Jonny Beyer

FISH BIOACCUMULATION AND BIOMARKERS IN ENVIRONMENTAL RISK ASSESSMENT: A REVIEW

In this review, a wide array of bioaccumulation markers and biomarkers, used to demonstrate exposure to and effects of environmental contaminants, has been discussed in relation to their feasibility in environmental risk assessment (ERA). Fish bioaccumulation markers may be applied in order to elucidate the aquatic behavior of environmental contaminants, as bioconcentrators to identify certain substances with low water levels and to assess exposure of aquatic organisms. Since it is virtually impossible to predict the fate of xenobiotic substances with simple partitioning models, the complexity of bioaccumulation should be considered, including toxicokinetics, metabolism, biota-sediment accumulation factors (BSAFs), organ-specific bioaccumulation and bound residues. Since it remains hard to accurately predict bioaccumulation in fish, even with highly sophisticated models, analyses of tissue levels are required. The most promising fish bioaccumulation markers are body burdens of persistent organic pollutants, like PCBs and DDTs. Since PCDD and PCDF levels in fish tissues are very low as compared with the sediment levels, their value as bioaccumulation markers remains questionable. Easily biodegradable compounds, such as PAHs and chlorinated phenols, do not tend to accumulate in fish tissues in quantities that reflect the exposure. Semipermeable membrane devices (SPMDs) have been successfully used to mimic bioaccumulation of hydrophobic organic substances in aquatic organisms. In order to assess exposure to or effects of environmental pollutants on aquatic ecosystems, the following suite of fish biomarkers may be examined: biotransformation enzymes (phase I and II), oxidative stress parameters, biotransformation products, stress proteins, metallothioneins (MTs), MXR proteins, hematological parameters, immunological parameters, reproductive and endocrine parameters, genotoxic parameters, neuromuscular parameters, physiological, histological and morphological parameters. All fish biomarkers are evaluated for their potential use in ERA programs, based upon six criteria that have been proposed in the present paper. This evaluation demonstrates that phase I enzymes (e.g. hepatic EROD and CYP1A), biotransformation products (e.g. biliary PAH metabolites), reproductive parameters (e.g. plasma VTG) and genotoxic parameters (e.g. hepatic DNA adducts) are currently the most valuable fish biomarkers for ERA. The use of biomonitoring methods in the control strategies for chemical pollution has several advantages over chemical monitoring. Many of the biological measurements form the only way of integrating effects on a large number of individual and interactive processes in aquatic organisms. Moreover, biological and biochemical effects may link the bioavailability of the compounds of interest with their concentration at target organs and intrinsic toxicity. The limitations of biomonitoring, such as confounding factors that are not related to pollution, should be carefully considered when interpreting biomarker data. Based upon this overview there is little doubt that measurements of bioaccumulation and biomarker responses in fish from contaminated sites offer great promises for providing information that can contribute to environmental monitoring programs designed for various aspects of ERA.

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.

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.

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.

Endocrine Modulation in Atlantic Cod (Gadus morhua L.) Exposed to Alkylphenols, Polyaromatic Hydrocarbons, Produced Water, and Dispersed Oil

Effluent from oil production activities contains chemicals that are suspected of inducing endocrine disruption in fish. In this study, Atlantic cod (Gadus morhua L.) were exposed to mixtures of low- and medium-molecular-weight alkylphenols (AP) (methyl- to heptylphenol), polycyclic aromatic hydrocarbons (PAH), diluted produced water, and dispersed oil for 15 d in a flow-through exposure system. Condition index (CI), hepatosomatic index (HSI), gonadosomatic index (GSI), concentration of the estrogenic biomarker vitellogenin (Vtg), and modulation of the total sex steroid-binding capacity in plasma were determined to assess whether these mixtures were capable of interfering with endocrine-regulated physiological processes in Atlantic cod. No marked differences in plasma Vtg levels were found between control and exposed groups of either males or females, possibly due to high intergroup variances and low sample numbers. An apparent numerical increase in the number of male and female fish with high plasma Vtg levels was, however, observed in some exposure groups compared to control. This purported weak estrogenic effect was several orders of magnitude lower than that observed for potent estrogens and suggested that the levels of estrogen receptor (ER) agonists were low. Exposure of female fish to a mixture of dispersed oil and a mixture of AP, PAH, and dispersed oil led to upregulation of the plasma total sex steroid-binding capacity, indicating interference with the normal blood steroid transport. No significant effects were seen for CI, HSI, and GSI, suggesting that the endocrine-disrupting potential was not sufficient to elicit effects on general physiological conditions and gonad development during this short exposure period.

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.

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.

Characterization of alkylphenol metabolites in fish bile by enzymatic treatment and HPLC-fluorescence analysis.

Alkylphenol (AP) metabolites were characterized in the bile of Atlantic cod (Gadus morhua L.) after exposure to nine individual compounds (10mg/kg fish), 2-methylphenol (2-MP), 4-methylphenol (4-MP), 3,5-dimethylphenol (3,5-DMP), 2,4,6-trimethylphenol (2,4,6-TMP), 4-tert-butylphenol (4-t-BP), 4-tert-butyl-2-methylphenol (4-t-B-2-MP), 4-n-pentylphenol (4-n-PP), 4-n-hexylphenol (4-n-HexP) and 4-n-heptylphenol (4-n-HepP), and a mixture (total dose; 13.5 mg/kg fish) of the nine APs by inter-muscular injection. The degree of alkylation ranged from methyl (C1) to heptyl (C7) and represents the types of APs present in produced water. Fish bile was collected on day 4 and 16 (exposure groups 2-MP, 3,5-DMP, 2,4,6-TMP and 4-t-B-2-MP) following exposure. Characterization of major metabolites was accomplished by enzymatic de-conjugation and analysis by high performance liquid chromatography connected to a fluorescence detector (HPLC-F) acquiring at ex/em 222/306 nm. Two solid phase extraction (SPE) columns were evaluated for clean-up of samples prior to analysis. Independent of alkyl homologue, the glucuronide conjugated APs were the most abundant metabolites (73-100%), whereas sulfates, glucosides and unchanged compounds were excreted in amounts of 0-21%, 0-6.1% and 0-6.3%, respectively. The total concentration of measured metabolites in the bile, determined as their respective APs after de-conjugation, increased with increasing degree of alkylation (3.2+/-2.6 microg/g bile for 2-MP and 571+/-81 microg/g bile for 4-n-HepP) after exposure to an equal dose of AP. Comparison of metabolite concentrations in bile sampled 4 and 16 days after exposure, showed that the levels of 2-MP, 2,4,6-TMP and 4-t-B-2-MP were reduced by 55%, 30% and 45%, respectively whereas 3,5-DMP increased by 25% (not significant). This study suggests that analysis of de-conjugated metabolites in fish bile can be used to monitor AP exposure to fish, due to the relatively high and persistent level of these compounds. However, although HPLC-F is suitable for laboratory exposures, it might not be sufficient selective for field studies.

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.

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.