Biomarker responses and accumulation of polycyclic aromatic hydrocarbons in Mytilus trossulus and Gammarus oceanicus during exposure to crude oil.

In the brackish water Baltic Sea, oil pollution is an ever-present and significant environmental threat mainly due to the continuously increasing volume of oil transport in the area. In this study, effects of exposure to crude oil on two common Baltic Sea species, the mussel Mytilus trossulus and the amphipod Gammarus oceanicus, were investigated. The species were exposed for various time periods (M. trossulus 4, 7, and 14 days, G. oceanicus 4 and 11 days) to three oil concentrations (0.003, 0.04, and 0.30 mg L-1 based on water measurements, nominally aimed at 0.015, 0.120, and 0.750 mg L-1) obtained by mechanical dispersion (oil droplets). Biological effects of oil exposure were examined using a battery of biomarkers consisting of enzymes of the antioxidant defense system (ADS), lipid peroxidation, phase II detoxification (glutathione S-transferase), neurotoxicity (acetylcholinesterase inhibition), and geno- and cytotoxicity (micronuclei and other nuclear deformities). In mussels, the results on biomarker responses were examined in connection with data on the tissue accumulation of polycyclic aromatic hydrocarbons (PAH). In M. trossulus, during the first 4 days of exposure the accumulation of all PAHs in the two highest exposure concentrations was high and was thereafter reduced significantly. Significant increase in ADS responses was observed in M. trossulus at 4 and 7 days of exposure. At day 14, significantly elevated levels of geno- and cytotoxicity were detected in mussels. In G. oceanicus, the ADS responses followed a similar pattern to those recorded in M. trossulus at day 4; however, in G. oceanicus, the elevated ADS response was still maintained at day 11. Conclusively, the results obtained show marked biomarker responses in both study species under conceivable, environmentally realistic oil-in-seawater concentrations during an oil spill, and in mussels, they are related to the observed tissue accumulation of oil-derived compounds.

Effects of chronic crude oil exposure on early developmental stages of the Northern krill (Meganyctiphanes norvegica).

Rising oil and gas activities in northern high latitudes have led to an increased risk of petroleum pollution in these ecosystems. Further, seasonal high UV radiation at high latitudes may elevate photo-enhanced toxicity of petroleum pollution to marine organisms. Zooplanktons are a key ecological component of northern ecosystems; therefore, it is important to assess their sensitivity to potential pollutants of oil and gas activity. As ontogenetic development may be particularly sensitive, the aim of this study was to examine the impact of chronic exposure to oil water dispersion (OWD) on development and feeding of early life stages of the Northern krill, Meganyctiphanes norvegica. In a range of experiments, embryonic, nonfeeding, and feeding larval stages were exposed to concentrations of between 0.01 and 0.1 mg/L of oil or photo-modified oil for 19 and 21 d. No significant effects on egg respiration, hatching success, development, length and larval survival were observed from these treatments. Similarly, evolution of fatty acid composition patterns during ontogenetic development was unaffected. The results indicates a high degree of resilience of these early developmental stages to such types and concentrations of pollutants. However, feeding and motility in later calyptopis-stage larvae were significantly impaired at exposure of 0.1 mg/L oil. Data indicate that feeding larval stage of krill was more sensitive to OWD than early nonfeeding life stages. This might be attributed to the narcotic effects of oil pollutants, their direct ingestion, or accumulated adverse effects over early development.

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

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

III: Use of biomarkers as Risk Indicators in Environmental Risk Assessment of oil based discharges offshore.

Offshore oil and gas activities are required not to cause adverse environmental effects, and risk based management has been established to meet environmental standards. In some risk assessment schemes, Risk Indicators (RIs) are parameters to monitor the development of risk affecting factors. RIs have not yet been established in the Environmental Risk Assessment procedures for management of oil based discharges offshore. This paper evaluates the usefulness of biomarkers as RIs, based on their properties, existing laboratory biomarker data and assessment methods. Data shows several correlations between oil concentrations and biomarker responses, and assessment principles exist that qualify biomarkers for integration into risk procedures. Different ways that these existing biomarkers and methods can be applied as RIs in a probabilistic risk assessment system when linked with whole organism responses are discussed. This can be a useful approach to integrate biomarkers into probabilistic risk assessment related to oil based discharges, representing a potential supplement to information that biomarkers already provide about environmental impact and risk related to these kind of discharges.

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

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

Effect of dispersed crude oil on the feeding activity, retention efficiency, and filtration rate of differently sized blue mussels (Mytilus edulis).

The use of physiological response endpoints in environmental monitoring represents an opportunity to provide an integrated picture of health status and ecological fitness of individuals, and may provide an indication of potential longer term effects on aquatic organisms in the environment. The feeding behavior response sensitivity of blue mussels (Mytilus edulis) of differing size to dispersed crude oil (DCO) was investigated in a lab exposure experiment. The ability of mussels to recover following a single exposure was also investigated, as well as the response to consecutive exposures, in order to assess the utility of employing the same individuals in chronic environmental monitoring. Feeding physiology was assessed by measuring retention efficiency and filtration rate of individual mussels in a live-algae feeding assay. In addition, the percentage of mussels actively filtering during testing was calculated. The feeding physiology parameters were sensitive and able to discriminate exposed mussels from controls. Further, data indicated that larger mussels appear more suitable in environmental monitoring, as these animals showed both sensitivity and an ability to adapt and recover from exposure while remaining sensitive to subsequent treatments. Smaller mussels were also sensitive to the measured endpoints, even if these animals suffered higher rates of mortality during the exposure. Finally, when exposed to the high concentration of DCO, mussels displayed a tendency to close the valves and terminate filtration.

Environmental impacts of produced water and drilling waste discharges from the Norwegian offshore petroleum industry.

Operational discharges of produced water and drill cuttings from offshore oil and gas platforms are a continuous source of contaminants to continental shelf ecosystems. This paper reviews recent research on the biological effects of such discharges with focus on the Norwegian Continental Shelf. The greatest concern is linked to effects of produced water. Alkylphenols (AP) and polyaromatic hydrocarbons (PAH) from produced water accumulate in cod and blue mussel caged near outlets, but are rapidly metabolized in cod. APs, naphtenic acids, and PAHs may disturb reproductive functions, and affect several chemical, biochemical and genetic biomarkers. Toxic concentrations seem restricted to <2 km distance. At the peak of discharge of oil-contaminated cuttings fauna disturbance was found at more than 5 km from some platforms, but is now seldom detected beyond 500 m. Water-based cuttings may seriously affect biomarkers in filter feeding bivalves, and cause elevated sediment oxygen consumption and mortality in benthic fauna. Effects levels occur within 0.5-1 km distance. The stress is mainly physical. The risk of widespread, long term impact from the operational discharges on populations and the ecosystem is presently considered low, but this cannot be verified from the published literature.

Environmental risk assessment of alkylphenols from offshore produced water on fish reproduction.

Concern has been raised over whether environmental release of alkylphenols (AP) in produced water (PW) discharges from the offshore oil industry could impose a risk to the reproduction of fish stocks in the North Sea. An environmental risk assessment (ERA) was performed to determine if environmental exposure to PW APs in North Sea fish populations is likely to be high enough to give effects on reproduction endpoints. The DREAM (Dose related Risk and Effect Assessment Model) software was used in the study and the inputs to the ERA model included PW discharge data, fate information of PW plumes, fish distribution information, as well as uptake and elimination information of PW APs. Toxicodynamic data from effect studies with Atlantic cod (Gadus morhua) exposed to APs were used to establish a conservative environmental risk threshold value for AP concentration in seawater. By using the DREAM software to 1) identify the areas of highest potential risk and 2) integrate fish movement and uptake/elimination rates of APs for the chosen areas we found that the environmental exposure of fish to APs from PW is most likely too low to affect reproduction in wild populations of fish in the North Sea. The implications related to risk management of offshore PW and uncertainties in the risk assessment performed are discussed.

Effects of chronic exposure to dispersed oil on selected reproductive processes in adult blue mussels (Mytilus edulis) and the consequences for the early life stages of their larvae.

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.

Alkylphenol metabolites in fish bile as biomarkers of exposure to offshore oil industry produced water in feral fish.

The measurement of low-concentration alkylphenol (AP) exposure in fish is relevant in connection with monitoring and risk assessment of offshore oil industry produced water (PW) discharges. Detection of AP markers in fish bile offers significantly greater sensitivity than detection of AP in tissues such as liver. Recent studies revealed that gas chromatography-mass spectrometry in electron ionization mode (GC-EI-MS) enabled a selective and sensitive analytical detection of PW AP in mixtures with unknown composition. A procedure consisting of enzymatic deconjugation of metabolites in fish bile followed by derivatization with bis(trimethylsilyl)trifluoroacetamide and then separation and quantification of derivatized AP using GC-EI-MS is presented. The use of this procedure as a possible recommended approach for assessment and biomonitoring of AP contamination in fish populations living down-current from offshore oil production fields is presented.

Chronic exposure of adults and embryos of Pandalus borealis to oil causes PAH accumulation, initiation of biomarker responses and an increase in larval mortality.

Adult shrimps (Pandalus borealis) and their embryos were exposed to an oil-water dispersion (OWD) at concentrations of 0.015, 0.06 and 0.25 mg/L using a continuous flow system. Lysosomal membrane stability was analysed in haemocytes using the neutral red retention assay and an alkaline unwinding assay was used to measure DNA damage in hepatopancreas tissue. Exposure to oil induced concentration and time dependent biomarker responses in adult shrimps together with the accumulation of PAH in their tissues. Oil exposure of shrimp embryos caused increased mortality in the resultant larvae, even if the larvae were kept in clean water after hatching. There were minor differences observed in larval stage development times in the first part of the experiments. The fatty acid composition of embryos exposed to oil was different to that of non-exposed larvae. PAH tissue concentration and biomarker responses correlated to the reduced survival of the shrimp larvae.

The Arctic is no longer put on ice: evaluation of Polar cod (Boreogadus saida) as a monitoring species of oil pollution in cold waters.

The withdrawing Arctic ice edge will facilitate future sea transport and exploration activities in the area, which calls for the establishment of relevant cold water monitoring species. The present study presents first results of field baseline levels for core oil pollution biomarkers in Polar cod (Boreogadussaida) sampled from pristine, Arctic waters. Furthermore, biomarker response levels were characterized in controlled laboratory exposure experiments running over 2 weeks. Fish exposed to a simulated petrogenic spill (1ppm dispersed, crude oil) exhibited elevated hepatic EROD activity, bile PAH-metabolites, and hepatic DNA-adducts, whereas male individuals exposed to simulated produced water (30ppb nonylphenol) exhibited a strong induction of plasma vitellogenin. In conclusion, the results demonstrated low and robust biomarker baseline levels that were clearly different from exposure responses. In combination with its high abundance and circumpolar distribution, the Polar cod seems well qualified for oil pollution monitoring in Arctic waters.

Development of a laboratory exposure system using marine fish to carry out realistic effect studies with produced water discharged from offshore oil production.

A biotest system for environmentally realistic exposure of fish to produced water (PW) was developed and tested. Authentic PW was collected at an oil production platform in the North Sea and preserved by freezing in multiple aliquots a 25L. After transport to the test laboratory onshore, daily PW aliquots were thawed, homogenised and administered to the test fish, Atlantic cod (Gadus morhua), in two diluted exposure concentrations, 0.1% and 0.5%, during a 15 d period, using a continuous flow-through exposure setup. Positive control groups were exposed to two crude oil treatments for comparison. Chemical analyses showed that alkylphenol (AP) and PAH concentrations in PW exposure waters were very low. Observations of significantly increased AP and PAH metabolite levels in PW exposed fish demonstrated the suitability of the biotest system for its use in biological exposure/effect studies of PW, and it also demonstrated the sensitivity of bile metabolites as PW exposure markers in fish. The relevance of the biotest system for PW effect studies and for validating modelled environmental risk estimates of PW dischargers from offshore oil production is discussed.

Relating biomarkers to whole-organism effects using species sensitivity distributions: a pilot study for marine species exposed to oil.

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.

Water column monitoring near oil installations in the North Sea 2001-2004.

Fisheries have been vital to coastal communities around the North Sea for centuries, but this semi-enclosed sea also receives large amounts of waste. It is therefore important to monitor and control inputs of contaminants into the North Sea. Inputs of effluents from offshore oil and gas production platforms (produced water) in the Norwegian sector have been monitored through an integrated chemical and biological effects programme since 2001. The programme has used caged Atlantic cod and blue mussels. PAH tissue residues in blue mussels and PAH bile metabolites in cod have confirmed exposure to effluents, but there was variation between years. Results for a range of biological effects methods reflected exposure gradients and indicated that exposure levels were low and caused minor environmental impact at the deployment locations. There is a need to develop methods that are sufficiently sensitive to components in produced water at levels found in marine ecosystems.

Total oxyradical scavenging capacity responses in Mytilus galloprovincialis transplanted into the Venice lagoon (Italy) to measure the biological impact of anthropogenic activities.

Oxidative stress related investigations to monitor the impact of the pollutant discharges into the Venice lagoon (Italy) originating from anthropogenic activities (raw sewage water, agricultural and industrial effluents, oil tanker traffic), on marine organisms have classically been carried out by analyzing specific, single antioxidants (i.e. catalase, superoxide dismutase). In this paper, two studies are reported where the total oxyradical scavenging capacity assay (TOSC) was selected and measured toward peroxyl, and hydroxyl free radicals, and peroxynitrite in mussels (Mytilus galloprovincialis) transplanted into the Venice city and throughout the lagoon to measure the biological effects of anthropogenic activities. In the first experiment, mussels from a clean site (farm) were transplanted to the urban area of Venice for 0, 1, 2 and 4 weeks; cytosolic TOSC toward peroxyl and hydroxyl free radicals, and peroxynitrite revealed that the transplantation process caused a stress (handling stress, anoxia, oxidative burst) resulting in a reduction of TOSC in both control and urban sites, therefore, preventing clear interpretation of the data after one week. At week 2, a significant TOSC reduction (P < 0.05) toward peroxyl and hydroxyl radicals in the urban site revealed that mussels experienced oxidative pressure exerted by pollutants. Most TOSC values returned to initial levels at week 4; however, TOSC induction was noticed in the control group toward peroxyl and hydroxyl radicals while in the exposed group it was not indicating an inhibition of the oxidative metabolism. In the second experiment, mussels were deployed at seven different sites throughout the lagoon. After five weeks of exposure, significant TOSC reduction was measured (P < 0.05) toward peroxyl for Palude della Rosa, Chioggia and Valle Millecampi, toward hydroxyl radicals for Valle Millecampi and Campalto and toward peroxynitrite for Valle Millecampi. Although these data indicate a depletion of the low molecular weight scavengers, additional biomarkers are needed to draw a conclusion on the health of the mussels. TOSC was proved to be an interesting health index parameter to measure pollution impact in a transplantation study provided that the mussels are exposed for two weeks and a control is run in parallel.

Detection of DNA damage in mussels and sea urchins exposed to crude oil using comet assay.

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.