Long-Term Influence of PCB- and PBDE-Spiked Microplastic Spheres Fed through Rotifers to Atlantic Cod (Gadus morhua) Larvae.

Omnipresent microplastics (MPs) in marine ecosystems are ingested at all trophic levels and may be a vector for the transfer of persistent organic pollutants (POPs) through the food web. We fed rotifers polyethylene MPs (1-4 µm) spiked with seven congeners of polychlorinated biphenyls (PCBs) and two congeners of polybrominated diphenyl ethers (PBDEs). In turn, these rotifers were fed to cod larvae from 2-30 days post-hatching (dph), while the control groups were fed rotifers without MPs. After 30 dph, all the groups were fed the same feed without MPs. Whole-body larvae were sampled at 30 and 60 dph, and four months later the skin of 10 g juveniles was sampled. The PCBs and PBDEs concentrations were significantly higher in MP larvae compared to the control larvae at 30 dph, but the significance dissipated at 60 dph. Expression of stress-related genes in cod larvae at 30 and 60 dph showed inconclusive minor random effects. The skin of MP juveniles showed disrupted epithelial integrity, fewer club cells and downregulation of a suite of genes involved in immunity, metabolism and the development of skin. Our study showed that POPs were transferred through the food web and accumulated in the larvae, but that the level of pollutants decreased once the exposure was ceased, possibly related to growth dilution. Considering the transcriptomic and histological findings, POPs spiked to MPs and/or MPs themselves may have long-term effects in the skin barrier defense system, immune response and epithelium integrity, which may potentially reduce the robustness and overall fitness of the fish.

Potential of Atlantic Codfish (Gadus morhua) Skin Collagen for Skincare Biomaterials.

Collagen is the major structural protein in extracellular matrix present in connective tissues, including skin, being considered a promising material for skin regeneration. Marine organisms have been attracting interest amongst the industry as an alternative collagen source. In the present work, Atlantic codfish skin collagen was analyzed, to evaluate its potential for skincare. The collagen was extracted from two different skin batches (food industry by-product) using acetic acid (ASColl), confirming the method reproducibility since no significant yield differences were observed. The extracts characterization confirmed a profile compatible with type I collagen, without significant differences between batches or with bovine skin collagen (a reference material in biomedicine). Thermal analyses suggested ASColl's native structure loss at 25 °C, and an inferior thermal stability to bovine skin collagen. No cytotoxicity was found for ASColl up to 10 mg/mL in keratinocytes (HaCaT cells). ASColl was used to develop membranes, which revealed smooth surfaces without significative morphological or biodegradability differences between batches. Their water absorption capacity and water contact angle indicated a hydrophilic feature. The metabolic activity and proliferation of HaCaT were improved by the membranes. Hence, ASColl membranes exhibited attractive characteristics to be applied in the biomedical and cosmeceutical field envisaging skincare.

Changes in the Molecular Characteristics of Bovine and Marine Collagen in the Presence of Proteolytic Enzymes as a Stage Used in Scaffold Formation.

Biopolymers, in particular collagen and fibrinogen, are the leading materials for use in tissue engineering. When developing technology for scaffold formation, it is important to understand the properties of the source materials as well as the mechanisms that determine the formation of the scaffold structures. Both factors influence the properties of scaffolds to a great extent. Our present work aimed to identify the features of the molecular characteristics of collagens of different species origin and the changes they undergo during the enzymatic hydrolysis used for the process of scaffold formation. For this study, we used the methods of gel-penetrating chromatography, dynamic light scattering, reading IR spectra, and scanning electron microscopy. It was found that cod collagen (CC) and bovine collagen (BC) have different initial molecular weight parameters, and that, during hydrolysis, the majority of either type of protein is hydrolyzed by the proteolytic enzymes within the first minute. The differently sourced collagen samples were also hydrolyzed with the formation of two low molecular fractions: Mw ~ 10 kDa and ~20 kDa. In the case of CC, the microstructure of the final scaffolds contained denser, closely spaced fibrillar areas, while the BC-sourced scaffolds had narrow, short fibrils composed of unbound fibers of hydrolyzed collagen in their structure.

Effect of gillnet twine thickness on capture pattern and efficiency in the Northeast-Arctic cod (Gadus morhua) fishery.

Gillnets are among the most common fishing gears worldwide. They are often made of thin twine, which is prone to wear and tear, limiting the lifespan of the gillnet. This increases gillnet turnover, and consequently increased risk of gear discarding, gear loss, ghost fishing and marine pollution. This might be mitigated by increasing twine thickness, and thereby breaking strength. However, the tolerable increase in thickness for gillnet durability without compromising the catch efficiency is unknown. Therefore, this study conducted gillnet fishing trials under commercial conditions in the Northeast-Arctic cod gillnet fishery analysing and comparing ways of capture and efficiency between gillnets with two different twine thicknesses for two different mesh sizes. The results demonstrated that a 30 % increase in breaking strength and twine stiffness did not affect catch performance. Therefore, thicker gillnet twine can potentially reduce marine litter by plastic debris from damaged and lost gears without compromising catch performance.

Application of NMR spectroscopy for the detection and quantification of phthalic acid in fish muscles: The case of Atlantic Cod from Norwegian Sea.

Plastic litter might contain phthalates that can be transferred to marine environment or can be introduced into the marine food chain. Phthalic acid is the final product of phthalate decomposition in marine organisms. Here we used NMR spectroscopy to determine and quantify phthalic acid and dimethyl phthalate in fish muscles. Spike-and-recovery experiments were carried out to confirm assignment of phthalates resonance signals in NMR spectra and to evaluate the method specificity, accuracy, and linearity. The LOQ and LOD of the rapid 1H NMR experiment with a standard setting were respectively 23.0 and 8.0 mg of phthalic acid in kg of fish muscles. Phthalic acid was detected in 13 out of 113 Atlantic cod and none in farmed Atlantic salmon from Norwegian sea.

Effects of weathered polyethylene microplastic ingestion on sexual maturation, fecundity and egg quality in maturing broodstock Atlantic cod Gadus morhua.

Microplastics (MPs) have become a global issue as they are omnipresent in the ocean. Fish ingesting MPs through feed could be affected in their physiological function, e.g., disrupted enzyme production and function, reduction of feeding and reproductive failure. This study assessed the effects of feed containing naturally weathered MPs from the Oslofjord (Norway) on the reproductive physiology of Atlantic cod (Gadus morhua). Farmed cod broodstock were fed either control (C-diet) or feeds containing 1% microplastic (MP-diet) starting nine months prior to spawning, from June until May. No major differences were found between diet groups in overall biometrics or gonad histology. Sex steroid levels (testosterone, 11-ketotestosterone and 17ß-estradiol) resulted in expected profiles increasing over time without any significant differences between treatments. Gene expression levels of the steroidogenic enzyme 20ß-hydroxysteroid dehydrogenase (20ß-hsd) and vitellogenin1 (vtg1) showed significant differences between dietary treatments with lower expression in the control group. This can be a direct effect of MPs, but endocrine disrupting effects of potentially leachable plastic additives cannot be completely ruled out. Thus, these enzymes could be indicators of exposure to contaminants that disrupt sexual maturation by affecting the production of primarily maturation-inducing steroid. Although the concentration of MPs employed in this study may not be high enough to elicit any observable short-term biological effects, the observed gene expression suggests that long-term consequences should be considered caused by an expected increase of MPs in marine environments.

Inferring fish behaviour at the trawl mouth from escape location.

In this study, we used escape location underneath the trawl to understand groundfish herding behaviour at the trawl mouth. Three collecting bags (port, center, starboard) were mounted under the trawl and behind the footgear to collect escapees. The escape-at-length of species that escaped into the center bag were compared to the two wing bags to infer fish response behaviour, herding behaviour, and swimming capacity at the trawl mouth. For roundfish, smaller-sized individuals escaped more in the center for both Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus), <20 and 11 cm, respectively, indicating that larger-sized fish were to a greater extent seeking to escape under the trawl at the wings, vs small fish being herded to the center and likely overrun due to reduced swimming capacity. For flatfish and monkfish (Lophius piscatorius), results varied. European plaice (Pleuronectes platessa), American plaice (Hippoglossoides platessoides), and monkfish were caught most often in the wings, though not significantly for American plaice. Catches of dab (Limanda limanda) between 18 and 27 cm were significantly higher in the center, with no difference for smaller and larger individuals. The differences between fish escape location likely result from a combination of varying herding behaviour, size, and swimming capacity. Here, we were able to show how these size-dependent behaviours relate to fish response behaviour, escape behaviour, size, and likely swimming capacity.

PCB-126 spiked to polyethylene microplastic ingested by juvenile Atlantic cod (Gadus morhua) accumulates in liver and muscle tissues.

In the present study, polyethylene (PE) microplastics (150-300 µm) were added to Atlantic cod (Gadus morhua) feeds at 1 %, either in their present form (Virgin PE) or spiked with PCB-126 (Spiked PE). The feeds were given to juvenile cod for a 4-week period. The fish grew from 11 to 23 g with no significant difference between dietary treatments. Cod fed spiked PE showed a significantly higher concentration of PCB-126 in liver and muscle samples compared to control and fish ingesting virgin PE. In accordance with the accumulation of PCB-126 in the liver, the expression of hepatic cyp1a was higher in cod fed spiked PE. Notably, we observed that spiked PE, as well as virgin PE, have an effect on skin. Overall changes indicated a reduced skin barrier in fish fed a diet containing PE. Indicating that PE itself through interaction with gut tissue may influence skin health in fish.

Retinoic acid induces two osteocalcin isoforms and inhibits markers of osteoclast activity in Atlantic cod (Gadus morhua) ex vivo cultured craniofacial tissues.

Nutritional status including vitamin A could explain some of the developmental deformities observed in cultivated teleosts, including Atlantic cod (Gadus morhua). In the present study we aimed to investigate the transcriptional effect of retinoic acid (RA) on bone related genes using Atlantic cod craniofacial explants tissue cultures. Two different osteoblast specific osteocalcin/bone gla protein isoforms were discovered in cod. Transcription of both isoforms was up-regulated following RA treatment of 65 dph cod lower jaw explants. In contrast, transcripts coding for genes related to bone resorption and osteoclast activity, matrix metalloproteinase 9 and cathepsin K were down-regulated following RA treatment. This could be linked to the decreased transcriptional ratio between receptor activator of nuclear factor kappa-B ligand rankl and osteoprotegerin observed in the same tissue samples. RA treatment of juvenile explants had no effect on runt-related transcription factor 2 and osterix mRNA levels. However, osterix was significantly down-regulated in 25 dph cod head explants following RA treatment. In situ hybridizations revealed differential spatial distribution of the two isoforms and the predominant expression of cathepsin K in bone surrounding tissues. The present study indicates that RA causes a shift in the balance between osteoclast activity and osteoblast activity in favor of the latter.

Antioxidants from barley husks impregnated in films of low-density polyethylene and their effect over lipid deterioration of frozen cod (Gadus morhua).

BACKGROUND: The changes in quality of cod fillets packaged in films with and without antioxidants during 12 months of frozen storage at - 20 °C were investigated in the present study. The following parameters were determined in order to study lipid hydrolysis and primary and secondary lipid oxidation in the samples during frozen storage: peroxide value, conjugated dienes, conjugated triene hydroperoxides, free fatty acids, totox value, thiobarbituric acid-reactive substances and p-anisidine value. RESULTS: Films containing antioxidants isolated from barley husks were effective in slowing down lipid hydrolysis and primary and secondary lipid oxidation processes. Secondary lipid oxidation reached maximum values in the 12th month of storage in control samples and samples packaged with antioxidant-containing film. Maximum lipid hydrolysis and lipid oxidation values for control cod samples were significantly higher than the maximum values found in samples packaged with antioxidant-containing film. CONCLUSION: The results confirm the efficacy of natural antioxidants derived from barley husks in slowing down lipid hydrolysis and increasing the oxidative stability of cod flesh. They also demonstrate the potential usefulness of natural antioxidants extracted from barley husks in the development of active packaging films for food preservation.

Comparison of the efficiency and modes of capture of biodegradable versus nylon gillnets in the Northeast Atlantic cod (Gadus morhua) fishery.

Modern gillnets are usually made of nylon with high breaking strength, suitable elasticity and durability making them an efficient fishing gear. Lost, abandoned, or discarded gillnets at sea cause plastic pollution and can continue capturing marine animals over long periods of time. Biodegradable materials are being developed to replace nylon in gillnets. However, biodegradable gillnets have shown reduced catch efficiency compared to the nylon gillnets which challenges their acceptance by the fishing sector. This study investigated catch efficiency and modes of capture between biodegradable and nylon gillnets in commercial cod (Gadus morhua) fishery. On average, new biodegradable gillnets caught 25% fewer cod compared to new nylon gillnets. The main capture modes were by the gills and by the body in used and new biodegradable gillnets, respectively. Differences in catch efficiency are related to specific modes of capture that may be related to differences in material properties.

Can biodegradable materials reduce plastic pollution without decreasing catch efficiency in longline fishery?

Longlining is a widely used fishing method. During longline fishing, some of the snoods connecting the hooks to the mainline are often lost at sea. Since snoods are made of nylon or polyester, lost snoods contribute to marine plastic pollution. Replacing nylon or polyester with a new material made of biodegradable plastics can potentially reduce macro- and microplastic pollution that is caused by lost snoods. In this study, we estimated the risk for snood loss in a longline fishery targeting haddock (Melanogrammus aeglefinus (Linnaeus, 1758)) and Atlantic cod (Gadus morhua (Linnaeus, 1758)) in Barents Sea. Further, we compared catch efficiency in this fishery for snoods made of biodegradable and nylon materials. No significant differences were found between the two materials. Therefore, catch efficiency does not represent a barrier for using biodegradable materials in snoods.

Effects of changing environmental conditions on plastic ingestion and feeding ecology of a benthopelagic fish (Gadus morhua) in the Southwest Baltic Sea.

This study documents how the abundance of microplastics (<5 mm) in the Atlantic cod, Gadus morhua, relates to the changes of the fish diet during years with contrasting levels of anoxia for example following years of low or high major Baltic inflows (MBI). A MultiNet Maxi trawl and CTD were deployed annually to collect microplastic samples alongside oxygen, temperature, and salinity conditions. Microplastics were homogenously distributed both within the water column and across years. Gadus morhua diet shifted from dominantly benthic invertebrates (61 %) under oxygenated conditions to dominantly Sprattus sprattus (81 %) under anoxic conditions. The proportion of G. morhua with microplastics in their digestive tract increased when they fed on pelagic fish (38 %) versus on benthic invertebrates (15 %). The proportion of S. sprattus which ingested microplastics (~18 %) did not vary. As anoxia at depth is expected to increase due to climate change, microplastic ingestion by G. morhua will potentially increase.

Plastic in digestive tracts and gills of cod and herring from the Baltic Sea.

Plastic litter is widespread on our planet and is recognized as a contaminant of high concern. Plastic ingestion and retention in gills were studied in two key Baltic fish species: herring (Clupea harengus L.) and cod (Gadus morhua L.). In total, 183 fish from the southern Baltic Sea were analysed. Plastic litter was found in digestive tracts of 12.7 and 14.8% of herrings and cods, respectively. In addition, gills were shown to constitute an important transfer route of plastic to cod (9.9% of cods) but not to herring thus likely reflecting species-specific differences in fish lifestyles and/or water filtering capacity. No more than one plastic item per individual was found except for three fish with two items in their stomachs. Dominant microplastics (<5 mm) (MPs) (88.6% of all items) composed of irregular fragments, fibres and foils (61.3%, 25.8% and 12.9% of all MPs, respectively) were followed by meso- (5-25 mm) and macro-plastics (>25 mm) (5.7% each of all items). Plastic ingestion and retention in gills did not affect fish body condition assessed by Fulton's K index, although herrings that ingested plastic particles beyond the microplastic size range (i.e. >5 mm) were characterized by the lowest condition indices. All plastic items recovered from fish exhibited clear signs of weathering and surface extended due to their roughness and fragmentation which may enhance both sorption properties for chemical compounds and/or their leaching. Nevertheless, chemical contaminant levels in herring muscles were not different than in a plastic-free group. The vast majority of fibres found in fish was identified as air-borne or procedural contamination and were excluded from further analyses. In addition, the vessel's paint dust found in fish stressed the need for strict quality assurance also during fish sampling.

High stability of bilayer nano-emulsions fabricated by Tween 20 and specific interfacial peptides.

To ensure emulsions to be continuously stable, the hydrolysates recovered from cod bones by papain acted as a natural surfactant to synthesize high-stability bilayer nano-emulsions. As assisted by Tween 20, the average diameter of synthesized nano-emulsion with enzymatic hydrolysate could exhibit stability between 300-400 nm under a broad range of pH (4-8), temperatures (30-90 °C) and salt concentration (25-250 mM). With the addition of the hydrolysates, the rate of increase of the TBARS value in the emulsion decreased. Moreover, the bilayer structure of the nano-emulsion was characterized under an atomic force microscopy and a cryo-scanning electron microscopy. Nano-LC-Q-TOF-MS was adopted to primarily identify peptides that contained hydrophobic and hydrophilic amino acids at the emulsion interface. Besides, the absorbed peptides on the interface of emulsion enhanced the stability of emulsion lipid oxidation.

The effect of long-term use on the catch efficiency of biodegradable gillnets.

The effect of long-term use on the catch efficiency of biodegradable gillnets was investigated during commercial fishing trials and in controlled lab aging tests. The relative catch efficiency between biodegradable and nylon gillnets was evaluated over three consecutive fishing seasons for Atlantic cod (Gadus morhua) in Norway. The biodegradable gillnets progressively lost catch efficiency over time, as they caught 18.4%, 40.2%, and 47.4% fewer fish than the nylon gillnets during the first, second, and third season, respectively. A 1000-hour aging test revealed that both materials began to degrade after just 200 h and that biodegradable gillnets degraded faster than the nylon gillnets. Infrared spectroscopy revealed that the chemical structure of the biodegradable polymer changed more than the nylon. Although less catch efficient than nylon gillnets, biodegradable gillnets have great potential for reducing both capture in lost fishing gear and plastic pollution at sea, which are major problems in fisheries worldwide.

Occurrence of plastics ingested by Atlantic cod (Gadus morhua) destined for human consumption (Fogo Island, Newfoundland and Labrador).

In the province of Newfoundland and Labrador, fishing is a core occupation and also a source of marine plastic pollution. To look at this relationship, we examined 216 gastrointestinal tracts of Atlantic cod (Gadus morhua) caught by commercial fishers at Fogo Island, Newfoundland and Labrador, Canada. We found three tracts contained plastic for a frequency of occurrence of 1.4%. While this result is consistent with other cod sampled in the province, this study found two gastrointestinal tracts contained intact bait bags, used in commercial pots, and the third tract contained a polypropylene thread, likely originating from fishing rope. Our findings demonstrate the frequency of plastic ingestion in this region is low, but fishing-gear related plastics represent a key source of marine plastics in the region that should be addressed.

Acid and enzymatic extraction of collagen from Atlantic cod (Gadus Morhua) swim bladders envisaging health-related applications.

Atlantic cod is processed industrially for food purposes, with several by-products being directed to animal feed and other ends. Looking particularly into swim bladders, the extraction of collagen can be a valuable strategy for by-product valorization, explored in the present work for the first time. Collagen was extracted using acetic acid (ASCsb) and pepsin (PSCsb) with yields of 5.72% (w/w) and 11.14% (w/w), respectively. SDS-PAGE profile showed that the extracts were compatible with type I collagen. FTIR, CD and XRD results suggest that the PSCsb structure underwent partial denaturation, with microDSC showing a band at 54 °C probably corresponding to a melting process, while ASCsb structure remained intact, with preserved triple helix and a denaturation temperature of 29.6 °C. Amino acid composition indicates that the total content of proline-like amino acids was 148/1000 residues for ASCsb and 141/1000 residues for PSCsb, with a hydroxylation degree of about 37%. The extracts exhibited a typical shear thinning behavior, interesting property regarding their further processing toward the development of biomaterials. In this regard, assessment of metabolic activity of human fibroblast cells cultured in the presence of collagen extracts with concentrations up to 3 mg/mL revealed the absence of cytotoxic behavior. Collagen extracts obtained from Atlantic cod swim bladders shown attractive properties regarding their use in cosmetic or biomedical applications.

Localization of mRNAs and proteins in methyl methacrylate-embedded tissues.

Precise localization of proteins and mRNA in histological sections is necessary for evaluating spatial gene expression patterns. Here we report sensitive detection of the gene products in fish tissues by immunohistochemistry (IHC) and in situ hybridization (ISH) assays on sections of whole specimens and vertebra embedded in methyl methacrylate (MMA) resin. This plastic resin favors easy preparation of various specimen types and enables preparation of large sections with well-preserved cell morphology. IHC analysis of the muscle regulatory factor MyoD in transverse sections of juvenile cod revealed MyoD-positive cells in the dorsolateral parts of the adaxial muscle. ISH revealed less spatially restricted signals of the bone morphogenic protein bmp4 in muscle and brain. To assess the applicability of ISH on sections of bony tissue, col1a1 and col2a1 expression was investigated in non-decalcified vertebra sections of Atlantic salmon. The former was identified in both chondrocytes and osteoblasts, whereas the latter was mostly evident in chondrocytes. We conclude that MMA resin offers easy preparation of large and problematic tissues and the possibility of carrying out both IHC and ISH analyses using standard protocols.

Embryonic exposure to produced water can cause cardiac toxicity and deformations in Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) larvae.

Regular discharges of produced water from the oil and gas industry represents the largest direct discharge of effluent into the marine environment worldwide. Organic compound classes typically reported in produced water include saturated hydrocarbons, monoaromatic and polyaromatic hydrocarbons (MAHs, PAHs) as well as oxygenated compounds, such as phenols, acids and ketones. This forms a cocktail of known and suspect toxicants, but limited knowledge is yet available on the sub-lethal toxicity of produced water to cold-water marine fish species. In the present work, we conducted a 4-day exposure of embryos of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) to produced water extracts equivalent to 1:50, 1:500 and 1:5000 times dilutions of raw effluent. No significant reduction in survival or hatching success was observed, however, for cod, hatching was initiated earlier for exposed embryos in a concentration-dependent manner. During recovery, significantly reduced embryonic heart rate was observed for both species. After hatch, larvae subjected to embryonic exposure to produced water extracts were smaller, and displayed signs of cardiotoxicity, jaw and craniofacial deformations. In order to improve risk assessment and regulation of produced water discharges, it is important to identify which produced water components contribute to these effects.