbon and DNA methylation pathways in Atlantic cod embryos.

One-carbon (1-C) metabolism is essential for normal embryonic development through its regulation of DNA methylation and cell proliferation. With consideration to the potential future anthropogenic oceanic warming, we studied the effects of both acute thermal stress and continuous thermal stress (10°C) during Atlantic cod embryo development on the expression levels of genes associated with the 1-C metabolism, including DNA methyltransferases. We conducted a phylogenetic analysis of vertebrate DNA methyltransferases to determine the number and similarity of DNMT found in Atlantic cod. This analysis revealed that Atlantic cod have one maintenance dnmt (dnmt1) and five de novo DNMTs (dnmt4, dnmt3, dnmt3b, dnmt3aa, dnmt3ab). Stage specific changes in expression levels occurred for all genes analyzed. The effect of acute thermal stress was evaluated during early development. Compared to controls these experiments showed significant alterations in expression levels of several genes, that in some instances were reversed at later stages of development. A significant effect of continuous thermal stress was found in gastrula embryos where lower mRNA expression levels of 1-C metabolism, de novo DNMTs and cell proliferation genes were detected. One exception was the maintenance DNMT, which was only sensitive to acute and not continuous thermal stress. DNA methylation status indicated that blastula embryos were hypomethylated compared to spermatozoa and late gastrula stages. In post-gastrula stage, however, continuous thermal stress resulted in a higher degree of DNA methylation compared to controls. These data reveal that the regulation of epigenetically important transcripts in the 1-C metabolism of Atlantic cod embryos is sensitive to thermal stress.

Gill epithelial cell line ASG-10 from Atlantic salmon as a new research tool for solving water quality challenges in aquaculture.

Here we evaluated the gill epithelial cell line ASG-10 from Atlantic salmon, as an in vitro model for research on known water quality challenges in aquaculture. Ammonia/ammonium (NH3/NH4+), a recognized challenge in water-intensive recirculating aquaculture systems (RAS), induced lysosomal vacuolization, reduced protein degradation and cell migration of the ASG-10 cells. Aluminium (Aln+), another challenge in freshwater aquaculture facilities had only minor effects. Next, we investigated the tolerance for direct water exposure of ASG-10. The cells tolerated water with osmolarity between 169 and 419 mOsmol/kg for 24 h. However, cells exposed for 3 h to water at 863 mOsmol/kg changed cellular morphology and induced gene expression related to stress (gpx1, casp3, hsp70), and after 24 h exposure cellular viability was severely reduced. Nevertheless, when the cells were grown in transwell inserts, they tolerated 863 mOsmol/kg for 3 h and induction of stress response associated genes was considerably reduced. Lastly, the ASG-10 cells were exposed to water samples, with no known quality issues, from different aquaculture facilities. The cells showed no differences in viability or morphology compared to their representative control. In conclusion, the ASG-10 cell line is a promising in vitro model to study water quality challenges and whole water samples.

Enhanced neurotoxic effect of PCB-153 when co-exposed with polystyrene nanoplastics in zebrafish larvae.

Nanoplastics (NPs) and persistent organic pollutants such as polychlorinated biphenyls (PCBs) are ubiquitous aquatic pollutants. The coexistence of these pollutants in the environment emphasises the need to study their combined toxicity. NPs can cross biological membranes and act as vectors for other pollutants, whereas PCBs are known for their ability to bioaccumulate and biomagnify. The present work aimed to study the combined toxicity of polystyrene NPs and PCB-153 using physiological (development, heart rate, respiration), behavioural (swimming behaviour) and molecular (transcriptome) endpoints in zebrafish larvae. The results show that exposure to NPs, PCB and their mixture significantly affected the development and respiration in zebrafish larvae. Larvae co-exposed to NPs and PCB exhibited significant hyperlocomotion, whereas no such effect was observed after exposure to NPs or PCB alone. The transcriptomic results revealed that NPs exposure significantly affected several pathways associated with DNA compaction and nucleosome assembly, whereas PCB exposure significantly affected critical neurogenic pathways. In contrast, co-exposure to NPs and PCB generated multi-faceted toxicity and suppressed neurobehavioural, immune-related and detoxification pathways. The study highlights the complex interplay between NPs and PCBs, and documents how the two toxicants in combination give a stronger effect than the single toxicants alone. Understanding the mixture toxicity of these two pollutants is important to assess the environmental risks and developing effective management strategies, ultimately safeguarding ecosystems and human health.

Effects of gadolinium (Gd) and a Gd-based contrast agent (GBCA) on early life stages of zebrafish (Danio rerio).

Gadolinium (Gd) is one of the rare earth elements (REY) and is widely used in magnetic resonance imaging (MRI) contrast agents. Anthropogenic Gd enrichment has frequently been found in wastewater treatment plant effluents in industrialised countries, rising concerns regarding effects on aquatic biota. This study investigates the acute toxicity and sublethal effects of Gd in two forms, as inorganic salt (GdCl3) and as Gd-based contrast agent (GBCA), on early life stages of zebrafish (Danio rerio). Nominal exposure concentrations ranged from 3 to 3000 µg L-1, with an exposure duration of 96 h. None of the two tested compounds were acutely toxic to embryos and larvae. Similarly, we did not observe any effects on larval development and locomotive behaviour. However, we found significant changes in the brain activity of larvae exposed to the highest concentrations of GdCl3 and the GBCA. Our findings show that Gd can have sublethal effects on developing fish at lower concentrations than reported previously, highlighting the necessity of investigating the long-term fate and effects of GBCAs released into the aquatic environment.

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.