Connecting gut microbiome changes with fish health conditions in juvenile Atlantic cod (Gadus morhua) exposed to dispersed crude oil.

Polycyclic aromatic hydrocarbons found in crude oil can impair fish health following sublethal exposure. However, the dysbiosis of microbial communities within the fish host and influence it has on the toxic response of fish following exposure has been less characterized, particularly in marine species. To better understand the effect of dispersed crude oil (DCO) on juvenile Atlantic cod (Gadus morhua) microbiota composition and potential targets of exposure within the gut, fish were exposed to 0.05 ppm DCO for 1, 3, 7, or 28 days and 16 S metagenomic and metatranscriptomic sequencing on the gut and RNA sequencing on intestinal content were conducted. In addition to assessing species composition, richness, and diversity from microbial gut community analysis and transcriptomic profiling, the functional capacity of the microbiome was determined. Mycoplasma and Aliivibrio were the two most abundant genera after DCO exposure and Photobacterium the most abundant genus in controls, after 28 days. Metagenomic profiles were only significantly different between treatments after a 28-day exposure. The top identified pathways were involved in energy and the biosynthesis of carbohydrates, fatty acids, amino acids, and cellular structure. Biological processes following fish transcriptomic profiling shared common pathways with microbial functional annotations such as energy, translation, amide biosynthetic process, and proteolysis. There were 58 differently expressed genes determined from metatranscriptomic profiling after 7 days of exposure. Predicted pathways that were altered included those involved in translation, signal transduction, and Wnt signaling. EIF2 signaling was consistently dysregulated following exposure to DCO, regardless of exposure duration, with impairments in IL-22 signaling and spermine and spermidine biosynthesis in fish after 28 days. Data were consistent with predictions of a potentially reduced immune response related to gastrointestinal disease. Herein, transcriptomic-level responses helped explain the relevance of differences in gut microbial communities in fish following DCO exposure.

DNA damage and health effects in juvenile haddock (Melanogrammus aeglefinus) exposed to PAHs associated with oil-polluted sediment or produced water.

The research objective was to study the presence of DNA damages in haddock exposed to petrogenic or pyrogenic polyaromatic hydrocarbons (PAHs) from different sources: 1) extracts of oil produced water (PW), dominated by 2-ring PAHs; 2) distillation fractions of crude oil (representing oil-based drilling mud), dominated by 3-ring PAHs; 3) heavy pyrogenic PAHs, mixture of 4/5/6-ring PAHs. The biological effect of the different PAH sources was studied by feeding juvenile haddock with low doses of PAHs (0.3-0.7 mg PAH/kg fish/day) for two months, followed by a two-months recovery. In addition to the oral exposure, a group of fish was exposed to 12 single compounds of PAHs (4/5/6-ring) via intraperitoneal injection. The main endpoint was the analysis of hepatic and intestinal DNA adducts. In addition, PAH burden in liver, bile metabolites, gene and protein expression of CYP1A, GST activity, lipid peroxidation, skeletal deformities and histopathology of livers were evaluated. Juvenile haddock responded quickly to both intraperitoneal injection and oral exposure of 4/5/6-ring PAHs. High levels of DNA adducts were detected in livers three days after the dose of the single compound exposure. Fish had also high levels of DNA adducts in liver after being fed with extracts dominated by 2-ring PAHs (a PW exposure scenario) and 3-ring PAHs (simulating an oil exposure scenario). Elevated levels of DNA adducts were observed in the liver of all exposed groups after the 2 months of recovery. High levels of DNA adduct were found also in the intestines of individuals exposed to oil or heavy PAHs, but not in the PW or control groups. This suggests that the intestinal barrier is very important for detoxification of orally exposures of PAHs.

Study of the plasma proteome of Atlantic cod (Gadus morhua): Changes due to crude oil exposure.

Oil contamination is an environmental issue of great concern and the necessity for background studies and monitoring programs to continuously evaluate the levels of oil pollution is required. In this study, Atlantic cod (Gadus morhua) were exposed to dispersed crude oil for 1 and 4 weeks to simulate environmental contamination. Fractionated plasma samples were then analysed by tandem mass spectrometry. In total, 717 proteins were identified and 10 new protein biomarker candidates were found. The significant proteome changes were related to the immune response by alterations in the levels of specific immunoglobulins, alpha-2-macroglobulin and galectin-3-binding proteins. After 4 weeks of oil exposure, a lowered level of a NLRC3-like protein was also observed. The results from this study provide insight into the Atlantic cod plasma proteome and into the toxicological effects and potential response mechanisms of short and long-term exposure to crude oil.

II. Species sensitivity distributions based on biomarkers and whole organism responses for integrated impact and risk assessment criteria.

The aim of this paper is to bridge gaps between biomarker and whole organism responses related to oil based offshore discharges. These biomarker bridges will facilitate acceptance criteria for biomarker data linked to environmental risk assessment and translate biomarker results to higher order effects. Biomarker based species sensitivity distributions (SSDbiomarkers) have been constructed for relevant groups of biomarkers based on laboratory data from oil exposures. SSD curves express the fraction of species responding to different types of biomarkers. They have been connected to SSDs for whole organism responses (WORs) constructed in order to relate the SSDbiomarkers to animal fitness parameters that are commonly used in environmental risk assessment. The resulting SSD curves show that biomarkers and WORs can be linked through their potentially affected fraction of species (PAF) distributions, enhancing the capability to monitor field parameters with better correlation to impact and risk assessment criteria and providing improved chemical/biological integration.

I: Biomarker quantification in fish exposed to crude oil as input to species sensitivity distributions and threshold values for environmental monitoring.

The aim of this study was to determine a suitable set of biomarker based methods for environmental monitoring in sub-arctic and temperate offshore areas using scientific knowledge on the sensitivity of fish species to dispersed crude oil. Threshold values for environmental monitoring and risk assessment were obtained based on a quantitative comparison of biomarker responses. Turbot, halibut, salmon and sprat were exposed for up to 8 weeks to five different sub-lethal concentrations of dispersed crude oil. Biomarkers assessing PAH metabolites, oxidative stress, detoxification system I activity, genotoxicity, immunotoxicity, endocrine disruption, general cellular stress and histological changes were measured. Results showed that PAH metabolites, CYP1A/EROD, DNA adducts and histopathology rendered the most robust results across the different fish species, both in terms of sensitivity and dose-responsiveness. The reported results contributed to forming links between biomonitoring and risk assessment procedures by using biomarker species sensitivity distributions.

Dietary fish protein hydrolysates containing bioactive motifs affect serum and adipose tissue fatty acid compositions, serum lipids, postprandial glucose regulation and growth in obese Zucker fa/fa rats.

The world's fisheries and aquaculture industries produce vast amounts of protein-containing by-products that can be enzymatically hydrolysed to smaller peptides and possibly be used as additives to functional foods and nutraceuticals targeted for patients with obesity-related metabolic disorders. To investigate the effects of fish protein hydrolysates on markers of metabolic disorders, obese Zucker fa/fa rats consumed diets with 75 % of protein from casein/whey (CAS) and 25 % from herring (HER) or salmon (SAL) protein hydrolysate from rest raw material, or 100 % protein from CAS for 4 weeks. The fatty acid compositions were similar in the experimental diets, and none of them contained any long-chain n-3 PUFA. Ratios of lysine:arginine and methionine:glycine were lower in HER and SAL diets when compared with CAS, and taurine was detected only in fish protein hydrolysate diets. Motifs with reported hypocholesterolemic or antidiabetic activities were identified in both fish protein hydrolysates. Rats fed HER diet had lower serum HDL-cholesterol and LDL-cholesterol, and higher serum TAG, MUFA and n-3:n-6 PUFA ratio compared with CAS-fed rats. SAL rats gained more weight and had better postprandial glucose regulation compared with CAS rats. Serum lipids and fatty acids were only marginally affected by SAL, but adipose tissue contained less total SFA and more total n-3 PUFA when compared with CAS. To conclude, diets containing hydrolysed rest raw material from herring or salmon proteins may affect growth, lipid metabolism, postprandial glucose regulation and fatty acid composition in serum and adipose tissue in obese Zucker rats.

Naphthalene biodegradation in temperate and arctic marine microcosms.

Naphthalene, the smallest polycyclic aromatic hydrocarbon (PAH), is found in abundance in crude oil, its major source in marine environments. PAH removal occurs via biodegradation, a key process determining their fate in the sea. Adequate estimation of PAH biodegradation rates is essential for environmental risk assessment and response planning using numerical models such as the oil spill contingency and response (OSCAR) model. Using naphthalene as a model compound, biodegradation rate, temperature response and bacterial community composition of seawaters from two climatically different areas (North Sea and Arctic Ocean) were studied and compared. Naphthalene degradation was followed by measuring oxygen consumption in closed bottles using the OxiTop(®) system. Microbial communities of untreated and naphthalene exposed samples were analysed by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. Three times higher naphthalene degradation rate coefficients were observed in arctic seawater samples compared to temperate, at all incubation temperatures. Rate coefficients at in situ temperatures were however, similar (0.048 day(-1) for temperate and 0.068 day(-1) for arctic). Naphthalene biodegradation rates decreased with similar Q10 ratios (3.3 and 3.5) in both seawaters. Using the temperature compensation method implemented in the OSCAR model, Q10 = 2, biodegradation in arctic seawater was underestimated when calculated from the measured temperate k1 value, showing that temperature difference alone could not predict biodegradation rates adequately. Temperate and arctic untreated seawater communities were different as revealed by pyrosequencing. Geographic origin of seawater affected the community composition of exposed samples.

Estimation of hydrocarbon biodegradation rates in marine environments: a critical review of the Q10 approach.

Offshore oil & gas industry is moving exploration and production activities into Arctic and deep water regions. Governmental regulations require environmental impact assessments before operations to evaluate the possible effects of accidental oil releases. These are often performed by numerical fate models, like the Oil Spill Contingency and Response (OSCAR) model, which has become an industry standard in Norway. In this model, biodegradation rates are adjusted to local conditions by temperature compensation according to a Q10 approach. Q10 is the multiplier by which rates of enzymatic reactions increase at a 10 °C temperature rise. Herein, this Q10 approach implemented in the OSCAR model is investigated based on published data and novel obtained results. Overall, biodegradation rate predictions calculated by temperature compensation are found to be questionable, and choosing one universal Q10 value is considered not feasible. The high variation in Q10 values is herein attributed to indirect effects of temperature.

Biomarker responses in Atlantic cod (Gadus morhua) exposed to produced water from a North Sea oil field: laboratory and field assessments.

Biological markers of produced water (PW) exposure were studied in Atlantic cod (Gadus morhua) in both laboratory and field experiments, using authentic PW from a North Sea oil field. In the laboratory study, the PW exposure yielded significantly elevated levels of metabolites of polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APs) in bile even at the lowest exposure dose (0.125% PW). Other biomarkers (hepatic CYP1A induction and DNA adduct formation) responded at 0.25% and 0.5% PW concentrations. In the field study, bile metabolite markers and hepatic CYP1A were clearly increased in fish caged close to the PW outfall. Induction of plasma vitellogenin was not found in laboratory or field exposures, suggesting that the levels of oestrogen agonists (such as APs) might not have been sufficient to elicit induction, under the present conditions. The applicability of the biomarkers for use in water column biomonitoring programs is discussed.

PAH body burden and biomarker responses in mussels (Mytilus edulis) exposed to produced water from a North Sea oil field: laboratory and field assessments.

In order to study the impact of produced water (PW) from a North Sea oil field on blue mussels (Mytilus edulis), chemical and biological markers were selected. A laboratory exposure (0.125%, 0.25% and 0.5% of PW) and a field study (6 stations 0.2-2 km from a PW discharge point) were conducted. In the laboratory study, PAH bioaccumulation increased in mussel soft tissue even at the lowest exposure dose. Micronuclei frequency demonstrated a dose-response pattern, whereas lysosomal membrane stability showed tendency towards a dose-response pattern. The same markers were assessed in the field study, biomarker analyses were consistent with the contamination level, as evaluated by mussel polycyclic aromatic hydrocarbons body burden. Overall, obtained results confirmed the value of an ecotoxicological approach for a scientifically sound characterisation of biological effects induced by offshore oilfield operational discharges.

Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions.

The aim of the present study was to standardize and to assess the predictive value of the cytogenetic analysis by MN test in fish erythrocytes as a biomarker for marine environmental contamination. MN frequency baseline in erythrocytes was evaluated in a number of fish species from a reference area (S. Teresa, La Spezia Gulf) and genotoxic potential of a number of common chemical contaminants and mixtures was determined in fish experimentally exposed in aquarium under controlled conditions. Fish (Scophthalmus maximus) were exposed for 3 weeks to 50 ppb of single chemicals (dialkyl phthalate, bisphenol A, tetrabromodiphenyl ether), 30 ppb nonylphenol and mixtures (North Sea oil and North Sea oil with alkylated phenols). Chromosomal damage was determined as micronuclei (MN) frequency in fish erythrocytes. Nuclear anomalies such as blebbed, notched and lobed nuclei were also recorded. Significant increase in MN frequency was observed in erythrocytes of fish exposed to bisphenol A and tetrabromodiphenylether. Chemical mixture North Sea oil+alkylated phenols induced the highest MN frequency (2.95 micronucleated cells/1000 cells compared to 1 MNcell/1000 cells in control animals). The study results revealed that micronucleus test, as an index of cumulative exposure, appears to be a sensitive model to evaluate genotoxic compounds in fish under controlled conditions.

The BEEP Stavanger Workshop: Mesocosm exposures.

Within the BEEP project (Biological Effects of Environmental Pollution in Marine Ecosystems) the Work Package 1 was addressed to the development of new and more sensitive biomarkers of exposure in several sentinel organisms. Within this framework, common mesocosm exposures of organic pollutants relevant for marine ecosystems were conducted in the facilities of Akvamiljø a/s (Stavanger, Norway). In the first experiment, Atlantic cod (Gadus morhua), turbot (Scophthalmus maximus) and shore crab (Carcinus maenas) were exposed to nonylphenol, North Sea crude oil and a combination of crude oil and alkylated phenols. Mussels (Mytilus edulis) were exposed to North Sea crude oil and a combination of crude oil, alkylated phenols and PAHs. In the second experiment, Atlantic cod, turbot, mussel and spider crab (Hyas araneus) were exposed to the plasticizers bisphenol A and diallyl phatalate and the brominated flame retardant BDE-47. The main purpose of the present study was to provide the 30 participating Institutes with samples which had been exposed to defined contaminant concentrations in a controlled laboratory exposure for 3 weeks. This paper describes the mesocosm experimental design, the transplantation and treatment of the organisms, and the contaminant exposures.