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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. publishedVersion

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 targetingL. salmonisDNA in environmental samples. Here, we developed and tested a newL. salmonisspecific 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 differentL. salmonisisolates. Seawater was spiked with known numbers of nauplii and copepodite free-swimming (planktonic) stages ofL. salmonisto 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 forL. salmonisdetection and quantification. The assay specificity was high and a high correlation between MGC and planktonic stages ofL. salmoniswas established in the laboratory conditions. In the field,L. salmonisDNA was consequently detected, but with MGC number below that expected for one copepodite or nauplii. We concluded that onlyL. salmonistissue or eDNA residues were detected. This novel study opens for a fully automatizedL. salmonisDNA quantification using ESP robotic to monitor the parasitic load, but the challenge remains the adequate sampling of a volume of seawater sufficiently large to be representative of outbreaks and load around fish farms.

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 NSCHigh and ISBRHigh compared to control. Mild inflammatory response reactions and tissue ultrastructural alterations in gill tissue were observed in both treatments. Signs of necrosis occurred in ISBRHigh. 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 NSCHigh. 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.

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.

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.

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. publishedVersion

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.

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.

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).

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.

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.

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.

Since the 1990s, electrochemical genosensors have emerged as a dynamic analytical technique to perform qualitative and quantitative detection of “various analytes” in the field of clinical diagnostics, food safety, and environmental contamination. The term genosensor describes an analytical device resulting from the integration of a sequence-specific probe (usually a short synthetic oligonucleotide, termed capture probe) and a signal transducer. The capture probe, immobilized onto the transducer surface, acts as the biorecognition molecule and recognizes the target DNA or RNA. In this chapter, the essential features for developing an electrochemical genosensor will be elucidated. Moreover, this chapter discusses the application of electrochemical genosensors to environmental contamination and surveillance especially in the field of aquaculture and e-DNA

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.

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.

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.

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.

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 scientific goal of the present work is the development of a new molecular biomarker as a biomonitoring tool for early assessment of the biological effect of complex mixtures of pollutants (especially polycyclic aromatic hydrocarbons) and validation of the approach by comparison with standard methodologies. Hence, a DNA‐based biosensor, which is able to detect the presence of compounds with affinity for DNA to measure their effect on the guanine base, was used in this research.

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.

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.

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.

Within the frame of a large environmental study, we report on a research program that investigated the potential for bioaccumulation and subsequent effect responses in several marine organisms exposed to chronic levels of dispersed crude oil. Body burden can be estimated from kinetic parameters (rate constants for uptake and elimination), and appropriate body burden-effect relationships may improve assessments of environmental risks or the potential for such outcomes following chronic discharges at sea. We conducted a series of experiments in a flow-through system to describe the bioaccumulation kinetics of polycyclic aromatic hydrocarbons (PAH) at low concentrations of dispersed crude oils. Mussels (Mytilus edulis) and juvenile turbot (Scophthalmus maximus) were exposed for periods ranging from 8 to 21 d. Postexposure, the organisms were kept for a period of 9 to 10 d in running seawater to study elimination processes. Rate constants of uptake (k1) and elimination (k2) of the PAHs during and following exposure were calculated using a first-order kinetic model that assumed a decrease of the substances in the environment over time. The estimated bioconcentration factor was calculated from the ratio of k1/k2. The kinetic parameters of two-, three-, and four-ring PAHs in mussel and fish are compared with estimates based on hydrophobicity alone, expressed by the octanol-water partition coefficient, Kow (partitioning theory). A combination of reduced bioavailability of PAHs from oil droplets and degradation processes of PAHs in body tissues seems to explain discrepancies between kinetic rates based on Kow and actual kinetic rates measured in fish. Mussels showed a pattern more in compliance with the partitioning theory.

The fate of hydrocarbons discharged at sea was studied in laboratory exposures examining the uptake of PAC (polyaromatic compounds) in marine organisms. Methodology and species selection were guided by the Northwest European (OSPAR) convention. The acute toxicity of poorly soluble substances is normally assessed by preparing water accommodated fractions (WAF). Following such tests we studied PAC uptake in unicellular algae, crustaceans and fish. Preparation of WAF was first improved because in the standard procedure, oil droplet size increased with oil loading and flask size. At similar exposure levels the proportion of large molecular weight PAC associated with algae increased when the density of algae decreased. However, as body size increased via crustaceans to fish more of the smaller molecular weight PAC were bioconqentrated. Organism density and type of organism thus influence the abundance of PAC in food chains.

Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (P40), we have analyzed the interaction between OmpA and macrophages. We report that Alexa488-labeled P40 binds (at 4°C) to murine and human macrophages in a dose-dependent manner and is rapidly internalized (at 37°C). No binding or internalization of the Alexa488-labeled glycophorin A control protein is observed under the same conditions. Furthermore, P40 up-regulates the production of IL-1β, IL-8, IL-10, IL-12, and TNF-α by human macrophages and of NO by the RAW 264.7 murine macrophage cell line. P40 also synergizes with IFN-γ and suboptimal concentrations of LPS to up-regulate the production of these mediators. In conclusion, P40 binds to and activates macrophages. These data suggest that recognition of OmpA by macrophages may be an initiating event in the antibacterial host response.

We have recently cloned a new protein, recombinant P40 (rP40). When tested in vivo after conjugation to a B-cell epitope, rP40 induces an important antibody response without the need for adjuvant. To characterize its potency, this carrier protein was coupled to a peptide derived from respiratory syncytial virus attachment G protein (G1′). After immunization of mice with the rP40-G1′ conjugate, strong antipeptide antibodies were detected, whereas peptide alone was not immunogenic. To emphasize the carrier properties of rP40, a polysaccharide derived from Haemophilus influenzae type b (Hib) was coupled to it. Immunoglobulin G responses against the Hib polysaccharide were observed after coupling to rP40. Interestingly, an antipeptide antibody response was observed despite preexisting anti-rP40 antibodies generated by preimmunization with rP40. In addition, rP40 compares well with the reference carrier protein, tetanus toxoid (TT), since antibody responses of equal intensity were observed when a peptide or a polysaccharide was coupled to TT and rP40. Moreover, rP40 had advantages compared to TT; e.g., it induced a mixed Th1/Th2 response, whereas TT induced only a Th2 profile. Together, the results indicate that rP40 is a novel carrier protein with potential for use as an alternative carrier for human vaccination.

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.

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. © 2009 SETAC.

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.

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.

Assessing the fate in marine biota of hydrocarbons derived from oil particles that are discharged during exploration and production is of relevant environmental concern. However, a rather complex experimental setup is required to carry out such investigations. In this study, a sophisticated tool, the continuous-flow system (CFS), was used to mimic dispersed oil exposure to marine biota. Polycyclic aromatic hydrocarbon (PAH) uptake was studied in two species, the blue mussel Mytilus edulis and juvenile of the turbot Scophthalmus maximus, and in semipermeable membrane devices (SPMD) exposed to crude oil dispersed in a flow-through system. After an exposure period of 8 to 21 d, elimination in organisms and devices was analyzed for 9 to 10 d following transfer to PAH-free seawater. Principal component analysis (PCA) revealed different PAH patterns. In mussel and SPMD, the PAH profiles were very close to that analyzed in seawater. Slight differences were, however, indicated for large molecules with log Kow above six. Nonachievement of steady-state concentration and bioavailability of PAH in oil droplets may account for these differences. The PAH composition in fish revealed only congeners with two to three aromatic rings. A combination of bioavailability and efficient metabolism of the larger PAH molecules may explain this pattern. The CFS made possible a better understanding of some critical factors governing bioconcentration in marine biota from dispersed oil. Yet the results illustrate that uptake of PAH from exposure to oil particles is complex and that different species may bioconcentrate different molecules depending on factors like life style and metabolic capability to degrade the potential harmful substances. Hence, risk assessment of the actual impact of discharges to marine biota should consider these essential biological and ecological factors.

We analyzed the interaction between a bacterial cell wall protein and dendritic cells (DCs). Outer membrane protein A from Klebsiella pneumoniae (kpOmpA) specifically bound to professional antigen presenting cells and was endocytosed by immature DCs via a receptor-dependent mechanism. kpOmpA signaled through Toll-like receptor 2, induced DCs to produce interleukin 12 and induced maturation of DCs. Whole antigen that was coupled to kpOmpA and injected into mice was taken up by DCs and delivered to the conventional cytosolic MHC class I presentation pathway. kpOmpA also primed antigen-specific CD8+ CTLs in the absence of CD4+ T cell help or adjuvant and elicited therapeutic immunity to antigen-expressing tumors. Thus, OmpA belongs to a class of proteins that are able to elicit CTL responses to exogenous antigen.

In order to perform environmental risk assessments with regard to oil contamination in the sea, it is important to obtain knowledge about threshold levels for possible adverse effects in marine organisms. With this objective in mind, selected biomarkers were studied in Atlantic cod (Gadus morhua) chronically exposed to mechanically dispersed crude oil. The fish were exposed for 30 days in a continuous flow system to nominal concentrations of 0.06, 0.25 and 1 ppm North Sea crude oil. Fish were sampled five times during the exposure period. In addition, the 1 ppm group and the control group were sampled 1 week after the end of exposure. Polyaromatic hydrocarbon (PAH) concentrations in the seawater were analysed regularly by direct fluorescence and, at one occasion, by gas chromatography with mass spectrographic detection (GC/MS) measurements. Liver samples were analysed for parent PAH levels by means of GC/MS measurements, and PAH metabolites in bile were analysed by means of fixed wavelength fluorescence. Cytochrome P450 induction in liver was estimated by ethoxyresorufin-O-deethylase (EROD) activity, and hepatic DNA adducts were analysed by the 32P-postlabelling assay. The parent PAH concentrations in liver showed peak levels 3 days after the start of exposure, followed by a reduction towards the end of the experiment. In contrast, the PAH metabolites in bile and EROD activity showed generally increasing levels throughout the whole exposure period, indicating an increased biotransformation efficiency. The level of DNA adducts in the 1 ppm group showed a stable increase during the entire exposure period. Only a slight, non-significant decrease in DNA adduct levels was observed after 7 days of recovery in clean water. Exposure-dependent responses were observed for all three biomarkers. The lowest nominal concentration of dispersed oil in water, 0.06 ppm, corresponded to a measured total PAH concentration in the water of 0.3 ppb. Atlantic cod exposed to this concentration showed increased levels of PAH metabolites in bile and a slight induction of CYP1A, as well as formation of DNA adducts when compared with control fish. Particularly noteworthy is the detection of DNA adducts at such a low exposure concentration of oil in water, which, to our knowledge, is a novel finding. These dose-response data may serve as useful contributions when assessing environmental risk with regard to marine oil pollution.

The amount of produced water from oil production is expected to increase in years to come, and presently most of it is released to sea. Oil residues are regarded as one of the main environmental hazards of produced water discharges. The presence of polyaromatic hydrocarbons (PAH) metabolites in bile, induction of detoxification enzymes (CYP1A) and the formation of DNA adducts in liver was studied in a laboratory experiment with Atlantic cod (Gadus morhua). Responses in these parameters (biomarkers) have been positively correlated with the occurrence of mutagenic and carcinogenic effects in fish. The laboratory study demonstrated that PAH from the oil were taken up, metabolised and bound to DNA molecules after a 30 days exposure to dispersed crude oil at nominal oil concentrations as low as 60 µg/l (ppb). These molecular responses are suggested as environmental risk parameters for potential adverse effects of produced water, and in particular effects of PAH. Atlantic cod, haddock (Melanogrammus aeglefinus) and long rough dab (Hippoglossoides platessoides) caught in an environmental monitoring survey in the North Sea, did not show detectable levels of PAH metabolites in bile nor induction of CYP1A, which could be associated to oil activity. DNA adduct formation was not investigated during this survey.

Klebsiella pneumoniae OmpA, the 40-kDa major protein of the outer membrane, was cloned and expressed in Escherichia coli. The recombinant protein was produced intracellularly in E. coli as inclusion bodies. Fusion of a short peptide to the N-terminus of native P40 facilitated high-level expression of the recombinant protein. Purified recombinant P40 was analyzed to verify purity and structural integrity. The molecular mass of purified recombinant P40 determined by electrospray mass spectrometry was 37,061 Da, in agreement with the theoretical mass deduced from the DNA sequence. Specific proliferation of recombinant-P40-primed murine lymph node cells in response to recombinant P40 stimulation in vitro indicated the presence of a T-cell epitope on recombinant P40. The induction of high serum antibody titers to a synthetic peptide derived from the attachment protein G of the respiratory syncytial virus when chemically coupled to recombinant P40 indicated that the protein had potent carrier properties.

A mixture of sialylglycoasparagines and sialylglycopeptides was successively incubated with lysosomal extracts, at two pH values, first at pH 7.5 and then at pH 4. The 1H-NMR analysis of the sialyloligosaccharides released during the enzymatic digestion demonstrates the sequential action of aspartylglucosaminidase and an endo-N-acetyl-beta-D-glucosaminidase which release sialyloligosaccharides identical to the reference sugars isolated from the urine of patients suffering from sialidosis. This process represents a new catabolic pathway for N-glycosyl-proteins which may account for the appearance of the oligosaccharides stored in tissues and urine of patients suffering from lysosomal diseases.