Ciona spp. and ascidians as bioindicator organisms for evaluating effects of endocrine disrupting chemicals: A discussion paper.

In context of testing, screening and monitoring of endocrine-disrupting (ED) type of environmental pollutants, tunicates could possibly represent a particularly interesting group of bioindicator organisms. These primitive chordates are already important model organisms within developmental and genomics research due to their central position in evolution and close relationship to vertebrates. The solitary ascidians, such as the genus Ciona spp. (vase tunicates), could possibly be extra feasible as ED bioindicators. They have a free-swimming, tadpole-like larval stage that develops extremely quickly (<20 h under favorable conditions), has a short life cycle (typically 2-3 months), are relatively easy to maintain in laboratory culture, have fully sequenced genomes, and transgenic embryos with 3D course data of the embryo ontogeny are available. In this article, we discuss possible roles of Ciona spp. (and other solitary ascidians) as ecotoxicological bioindicator organisms in general but perhaps especially for effect studies of contaminants with presumed endocrine disrupting modes of action.

The ecotoxicology of marine tributyltin (TBT) hotspots: A review.

Tributyltin (TBT) was widely used as a highly efficient biocide in antifouling paints for ship and boat hulls. Eventually, TBT containing paints became globally banned when TBT was found to cause widespread contamination and non-target adverse effects in sensitive species, with induced pseudohermaphroditism in female neogastropods (imposex) being the best-known example. In this review, we address the history and the status of knowledge regarding TBT pollution and marine TBT hotspots, with a special emphasis on the Norwegian coastline. The review also presents a brief update on knowledge of TBT toxicity in various marine species and humans, highlighting the current understanding of toxicity mechanisms relevant for causing endocrine disruption in marine species. Despite observations of reduced TBT sediment concentrations in many marine sediments over the recent decades, contaminant hotspots are still prevalent worldwide. Consequently, efforts to monitor TBT levels and assessment of potential effects in sentinel species being potentially susceptible to TBT in these locations are still highly warranted.

Environmental effects of offshore produced water discharges: A review focused on the Norwegian continental shelf.

Produced water (PW), a large byproduct of offshore oil and gas extraction, is reinjected to formations or discharged to the sea after treatment. The discharges contain dispersed crude oil, polycyclic aromatic hydrocarbons (PAHs), alkylphenols (APs), metals, and many other constituents of environmental relevance. Risk-based regulation, greener offshore chemicals and improved cleaning systems have reduced environmental risks of PW discharges, but PW is still the largest operational source of oil pollution to the sea from the offshore petroleum industry. Monitoring surveys find detectable exposures in caged mussel and fish several km downstream from PW outfalls, but biomarkers indicate only mild acute effects in these sentinels. On the other hand, increased concentrations of DNA adducts are found repeatedly in benthic fish populations, especially in haddock. It is uncertain whether increased adducts could be a long-term effect of sediment contamination due to ongoing PW discharges, or earlier discharges of oil-containing drilling waste. Another concern is uncertainty regarding the possible effect of PW discharges in the sub-Arctic Southern Barents Sea. So far, research suggests that sub-arctic species are largely comparable to temperate species in their sensitivity to PW exposure. Larval deformities and cardiac toxicity in fish early life stages are among the biomarkers and adverse outcome pathways that currently receive much attention in PW effect research. Herein, we summarize the accumulated ecotoxicological knowledge of offshore PW discharges and highlight some key remaining knowledge needs.

Levels and trends of tributyltin (TBT) and imposex in dogwhelk (Nucella lapillus) along the Norwegian coastline from 1991 to 2017.

The banning of organotin biocides, such as tributyltin (TBT), from use in marine antifouling paints is now leading to reproductive health recovery in marine gastropod populations all over the world. TBT induces so-called imposex (superimposition of male sexual characters onto females) in certain marine gastropods, such as the common dogwhelk Nucella lapillus. In this publication, the results of the Norwegian TBT and imposex monitoring in N. lapillus from the period 1991-2017 are presented. Significantly higher levels of TBT and imposex were measured in coastal areas close to shipping lanes along most of the coast prior to 2008 than afterwards. Levels started declining after restrictions were imposed on the use of TBT in all antifouling paint applications, with a total ban in 2008. In 2017, no sign of imposex was found in N. lapillus in any of the monitoring stations along the Norwegian coastline. Based on monitoring data shown herein, the importance of long-term biomonitoring and international chemical regulations, as well as the TBT and imposex story in general, are discussed.

Mytilus spp. as sentinels for monitoring microplastic pollution in Norwegian coastal waters: A qualitative and quantitative study.

Microplastic (MP) contamination is ubiquitous in the environment and many species worldwide have been shown to contain MP. The ecological impact of MP pollution is still unknown, thus there is an urgent need for more knowledge. One key task is to identify species suitable as sentinels for monitoring in key eco-compartments, such as coastal waters. In Norway, mussels (Mytilus spp.) have been monitored for hazardous contaminants through OSPAR since 1981. Norway has the longest coastline in Europe and adding MP to the Norwegian Mussel Watch is therefore important in a European and global context. The present study reports MP data in mussels (332 specimens) collected from multiple sites (n = 15) spanning the whole Norwegian coastline. MPs were detected at all locations, except at one site on the west coast. Among the most surprising findings, mussels from the Barents Sea coastline in the Finnmark region, contained significantly more MPs than mussels from most of the southern part of the country, despite the latter sites being located much closer to major urban areas. Only mussels from a site located very close to Oslo, the capital, contained levels similar to those observed in the remote site in Finnmark. In total an average of 1.5 (±2.3) particles ind-1 and 0.97 (±2.61) particles w.w. g-1 was found. The most common MPs were <1 mm in size, and fibres accounted for 83% of particles identified, although there was inter-site variability. Thirteen different polymeric groups were identified; cellulosic being the most common and black rubbery particles being the second. This study suggests Mytilus spp. are suitable for semi-quantitative and qualitatively monitoring of MPs in coastal waters. However, some uncertainties remain including mussel size as a confounding factor that may influence ingestion, the role of depuration and other fate related processes, and this call for further research.

Blue mussels (Mytilus edulis spp.) as sentinel organisms in coastal pollution monitoring: A review.

The blue mussel (Mytilus spp.) is widely used as a bioindicator for monitoring of coastal water pollution (mussel watch programs). Herein we provide a review of this study field with emphasis on: the suitability of Mytilus spp. as environmental sentinels; uptake and bioaccumulation patterns of key pollutant classes; the use of Mytilus spp. in mussel watch programs; recent trends in Norwegian mussel monitoring; environmental quality standards and background concentrations of key contaminants; pollutant effect biomarkers; confounding factors; particulate contaminants (microplastics, engineered nanomaterials); climate change; harmonization of monitoring procedures; and the use of deployed mussels (transplant caging) in pollution monitoring. Lastly, the overall state of the art of blue mussel pollution monitoring is discussed and some important issues for future research and development are highlighted.

Comparison of caged and native blue mussels (Mytilus edulis spp.) for environmental monitoring of PAH, PCB and trace metals.

Contaminant bioaccumulation was studied in blue mussels (Mytilus edulis spp.) using the harbor waters of Kristiansand (Norway) as a case study. A suite of chemical contaminants (trace metals, PAHs and PCBs) was analyzed in caged and native mussels as well as in passive samplers (Diffusive Gradients in Thin films (DGT)-devices and silicone rubbers) placed alongside the mussels for estimation of contaminant concentrations in water and uptake rates and bioaccumulation factors (BAFs) in mussels during a six-months deployment period. Estimated logBAFs were in the ranges 2.3-5.5, 3.8-5.2 and 3.2-4.4 for metals, PCBs and PAHs, respectively. Contaminant levels in caged mussels increased rapidly to stable levels for trace metals, whereas for hydrophobic organic contaminants the increase was steady but slow and for many compounds did not reach the levels observed in native mussels. Some key issues related to mussel caging design, such as mussel deployment time and confounding influence from seasonal fluctuations, are discussed herein.

Environmental effects of the Deepwater Horizon oil spill: A review.

The Deepwater Horizon oil spill constituted an ecosystem-level injury in the northern Gulf of Mexico. Much oil spread at 1100-1300m depth, contaminating and affecting deepwater habitats. Factors such as oil-biodegradation, ocean currents and response measures (dispersants, burning) reduced coastal oiling. Still, >2100km of shoreline and many coastal habitats were affected. Research demonstrates that oiling caused a wide range of biological effects, although worst-case impact scenarios did not materialize. Biomarkers in individual organisms were more informative about oiling stress than population and community indices. Salt marshes and seabird populations were hard hit, but were also quite resilient to oiling effects. Monitoring demonstrated little contamination of seafood. Certain impacts are still understudied, such as effects on seagrass communities. Concerns of long-term impacts remain for large fish species, deep-sea corals, sea turtles and cetaceans. These species and their habitats should continue to receive attention (monitoring and research) for years to come.

Environmental risk assessment of combined effects in aquatic ecotoxicology: a discussion paper.

Environmental regulatory edicts within the EU, such as the regulatory framework for chemicals REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals), the Water Framework Directive (WFD), and the Marine Strategy Framework Directive (MSFD) focus mainly on toxicity assessment of individual chemicals although the effect of contaminant mixtures is a matter of increasing concern. This discussion paper provides an overview of the field of combined effects in aquatic ecotoxicology and addresses some of the major challenges related to assessment of combined effects in connection with environmental risk assessment (ERA) and regulation. Potentials and obstacles related to different experimental, modelling and predictive ERA approaches are described. On-going ERA guideline and manual developments in Europe aiming to incorporate combined effects of contaminants, the use of different experimental approaches for providing combined effect data, the involvement of biomarkers to characterize Mode of Action and toxicity pathways and efforts to identify relevant risk scenarios related to combined effects are discussed.

Environmental harm assessment of a wastewater discharge from Hammerfest LNG: a study with biomarkers in mussels (Mytilus sp.) and Atlantic cod (Gadus morhua).

Biologically treated wastewater (WW) from the Hammerfest LNG (liquefied natural gas) plant is discharged to the sea. A study using biomarkers in mussels and Atlantic cod was performed to examine whether this discharge meets a zero harmful emission requirement. Caging of mussels close to the outfall and exposure of mussels and fish to WW in the laboratory were conducted, and a suite of contaminant responsive markers was assessed in exposed animals. In mussels the markers included chemical contaminant levels, haemocyte lysosomal instability and nucleus integrity, cellular energy allocation, digestive gland and gonad histopathology and shell-opening behaviour. In fish, biliary PAH metabolites and gill histopathology biomarkers were measured. A consistent cause-effect relationship between WW treatments and markers measured in test animals was not found. The results therefore indicate that the WW emission is unlikely to represent a significant stress factor for the local marine environment under the conditions studied.

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.

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

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

Biomarkers in natural fish populations indicate adverse biological effects of offshore oil production.

BACKGROUND: Despite the growing awareness of the necessity of a sustainable development, the global economy continues to depend largely on the consumption of non-renewable energy resources. One such energy resource is fossil oil extracted from the seabed at offshore oil platforms. This type of oil production causes continuous environmental pollution from drilling waste, discharge of large amounts of produced water, and accidental spills. METHODS AND PRINCIPAL FINDINGS: Samples from natural populations of haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua) in two North Sea areas with extensive oil production were investigated. Exposure to and uptake of polycyclic aromatic hydrocarbons (PAHs) were demonstrated, and biomarker analyses revealed adverse biological effects, including induction of biotransformation enzymes, oxidative stress, altered fatty acid composition, and genotoxicity. Genotoxicity was reflected by a hepatic DNA adduct pattern typical for exposure to a mixture of PAHs. Control material was collected from a North Sea area without oil production and from remote Icelandic waters. The difference between the two control areas indicates significant background pollution in the North Sea. CONCLUSION: It is most remarkable to obtain biomarker responses in natural fish populations in the open sea that are similar to the biomarker responses in fish from highly polluted areas close to a point source. Risk assessment of various threats to the marine fish populations in the North Sea, such as overfishing, global warming, and eutrophication, should also take into account the ecologically relevant impact of offshore oil production.

High matrix interference affecting detection of PAH metabolites in bile of Atlantic hagfish (Myxine glutinosa) used for biomonitoring of deep-water oil production.

The characteristic biology and wide distribution of hagfish species makes them relevant for use in pollution biomonitoring at great water depths, particularly in regions where deep-water oil production may take place. The exposure of fish to petrogenic contaminants can normally be detected from the level of polycyclic aromatic hydrocarbon (PAH) metabolites in bile fluid. Some of these metabolites are strong fluorophores, allowing analytical detection by means of simple fluorometric techniques such as fixed wavelength fluorescence (FF) and synchronous fluorescence scanning (SFS). In the present study bile from Atlantic hagfish (Myxine glutinosa) collected in pristine areas (Barents Sea and southwestern Norway) displayed strong bile fluorescence levels, suggesting the presence of PAH contaminants. However, gas-chromatography-mass spectrometry (GC-MS) analyses ruled out PAHs as the origin for this fluorescence signal. Rather, the bile of Myxine contains components resulting in unusually strong background fluorescence interfering at the wavelength pairs used for detection of PAH metabolites. Possible background for the observed matrix interference and implications for detection of PAH metabolites in hagfish is discussed.

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.

Analytical methods for determining metabolites of polycyclic aromatic hydrocarbon (PAH) pollutants in fish bile: A review.

The determination of polycyclic aromatic hydrocarbon (PAH) metabolites in bile can serve as a tool for assessing environmental PAH exposure in fish. Biliary PAH metabolite levels can be measured using several analytical methods, including simple fluorescence assays (fixed fluorescence detection or synchronous fluorescence spectrometry); high-performance liquid chromatography with fluorescence detection (HPLC-F); gas chromatography-mass spectrometry (GC-MS) after deconjugation, extraction and derivatization of the bile sample, and finally by advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) methods. The method alternatives are highly different both with regard to their analytical performance towards different PAH metabolite structures as well as in general technical demands and their suitability for different monitoring strategies. In the present review, the state-of-the-art for these different analytical methods is presented and the advantages and limitations of each approach as well as aspects related to analytical quality control and inter-laboratory comparability of data and availability of certified reference materials are discussed.

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.

Uptake and tissue distribution of C4-C7 alkylphenols in Atlantic cod (Gadus morhua): relevance for biomonitoring of produced water discharges from oil production.

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.