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Monitoring environmental status through molecular investigation of microorganisms in the marine environment is suggested as a potentially very effective method for biomonitoring, with great potential for automation. There are several hurdles to that approach with regards to primer design, variability across geographical locations, seasons, and type of environmental pollution. Here, qPCR analysis of genes involved in the initial activation of aliphatic and aromatic hydrocarbons were used in a laboratory setup mimicking realistic oil leakage at sea. Seawater incubation experiments were carried out under two different seasons with two different oil types. Degenerate primers targeting initial oxygenases (alkane 1-monooxygenase; alkB and aromatic-ring hydroxylating dioxygenase; ARHD) were employed in qPCR assays to quantify the abundance of genes essential for oil degradation. Shotgun metagenomics was used to map the overall community dynamics and the diversity of alkB and ARHD genes represented in the microbial community. The amplicons generated through the qPCR assays were sequenced to reveal the diversity of oil-degradation related genes captured by the degenerate primers. We identified a major mismatch between the taxonomic diversity of alkB and ARHD genes amplified by the degenerate primers and those identified through shotgun metagenomics. More specifically, the designed primers did not amplify the alkB genes of the two most abundant alkane degraders that bloomed in the experiments, Oceanobacter and Oleispira. The relative abundance of alkB sequences from shotgun metagenomics and 16S rRNA-based Oleispira-specific qPCR assay were better signals for oil in water than the tested qPCR alkB assay. The ARHD assay showed a good agreement with PAHs degradation despite covering only 25% of the top 100 ARHD genes and missing several abundant Cycloclasticus sequences that were present in the metagenome. We conclude that further improvement of the degenerate primer approach is needed to rely on the use of oxygenase-related qPCR assays for oil leakage detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Oil spill clean-up measures using in situ burning can potentially result in seafloor contamination affecting benthic organisms. To mimic realistic exposure and measure effects, ovigerous Northern shrimp were continuously exposed for two weeks to the water-soluble fraction of oil coated on gravel followed by two weeks in clean seawater. North Sea crude oil (NSC) and field generated in situ burn residue (ISBR) of NSC were used (Low: 3 g/kg gravel, Medium: 6 g/kg gravel and High: 12 g/kg gravel). The concentrations of polyaromatic hydrocarbons (PAHs) in the water resulting from NSC were higher compared to ISBR. No mortality was observed in any treatment and overall moderate sublethal effects were found, mostly after exposure to NSC. Feeding was temporarily reduced at higher concentrations of NSC. PAH levels in hepatopancreas tissue were significantly elevated following exposure and still significantly higher at the end of the experiment in NSC High and ISBR High compared to control. Mild inflammatory response reactions and tissue ultrastructural alterations in gill tissue were observed in both treatments. Signs of necrosis occurred in ISBR High . No change in shrimp locomotory activity was noted from NSC exposure. However, ISBR exposure increased activity temporarily. Larvae exposed as pleopod-attached embryos showed significant delay in development from stage I to stage II after exposure to NSC High . Based on this study, oil-contaminated seafloor resulting from in situ burning clean-up actions does not appear to cause serious effects on bottom-living shrimp., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Environmental DNA (eDNA) is a novel, non-invasive sampling procedure that allows the obtaining of genetic material directly from environmental samples without any evidence of biological sources. The eDNA methodology can greatly benefit from coupling it to reliable, portable and cost-effective tools able to perform decentralized measurements directly at the site of need and in resource-limited settings. Herein, we report a simple method for the selective analysis of eDNA using a magneto-assay with electrochemical detection. The proposed method involves the polymerase chain-reaction (PCR) amplification of mitochondrial eDNA of parasitic Salmon lice (Lepeophtheirus salmonis), extracted from seawater samples. The eDNA sequence was targeted via sandwich hybridization onto magnetic beads and enzymatic labeling was performed to obtain an electroactive product measured by differential pulse voltammetry. Quality by Design (QbD), a recent concept of science- and risk-oriented quality paradigm, was used for the optimization of the different parameters of the assay. Response surface methodology and Monte Carlo simulations were performed to define the method operable design region. The optimized electrochemical magneto-assay attained a limit of detection of 2.9 amol μL -1 of the short synthetic sea louse DNA analogue (43 bp). In addition, robustness testing using a further experimental design approach was performed for monitoring eDNA amplicons. Seawater samples spiked with individuals of free-swimming L. salmonis copepodite stages and seawater collected from tanks with sea lice-infested fish were analyzed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In situ burning (ISB) is an oil spill clean-up option used by oil spill responders to mitigate impacts on the marine environment. Despite advantages such as high efficiency and potential applicability for challenging areas such as the Arctic, the actual environmental side effects are still uncertain. Acute and sublethal effects of the water accommodated fractions (WAFs from 25 g oil/L seawater) of a pre-weathered North Sea crude (Oseberg Blend 200 °C+) and field generated ISB residue were evaluated on Northern shrimp (Pandalus borealis) larvae. The larvae were first exposed for 96 h to a serial dilution of seven concentrations, and then maintained for two weeks in clean seawater post-exposure. No acute (mortality) or sublethal effects (feeding, development, or growth) were detected in any of the ISB residue concentrations. Significant larvae mortality was found in the three highest concentrations of crude oil (96-h LC50:469 μg/L total petroleum hydrocarbon) but no sublethal effects were found in the surviving larvae post-exposure. This study indicates that applying ISB could mitigate acute impacts of spilled oil on shrimp larvae., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Today's benthic offshore biological monitoring of oil & gas (O&G) activities relies on macrofauna taxa enumeration. For the future, analysis of DNA isolated directly from sediments holds great potential for multi-trophic biodiversity surveys and the monitoring of a larger spectrum of benthic taxa, including micro-fauna. Here, we evaluate more specifically the potential of microfauna-specific gene quantification in relation to both petroleum-related discharge compounds and other seafloor environmental properties. We carried out this evaluation using sediment samples collected at drilling Region III on the Norwegian continental shelf where DNA metabarcoding of eukaryotic diversity was already performed. Generally, the quantification of microfauna indicator taxa related well to the gradient of contamination on the seafloor. Contrary to eukaryotic Euplotida, metabarcoding data and qPCR numbers for indicative prokaryotic taxa showed the same relationship to offshore contaminants (both showed positive relationship). We found absolute numbers of SSU rRNA gene copies of (1) Dinophyceae, Bacillariophyceae and Alcanivorax were correlated with the level of petroleum-related compounds but not with other environmental variables, (2) bacteria closely related to Shewanella were correlated with the concentration of Ba, PAH, as well to percent of gravel, (3) Desulfobacteriales correlated with petroleum-related contaminants, but as well with percent of gravel and grain size. Findings from our study suggest that biomonitoring surveys of O&G activities on benthos could benefit from quantification of specific micro-fauna indicators that is simpler and faster than the methods currently used for impact assessment of benthos., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The application of chemical dispersants is one option of oil spill response (OSR). Here, Northern shrimp (Pandalus borealis) larvae were experimentally exposed for short periods (6 h and 1 h) to a realistic concentration of chemically dispersed oil (CDO) (~10 mg L -1 THC), mechanically dispersed oil (MDO) (~7 mg L -1 THC), and dispersant only (D). A control (C) with seawater served as reference. Short-term effects on survival and feeding were examined right after exposure and longer-term consequences on survival, feeding, growth and development following 30 days of recovery. Both exposure durations provoked long lasting effects on larval fitness, with 1 h exposure leading to minor effects on most of the selected endpoints. The 6 h exposure affected all endpoints with more adverse impacts after exposure to CDO. This study provides important data for assessing the best OSR option relevant to NEBA (Net Environmental Benefit Analysis)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In this paper, a molecular analytical approach for detecting hydrocarbonoclastic bacteria in water is suggested as a proxy measurement for tracking petroleum discharges in industrialized or pristine aquatic environments. This approach is tested for general application in cold marine regions (freezing to 5 °C). We used amplicon sequencing and qPCR to quantify 16S rRNA and GyrB genes from oleophilic bacteria in seawater samples from two different crude oil enrichments. The first experiment was conducted in a controlled environment using laboratory conditions and natural North Sea fjord seawater (NSC) at a constant temperature of 5 °C. The second was performed in the field with natural Arctic seawater (ARC) and outdoor temperature conditions from -7 °C to around 4 °C. Although the experimental conditions for NSC and ARC differed, the temporal changes in bacterial communities were comparable and reflected oil biotransformation processes. The common bacterial OTUs for NSC and ARC had the highest identity to Colwellia rossensis and Oleispira antarctica rRNA sequences and were enriched within a few days in both conditions. Other typical oil degrading bacteria such as Alcanivorax (n-alkane degrader) and Cycloclasticus (polycyclic aromatic hydrocarbons degrader) were rapidly enriched only in NSC conditions. Both the strong correlation between Oleispira SSU gene copies and oil concentration, and the specificity of the Oleispira assay suggest that this organism is a robust bioindicator for seawater contaminated by petroleum in cold water environments. Further optimization for automation of the Oleispira assay for in situ analysis with a genosensing device is underway. The assay for Colwellia quantification requires more specificity to fewer Colwellia OTUs and a well-established dose-response relationship before those taxa are used for oil tracking purposes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Knowledge of key species sensitivity for oil spill response (OSR) options is needed to support decision-making and mitigate impact on sensitive life stages of keystone species. Here, Northern shrimp (Pandalus borealis) larvae were exposed for 24 h to a gradient (H-High, M-Medium: 10 times dilution and L-Low: 100 times dilution) of mechanically- (MDO) (H < 6 mg/L total hydrocarbon content) and chemically- (CDO) dispersed oil (Slickgone NS, H < 20 mg/L total hydrocarbon content), followed by a recovery period. Larval mortality, feeding rate and development were evaluated. Overall, the results show that 24 h exposure to field-realistic concentrations of CDO lead to lower survival, reduced feeding rate and slower larval development in P. borealis larvae compared to MDO. These effects persisted during recovery, indicating a higher vulnerability with dispersant use and the need for longer observation periods post-exposure to fully evaluate the consequences for sensitive life-stages from OSR., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mussels (Mytilus edulis) were continuously exposed to dispersed crude oil (0.015-0.25mg/l) for 7 months covering the whole gamete development cycle. After 1 month exposure to 0.25 mg oil/l, the level of alkali-labile phosphates (ALP) and the volume density of atretic oocytes in female gonads were higher than those in the gonads of control females, indicating that oil affected the level of vitellogenin-like proteins and gamete development. Spawning of mussels was induced after 7 months oil exposure. Parental oil exposure did not affect subsequent fertilization success in clean seawater but this was reduced in 0.25 mg oil/l. Parental exposure to 0.25 mg oil/l caused both slow development and a higher percentage of abnormalities in D-shell larvae 2 days post-fertilization; reduced growth 7 days post-fertilization. These effects were greatly enhanced when larval stages were maintained at 0.25 mg oil/l. Similar studies are warranted for risk assessment prognosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The sensitivity of different tissues for assessment of chronic low-dose environmental exposure of fish to alkylphenols (APs) was investigated. We exposed Atlantic cod (Gadus morhua) in the laboratory to tritium labelled 4-tert-butylphenol, 4n-pentylphenol, 4n-hexylphenol, and 4n-heptylphenol via seawater (8 ng/l) and via contaminated feed (5 microg/kg fish per day). Measurements of different fish tissues during eight days of exposure and eight subsequent days of recovery revealed that APs administered via spiked seawater were readily taken up whereas the uptake was far less efficient when APs were administered in spiked feed. AP residues were mainly located in the bile fluid whereas the concentrations in liver were very low, indicating a rapid excretion and the liver-bile axis to be the major route of elimination. The biological half-life of APs in the exposed cod was short, between 10 and 20 h. Our study shows that in connection with biomonitoring of AP exposure in fish, assessment of AP metabolites in bile fluid is a more sensitive tool than detection of parent AP levels in liver or other internal tissues.

The single-cell microgel electrophoresis assay or the comet assay was used to evaluate DNA damage of dispersed crude oil on sea urchins (Strongylocentrotus droebachiensis) and mussels (Mytilus edulis L.). Sea urchins were exposed to 0.06 and 0.25 mg/L dispersed crude oil in a continuous flow system, while the mussels were exposed to 0.015, 0.06 and 0.25 mg/L dispersed crude oil. Sea urchin coelomocytes and mussel haemocytes were sampled after 4 and 5 weeks exposure, respectively. In the sea urchin coelomocytes, there was a significant concentration-related increase in the percentage of DNA in comet tail. In mussel haemocytes, there was a significantly higher percentage of DNA in comet tail for all treatments compared to the control. The responses were concentration-related up to 0.06 mg/L oil. The two highest exposure concentrations of mussels were not significantly different from each other. These results indicate that the comet assay can be used for biomonitoring of DNA damage in marine invertebrates following oil contamination.