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The naturally occurring ectoparasite salmon lice (Lepeophtherirus salmonis) poses a great challenge for the salmon farming industry, as well as for wild salmonids in the Northern hemisphere. To better control the infestation pressure and protect the production, there is a need to provide fish farmers with sensitive and efficient tools for rapid early detection and monitoring of the parasitic load. This can be achieved by targeting L. salmonis DNA in environmental samples. Here, we developed and tested a new L. salmonis specific DNA-based assay (qPCR assay) for detection and quantification from seawater samples using an analytical pipeline compatible with the Environmental Sample Processor (ESP) for autonomous water sample analysis of gene targets. Specificity of the L. salmonis qPCR assay was demonstrated through in-silico DNA analyses covering sequences of different L. salmonis isolates. Seawater was spiked with known numbers of nauplii and copepodite free-swimming (planktonic) stages of L. salmonis to investigate the relationship with the number of marker gene copies (MGC). Finally, field samples collected at different times of the year in the vicinity of a salmon production farm in Western Norway were analyzed for L. salmonis detection and quantification. The assay specificity was high and a high correlation between MGC and planktonic stages of L. salmonis was established in the laboratory conditions. In the field, L. salmonis DNA was consequently detected, but with MGC number below that expected for one copepodite or nauplii. We concluded that only L. salmonis tissue or eDNA residues were detected. This novel study opens for a fully automatized L. salmonis DNA quantification using ESP robotic to monitor the parasitic load, but challenges remain to exactly transfer information about eDNA quantities to decisions by the farmers and possible interventions., Competing Interests: The authors have declared that no competing interests exist.

Hyperspectral imaging (HSI) is a promising technology for environmental monitoring with a lot of undeveloped potential due to the high dimensionality and complexity of the data. If temporal effects are studied, such as in a monitoring context, the analysis becomes more challenging as time is added to the dimensions of space (image coordinates) and wavelengths. We conducted a series of laboratory experiments to investigate the impact of different stressor exposure patterns on the spectrum of the cold water coral Desmophyllum pertusum. 65 coral samples were divided into 12 groups, each group being exposed to different types and levels of particles. Hyperspectral images of the coral samples were collected at four time points from prior to exposure to 6 weeks after exposure. To investigate the relationships between the corals' spectral signatures and controlled experimental parameters, a new software tool for interactive visual exploration was developed and applied, the HypIX (Hyperspectral Image eXplorer) web tool. HypIX combines principles from exploratory data analysis, information visualization and machine learning-based dimension reduction. This combination enables users to select regions of interest (ROI) in all dimensions (2D space, time point and spectrum) for a flexible integrated inspection. We propose two HypIX workflows to find relationships in time series of hyperspectral datasets, namely morphology-based filtering workflow and embedded driven response analysis workflow. With these HypIX workflows three users identified different temporal and spatial patterns in the spectrum of corals exposed to different particle stressor conditions. Corals exposed to particles tended to have a larger change rate than control corals, which was evident as a shifted spectrum. The responses, however, were not uniform for coral samples undergoing the same exposure treatments, indicating individual tolerance levels. We also observed a good inter-observer agreement between the three HyPIX users, indicating that the proposed workflow can be applied to obtain reproducible HSI analysis results., Competing Interests: The study was financed by Equinor. Equinor Ventures is one of the main shareholders of Ecotone AS. IMH is a minor shareholder of Ecotone AS. Ecotone AS is the owner of patent no. NO/EP2286194 titled “Underwater Hyperspectral Imaging“. Ecotone sells scientific instruments for underwater use under the product name Underwater Hyperspectral Imager (UHI). Ecotone AS has two pending patent applications, IMH is involved as inventor. Equinor funded a project at the Biodata Mining Group, but these funding was in no way linked to the outcome of this study. This does not alter our adherence to all the policies on sharing data and materials. All other authors declare no competing interests.

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.)

Cold-water coral (CWC) reefs are numerous and widespread along the Norwegian continental shelf where oil and gas industry operate. Uncertainties exist regarding their impacts from operational discharges to drilling. Effect thresholds obtained from near-realistic exposure of suspended particle concentrations for use in coral risk modeling are particularly needed. Here, nubbins of Desmophyllum pertusum (Lophelia pertusa) were exposed shortly (5 days, 4h repeated pulses) to suspended particles (bentonite BE; barite BA, and drill cuttings DC) in the range of ~ 4 to ~ 60 mg.l-1 (actual concentration). Physiological responses (respiration rate, growth rate, mucus-related particulate organic carbon OC and particulate organic nitrogen ON) and polyp mortality were then measured 2 and 6 weeks post-exposure to assess long-term effects. Respiration and growth rates were not significantly different in any of the treatments tested compared to control. OC production was not affected in any treatment, but a significant increase of OC:ON in mucus produced by BE-exposed (23 and 48 mg.l-1) corals was revealed 2 weeks after exposure. Polyp mortality increased significantly at the two highest DC doses (19 and 49 mg.l-1) 2 and 6 weeks post-exposure but no significant difference was observed in any of the other treatments compared to the control. These findings are adding new knowledge on coral resilience to short realistic exposure of suspended drill particles and indicate overall a risk for long-term effects at a threshold of ~20 mg.l-1., Competing Interests: This study and the writing of the manuscript was financed by Equinor AS. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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 use of natural marine bacteria as "oil sensors" for the detection of pollution events can be suggested as a novel way of monitoring oil occurrence at sea. Nucleic acid-based devices generically called genosensors are emerging as potentially promising tools for in situ detection of specific microbial marker genes suited for that purpose. Functional marker genes are particularly interesting as targets for oil-related genosensing but their identification remains a challenge. Here, seawater samples, collected in tanks with oil addition mimicking a realistic oil spill scenario, were filtered and archived by the Environmental Sample Processor (ESP), a fully robotized genosensor, and the samples were then used for post-retrieval metatranscriptomic analysis. After extraction, RNA from ESP-archived samples at start, Day 4 and Day 7 of the experiment was used for sequencing. Metatranscriptomics revealed that several KEGG pathways were significantly enriched in samples exposed to oil. However, these pathways were highly expressed also in the non-oil-exposed water samples, most likely as a result of the release of natural organic matter from decaying phytoplankton. Temporary peaks of aliphatic alcohol and aldehyde dehydrogenases and monoaromatic ring-degrading enzymes (e.g., ben , box , and dmp clusters) were observed on Day 4 in both control and oil-exposed and non-exposed tanks. Few alkane 1-monooxygenase genes were upregulated on oil, mostly transcribed by families Porticoccaceae and Rhodobacteraceae , together with aromatic ring-hydroxylating dioxygenases, mostly transcribed by Rhodobacteraceae . Few transcripts from obligate hydrocarbonoclastic genera of Alcanivorax , Oleispira and Cycloclasticus were significantly enriched in the oil-treated exposed tank in comparison to control the non-exposed tank, and these were mostly transporters and genes involved in nitrogen and phosphorous acquisition. This study highlights the importance of seasonality, i.e., phytoplankton occurrence and senescence leading to organic compound release which can be used preferentially by bacteria over oil compounds, delaying the latter process. As a result, such seasonal effect can reduce the sensitivity of genosensing tools employing bacterial functional genes to sense oil. A better understanding of the use of natural organic matter by bacteria involved in oil-biodegradation is needed to develop an array of functional markers enabling the rapid and specific in situ detection of anthropogenic pollution., Competing Interests: The authors declare no conflict of interest.

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.)

Ocean warming (OW) and acidification (OA) are key features of global change and are predicted to have negative consequences for marine species and ecosystems. At a smaller scale increasing oil and gas activities at northern high latitudes could lead to greater risk of petroleum pollution, potentially exacerbating the effects of such global stressors. However, knowledge of combined effects is limited. This study employed a scenario-based, collapsed design to investigate the impact of one local acute stressor (North Sea crude oil) and two chronic global drivers (pH for OA and temperature for OW), alone or in combination on aspects of the biology of larval stages of two key invertebrates: the northern shrimp (Pandalus borealis) and the green sea urchin (Strongylocentrotus droebachiensis). Both local and global drivers had negative effects on survival, development and growth of the larval stages. These effects were species- and stage-dependent. No statistical interactions were observed between local and global drivers and the combined effects of the two drivers were approximately equal to the sum of their separate effects. This study highlights the importance of adjusting regulation associated with oil spill prevention to maximize the resilience of marine organisms to predicted future global conditions.

A portable surface plasmon resonance (SPR) instrument was tested for the first time for the detection of oligonucleotide sequences derived from the 16S rRNA gene of Oleispira antarctica RB-8, a bioindicator species of marine oil contamination, using morpholino-functionalized sensor surfaces. We evaluated the stability and specificity of morpholino coated sensor surfaces and tested two signal amplification regimes: (1) sequential injection of sample followed by magnetic bead amplifier and (2) a single injection of magnetic bead captured oligo. We found that the sensor surfaces could be regenerated for at least 85 consecutive sample injections without significant loss of signal intensity. Regarding specificity, the assay clearly differentiated analytes with only one or two mismatches. Signal intensities of mismatch oligos were lower than the exact match target at identical concentrations down to 200 nM, in standard phosphate buffered saline with 0.1 % Tween-20 added. Signal amplification was achieved with both strategies; however, significantly higher response was observed with the sequential approach (up to 16-fold), where first the binding of biotin-probe-labeled target oligo took place on the sensor surface, followed by the binding of the streptavidin magnetic beads onto the immobilized targets. Our experiments so far indicate that a simple coating procedure in combination with a relatively cost-efficient magnetic-bead-based signal amplification will provide robust SPR based nucleic acid sensing down to 0.5 nM of a 45-nucleotide long oligo target (7.2 ng/mL).

This study aimed at providing confidence in the predictability of the impacts of drill cuttings (DC) discharge on the cold-water coral Lophelia pertusa. L. pertusa was exposed to DC from offshore exploration in the lab with the goal to assess precautionary thresholds of effects. Two exposure scenarios with DC were tested: a long-term (LT) pulsed exposure (12 weeks, peak concentrations: 2-50 mg/L, mean concentrations: 1-25 mg/L) and a short-term (ST) continuous exposure (2.5 weeks, mean concentrations: 4-42 mg/L). After exposure, a recovery period of 16 and 4 weeks was maintained in LT and ST, respectively. While there was an assumption that DC might result in an increase in respiration, decrease in growth, enhanced mucus production, reduced fatty acid content, only a significant rise was noted in skeleton growth at DC 4 mg/L and a significant increase of mucus particulate organic carbon at 25 mg/L at end of the exposure. DC did not markedly reduce prey capture rate consecutive to DC exposure. However, the effect of DC produced an increase of coral polyp activity during exposure and a return to pre-exposure conditions after cessation of DC, and coenosarc was smothered from DC even after a long recovery period (4 weeks). Overall, a DC concentration of 10 mg/L seems to represent a threshold above which changes in coral conditions were observed however with no apparent physiological consequences for the coral within the experimental time scale.

The results of marine bacterial community succession from a short-term study of seawater incubations at 4°C to North Sea crude oil are presented herein. Oil was used alone (O) or in combination with a dispersant (OD). Marine bacterial communities resulting from these incubations were characterized by a fingerprinting analysis and pyrosequencing of the 16S rRNA gene with the aim of 1) revealing differences in bacterial communities between the control, O treatment, and OD treatment and 2) identifying the operational taxonomic units (OTUs) of early responders in order to define the bacterial gene markers of oil pollution for in situ monitoring.After an incubation for 1 d, the distribution of the individual ribotypes of bacterial communities in control and oil-treated (O and OD) tanks differed. Differences related to the structures of bacterial communities were observed at later stages of the incubation. Among the early responders identified (Pseudoalteromonas, Sulfitobacter, Vibrio, Pseudomonas, Glaciecola, Neptunomonas, Methylophaga, and Pseudofulvibacter), genera that utilize a disintegrated biomass or hydrocarbons as well as biosurfactant producers were detected. None of these genera included obligate hydrocarbonoclastic bacteria (OHCB). After an incubation for 1 d, the abundances of Glaciecola and Pseudofulvibacter were approximately 30-fold higher in the OD and O tanks than in the control tank. OTUs assigned to the Glaciecola genus were represented more in the OD tank, while those of Pseudofulvibacter were represented more in the O tank. We also found that 2 to 3% of the structural community shift originated from the bacterial community in the oil itself, with Polaribacter being a dominant bacterium.

Despite the importance of the cold-water coral Lophelia pertusa to deep-sea reef ecosystem functioning, current knowledge of key physiological responses to available food resources is scarce. Scenarios with varying food density may help to understand how corals deal with seasonal variations in the dark ocean and might be used to study consequences of anthropogenic activities potentially affecting food availability. Thus, the physiological responses of L. pertusa to varying food (Artemia salina nauplii) concentration, ranging from 20% to 300% of carbon equivalent turned over by basal coral respiration, were investigated. A starvation group was also included. Measurements of respiration, growth, mucus production, and energy reserves (storage fatty acids) were performed at several time intervals over 26 weeks. In general, data showed a stronger effect of experimental time on measured responses, but no significant influence of food density treatment. In starved corals, respiration rate declined to 52% of initial respiration, while skeleton growth rate was maintained at the same rate as Artemia-fed corals throughout the investigation. Mucus production measured as the sum of dissolved organic carbon (DOC) and particulate organic carbon (POC) was also similar across food treatments, but POC production exceeded that of DOC at the highest food density. No marked effect was observed on storage fatty acids. These results confirm that L. pertusa is highly resilient to environmental conditions with suboptimal food densities over a time scale of months. Regulation of several physiological processes, including respiration and mucus production, possibly in combination with an opportunistic feeding strategy, contributed to this tolerance to maintain viable corals. Thus, it appears that L. pertusa is well adapted to life in the deep sea.

Lophelia pertusa is the dominant reef-building organism of cold-water coral reefs, and is known to produce significant amounts of mucus, which could involve an important metabolic cost. Mucus is involved in particle removal and feeding processes, yet the triggers and dynamics of mucus production are currently still poorly described because the existing tools to study these processes are not appropriate. Using a novel microscopic technique-digital holographic microscopy (DHM)-we studied the mucus release of L. pertusa under various experimental conditions. DHM technology permits μm-scale observations and allows the visualization of transparent mucoid substances in real time without staining. Fragments of L. pertusa were first maintained in flow-through chambers without stressors and imaged with DHM, then exposed to various stressors (suspended particles, particulate food and air exposure) and re-imaged. Under non-stressed conditions no release of mucus was observed, whilst mucus strings and sheaths were produced in response to suspended particles (activated charcoal and drill cuttings sediment) i.e. in a stressed condition. Mucus strings and so-called 'string balls' were also observed in response to exposure to particulate food (brine shrimp Artemia salina). Upon air-exposure, mucus production was clearly visible once the fragments were returned to the flow chamber. Distinct optical properties such as optical path length difference (OPD) were measured with DHM in response to the various stimuli suggesting that different mucus types are produced by L. pertusa. Mucus produced to reject particles is similar in refractive index to the surrounding seawater, suggesting that the energy content of this mucus is low. In contrast, mucus produced in response to either food particle addition or air exposure had a higher refractive index, suggesting a higher metabolic investment in the production of these mucoid substances. This paper shows for the first time the potential of DHM technology for the detection, characterization and quantification of mucus production through OPD measurements in L. pertusa.

For the environmental monitoring of coral, mucus appears to be an appropriate biological matrix due to its array of functions in coral biology and the non-intrusive manner in which it can be collected. The aim of the present study was to evaluate the feasibility of using mucus of the stony coral Lophelia pertusa (L. pertusa) as an analytical matrix for discovery of biomarkers used for environmental monitoring. More specifically, to assess whether a mass-spectrometry-based proteomic approach can be applied to characterize the protein composition of coral mucus and changes related to petroleum discharges at the seafloor. Surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) screening analyses of orange and white L. pertusa showed that the mucosal protein composition varies significantly with color phenotype, a pattern not reported prior to this study. Hence, to reduce variability from phenotype difference, L. pertusa white individuals only were selected to characterize in more detail the basal protein composition in mucus using liquid chromatography, mass spectrometry, mass spectrometry (LC-MS/MS). In total, 297 proteins were identified in L. pertusa mucus of unexposed coral individuals. Individuals exposed to drill cuttings in the range 2 to 12 mg/L showed modifications in coral mucus protein composition compared to unexposed corals. Although the results were somewhat inconsistent between individuals and require further validation in both the lab and the field, this study demonstrated preliminary encouraging results for discovery of protein markers in coral mucus that might provide more comprehensive insight into potential consequences attributed to anthropogenic stressors and may be used in future monitoring of coral health.

Biomarkers have the potential to be used to assess the impact of anthropogenic discharges in marine waters. We have used a suite of biomarkers spanning from enzymatic to histopathological alterations and general stress responses to assess the short- and long-term impact on mussels Mytilus edulis of heavy fuel oil no. 6 and styrene. Mussels were exposed for 5 months, with a refilling of the exposure system, to a water soluble fraction of heavy fuel and, then, kept for a month in clean water for recovery. In a second experiment, mussels were exposed to styrene for 19 days and maintained in clean water for up to 4 months. Chemical body tissue levels reflected the weathering processes of these compounds. Acyl-CoA oxidase activity was induced in oil-exposed mussels after refilling, whereas styrene inhibited it after 19 days of exposure and after 2 weeks in clean water. Gamete development and alkali-labile phosphate levels suggest that neither oil nor styrene behaved as endocrine disruptors. Neutral red retention time was lower in treated groups than in controls. Lysosomal membrane stability was significantly reduced in exposed groups and recovered after withdrawal of oil but not after removal of styrene. Neither oil nor styrene exposure affected the condition index except for the reduction seen in mussels exposed to oil for 1 month. Biomarker response index discriminated exposed mussels, which showed higher values, and returned to control levels after recovery. Results obtained from these pilot experiments can help to identify relevant monitoring tools to assess the impact of oil and chemicals in marine spill scenarios.

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.)

Ocean acidification (OA) resulting from anthropogenic emissions of carbon dioxide (CO(2)) has already lowered and is predicted to further lower surface ocean pH. There is a particular need to study effects of OA on organisms living in cold-water environments due to the higher solubility of CO(2) at lower temperatures. Mussel larvae (Mytilus edulis) and shrimp larvae (Pandalus borealis) were kept under an ocean acidification scenario predicted for the year 2100 (pH 7.6) and compared against identical batches of organisms held under the current oceanic pH of 8.1, which acted as a control. The temperature was held at a constant 10°C in the mussel experiment and at 5°C in the shrimp experiment. There was no marked effect on fertilization success, development time, or abnormality to the D-shell stage, or on feeding of mussel larvae in the low-pH (pH 7.6) treatment. Mytilus edulis larvae were still able to develop a shell in seawater undersaturated with respect to aragonite (a mineral form of CaCO(3)), but the size of low-pH larvae was significantly smaller than in the control. After 2 mo of exposure the mussels were 28% smaller in the pH 7.6 treatment than in the control. The experiment with Pandalus borealis larvae ran from 1 through 35 days post hatch. Survival of shrimp larvae was not reduced after 5 wk of exposure to pH 7.6, but a significant delay in zoeal progression (development time) was observed.

Polar marine surface waters are characterized by high levels of dissolved oxygen, seasonally intense UV irradiance and high levels of dissolved organic carbon. Therefore, the Arctic sea-ice habitat is regarded as a strongly pro-oxidant environment, even though its significant ice cover protects the ice-associated (=sympagic) fauna from direct irradiation to a large extent. In order to investigate the level of resistance to oxyradical stress, we sampled the sympagic amphipod species Gammarus wilkitzkii during both winter and summer conditions, as well as exposed specimens to simulated levels of near-natural and elevated levels of UV irradiation. Results showed that this amphipod species possessed a much stronger antioxidant capacity during summer than during winter. Also, the experimental UV exposure showed a depletion in antioxidant defences, indicating a negative effect of UV exposure on the total oxyradical scavenging capacity. Another sympagic organism, Onisimus nanseni, was sampled during summer conditions. When compared to G. wilkitzkii, the species showed even higher antioxidant scavenging capacity.

Biomarkers are widely used to measure environmental impacts on marine species. For many biomarkers, it is not clear how the signal levels relate to effects on the whole organism. This paper shows how species sensitivity distributions (SSDs) can be applied to evaluate multiple biomarker responses in species assemblages. To our knowledge, the present study compared for the first time SSDs based on biomarker response levels for marine species to a SSD for whole-organism responses. The comparison indicates that for exposure to dispersed oil in the marine environment, the selected biomarkers were, on average, 35- to 50-fold more sensitive than the whole-organism effect. At the 5% hazardous concentration derived from the SSD for whole-organism effects, which is a conservative threshold level, the potentially affected fraction of species showing biomarker response corresponds to approximately 80%. Variation in species sensitivity, expressed either as biomarker or as whole-organism response levels, were similar. Although uncertainties exist, the link between biomarkers and risk assessment presented here provides a preliminary guideline for deciding when biomarker responses likely are hazardous and, therefore, require further investigation.

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.

Despite the extensive transport of chemicals at sea, there is current lack of knowledge of the fate and effects of many of them on the marine biota. The current regulation that follows the GESAMP-MARPOL classification is mainly based on ecotoxicity assessment from fresh water based studies. Repetitive spills in marine coastal environment from tanker ship loaded with several thousand tonnes of chemicals raised concern about whether the existing freshwater data location can be used to predict the behaviour and the environmental effects of contaminants in marine surroundings. There is a general lack of information of the fate of chemicals at sea. A deviating pattern in marine environment from that in freshwater may have significant consequences for the counteracting actions taken to fight the spill, on staff working on the site of spill as well as on marine life present in the vicinity of the accident. In the present article, an environmental effect study of styrene was conducted as part of the ECOPEL program. We report some biological effects of styrene in laboratory-exposed marine organisms. Styrene was continuously supplied at a nominal concentration of 2mg L(-1) over 7 days to both mussels (Mytilus edulis) and fish (Symphodus mellops). At the end of this period, DNA damage was assessed by the Comet assay performed on blood (fish) and haemolymph (mussel) cells. In mussels, the lysosomal membrane stability was additionally assessed by the neutral red retention time assay (NRRT). Significant biological responses were observed over the studied period in both organisms with these two tests. Hence, the results favour the use of a biomarker-based approach to assess the health conditions in case of spill.

Blood plasma and serum are very useful samples for the detection, identification and quantitation of proteins associated with both health and disease. However, analysis of plasma and serum is a challenge because traces of interesting polypeptides and proteins can be dominated by the very high concentration of albumin present. Albumin may be depleted by adsorption to immunoaffinity columns or to columns containing dyes such as Cibacron Blue, or by ultrafiltration, but these methods are far from ideal. We describe a new peptide-based affinity medium which is effective for removing albumin and is very specific. The albumin-binding capacity is at least 14 mg per mL of gel. The material may be reused hundreds of times after a simple regeneration step involving NaOH, with full retention of specificity and capacity. The material was tested with human and monkey plasma and serum and rat serum, and has been used to deplete litre volumes of human plasma. The development of other peptide-based affinity media to deplete abundant proteins is briefly discussed.

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.

We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g. by taking advantage of the powerful laboratory information management system we developed. The differential capacity of our platform is validated by detecting known markers in a real sample and by a spiking experiment. We introduce an innovative two-dimensional (2-D) plot for displaying identification results combined with chromatographic data. This 2-D plot is very convenient for detecting differential proteins. We also adapt standard multivariate statistical techniques to show that peptide identification scores can be used for reliable and sensitive differential studies. The interest of the protein separation approach we generally apply is justified by numerous statistics, complemented by a comparison with a simple shotgun analysis performed on a small volume sample. By introducing an automatic integration step after mass spectrometry data identification, we are able to search numerous databases systematically, including the human genome and expressed sequence tags. Finally, we explain how rigorous data processing can be combined with the work of human experts to set high quality standards, and hence obtain reliable (false positive < 0.35%) and nonredundant protein identifications.

Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.

Sheepshead minnows (Cyprinodon variegatus) were continuously exposed to two concentrations of polycyclic aromatic hydrocarbons (PAHs) dissolved in seawater (sigma PAH = 7.57 and 72.31 microg/L) for 36 d, followed by 8 d of depuration. The PAHs studied were naphthalene (NPH or C0-NPH), phenanthrene (PHE or C0-PHE), pyrene (PYR), 2-methylnaphthalene (C1-NPH), 1,3-dimethylnaphthalene (C2-NPH), 2-isopropylnaphthalene (C3-NPH), 9-methylphenanthrene (C1-PHE), and 9-ethylphenanthrene (C2-PHE). Uptake rate constants (k1) for NPHs increased with increasing degree of alkylation and log value of the octanol/water partition coefficient (Kow), whereas k1 values for three- and four-ring PAHs were lower despite their high log Kow values. Elimination rate constants (k2) for the homologue series of NPHs and PHEs generally increased with decreasing degree of alkylation and log Kow values. However, the depuration time did not directly correlate with the molecular size for nonalkylated PAHs. Bioconcentration factors (BCFs) were estimated from the ratio of k1 to k2 and also directly from PAH concentrations in fish tissue and water samples, and the factors generated by the two methods were very similar. A significant positive correlation was determined between log BCFs and log Kow values for the series of C0- through C3-NPH at both low (r2 = 0.985, p = 0.0077) and high (r2 = 0.956, p = 0.022) exposures, although this correlation was not determined for all the PAHs studied. As a result of increased metabolism and/ or reduced bioavailability with increasing lipophilic character, the estimated BCFs for C0- through C2-PHE and PYR were generally lower than those for C0- through C3-NPH. The two exposure levels revealed minor variations in k1 and k2 values for parent PAHs and in the temporal pattern of PAH metabolite concentrations in bile. The present results indicate that the presence and nature of alkyl groups have a significant influence on the kinetics and metabolism of PAHs in fish.