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Integrated Biomarkers in Aquatic Organisms as a Tool for Biomonitoring Environmental Pollution and Improved Ecological Risk Assessment
Prof. A. Valavanidis & Dr. Thomie Vlachogianni Department of Chemistry, University of Athens, University Campus Zografou, 15784 Athens, Greece E-mail: valavanidis@chem.uoa.gr & thvlach@chem.uoa.gr
Abstract Environmental pollution has changed dramatically in recent decades. This is an overview of recent advances in the use and significance of molecular biomarkers in environmental and human health risk assessment. Also, here we focused in aquatic organisms as diagnostic tools for aquatic pollution and ecological risk assessment in ecotoxicological studies. The use of molecular biomarkers in aquatic organisms is very important in order to address the broad spectrum of industrial, agricultural, commercial and domestic chemicals entering the environment and especially the aquatic environment and being taken up into the tissues of aquatic organisms. The biomarkers can act as prognostic tools for increased levels of pollution. Biomarkers can be defined as functional measures of exposure to chemical and physical pollutants at the suborganism, physiological and behavioral level. This encompasses molecular, cellular, genetic, immunological and physiological measures. The process of ecological risk assessment is continually developing in ecotoxicological studies in order to address changing needs and diverse toxicological issues. Risk assessment methods are designed to provide a quantitative estimate of the probability of an adverse effect occurring as a consequence of environmental pollution of a diverse mixture of chemical pollutants which can act also in a synergistic way. Among the various types of biomarkers the following have received special attention in ecotoxicological studies: cytochrome P450 (indicator of exposure to organic contaminants PAHs, PCBs, etc), DNA-damage (strand breaks) and bulky DNA-adducts due to exposure to mutagenic inorganic and organic xenobiotics, inhibition of acetylcholinesterase (AChE) activity (organophosphorous, carbamates, Cd, PB, Cu, etc), metallothionein synthesis in hepatic and other tissues (exposure to metals Zn, Cu, Cd, Hg, Fe, etc), antioxidant enzymes (superoxide dismuatse, catalase, glutathione transferase) (exposure to ROS , free radicals, pollutants causing oxidative stress, lipid peroxidation (oxidants, metals, etc), and vitellogenin induction (oestrogenic substances). Ecotoxicological studies are using biomarkers in order to establish the risk of environmental pollution to key components in the ecosystem. The basic assumption is that by monitoring the adverse consequences for species occupying critical positions at the trophic level of the ecosystem. In this way the results might provide insight into the integrity of the ecosystem as a whole.

Measurements of toxicity in sensitive species (sentinel species) can be used as an early warning of population decline and as an ecologically relevant endpoint. Ecological risk assessment must aim in the preservation of the integrity of the ecosystem.
Keywords: biomarker; ecological risk assessment; environmental pollution; toxic pollutants; ecosystem; aquatic organisms; ecotoxicology.

Introduction Environmental pollution has increased substantially in the last decades due to a great number of industrial, agricultural, commercial and domestic waste, effluents and emissions as well as hazardous substances. The majority of environmental pollutants are threatening initially human and environmental health but also the integrity and function of ecosystems.1,2 Aquatic pollution is the predominant form of pollution since the majority of chemical pollutants are entering seas, rivers, lakes and wetlands. Marine and coastal ecosystems are characterized by their complexity and their sensitivity to various inorganic and organic pollutants.3-5 Integrated risk assessment for environmental and ecological issues is the most valuable and can give a balanced view. The study of environmental and human health issues can achieve more realistic results if human and ecological risk assessment were combined. Integration may provide early signs of previously unidentified risks, with both human and wildlife species in the role of sentinel species. Integration of studies can enhance quality of measurements, costeffectiveness and predictive capability.6-9 The need for integrated assessment of human and environmental risks on an international scale has been promulgated by four very important global partners: The WHO through its International Programme on Chemical Safety (IPCS), the central authority on environmental protection in the USA, the Environmental Protection Agency (EPA), the European Union through its Environmental Protection Agency and the numerous Environmental Directives, and the OECD (Organization for Economic Co-operation and Development, Paris) which deals with global issues of chemical safety and chemical industry. The aim is to improve quality of environmental measurements, include important biomarkers, improve risk assessment and management and formulate policy implementation.10-12 Integrated risk assessment and biomarkers The priority research areas for integration of human and ecological risk assessment were identified and included various biomarkers in the risk assessment process.13 Table 1. Priority research areas for integration of human and ecological risk assessment. 1. 2. 3. 4 5. Exchange of information and improved researchers in health and in ecology areas communication between

Harmonization of exposure characterization, biomonitoring methods and models for assessment New methods to facilitate comparison of risks for the various endpoints of risk assessment in studies Development of common endpoints of risk assessment across taxa Better understanding of mechanisms of toxic effects at multiple scales of biological organization

By analyzing more carefully the recommendations, it becomes obvious that biomarkers must be included in the risk assessment process in order to improve integration of risk assessment. Biomarkers are defined as biologically functional measures due to exposure to pollutants or stressors, which are expressed at the sub-organismal, physiological or behavioural level. Functional measures are referring to molecular, cellular, genetic, immunological and physiological changes in living organisms.

The classification of biomarkers has been advocated by the National Research Council of the USA 1987.14 Biomarkers are measurable internal indicators of changes in organisms at the molecular or cellular level which can offer great potential to understand environmentally mediated disease and to improve the process of risk assessment. A valid biomarker could also be considered a key event linking a specific environmental exposure to a health outcome. In 1989 NRC published two monographs on biomarkers or biologic markers.15,16 In 1999 the U.S. EPA held a workshop on Biomarkers: Taking Stock, An EPA/NIEHS in-House Workshop on Applying Biomarker Research.17 Biomarkers can be classified into 4 major categories following the exposure to the appearance of clinical phenomena and diseases. There is considerable overlap between each classification: a) internal dose [how much of a chemical substance or mixture an organism takes internally (mg/kg of weight) after exposure to air, water or soil. Blood or tissue concentrations of a pollutant or its metabolites may be used to estimate exposure], b) biologically effective dose (chemicals entering a biological system can metabolize, accumulate of bioconcentrate at certain tissues or organs, so the effective dose which can alter enzyme activity or some other vital biological function, for example, may indicate biologically effective dose), c) pre-clinical biological effects (pathological lesions can provide evidence for adverse effects or altered structure or function of biological system), d) susceptibility [can be a series of biological effects, such as early biological effect (enzyme, system, biomolecules), altered biological structure/function, adverse health effect or disease, neurodegeneration etc].13 Biomarkers in health and ecological risk assessment is a recent development because of advances in molecular biology and high performance and quality of analytical science (chromatographic techniques, spectroscopic and spectrometric techniques). New, more sensitive molecular and genetic biomarkers of exposure, susceptibility and adverse effect have been developed and formulated to deal with a great variety of environmental, toxicological and ecotoxicological problems. Also, new diagnostic techniques, originally developed for human diseases, can be adapted and applied to environmental problems.18-20 Case studies for typical examples of chemical pollutants and integrated risk assessment have been published in recent years. Dioxins and polycyclic aromatic hydrocarbons (Ps), which are well known as persistent and carcinogenic organic pollutants and can cause adverse health effects and environmental problems, have been investigated as a case study.21 Organotin compounds and phosphorous pesticides provided another set of important environmental pollutants for integrated risk assessment.22,23 Integrated risk assessment for trichloroethylene in groundwater was used to overcome the uncertainties in health riks.24 In recent years there are more studies of integrated risk assessment and its application in a variety of environmental and human health problems by dangerous chemicals.25-28 Two very well studied biomarkers for integrated risk assessment concerned PAHs and organophosphorous pesticides. In the case of PAHs very sensitive biomarkers of exposure and adverse effects have been reported to humans (especially with regard to their carcinogenicity).29 Similar biomarkers have been successfully adapted and used to mammals and fish.30 Organophosphorous pesticides exert acute toxicity by inhibiting acetylcholinesterase (AChE) in the neuromuscular junction, causing intoxication and respiratory failure. Measurements of peripheral AChE is a useful dose-dependent means for monitoring pesticide exposure in humans and other vertebrates.31 The biomarker AChE can be used in the mussel Mytilus edulis for the evaluation in laboratory experiments. In humans the AChE is monitored in blood samples.32 Marine Pollution, Marine Organisms and Biomarkers Marine pollution is an area of global concern owing to the large number of pollutants, their increased toxicity and persistence in the aquatic environment. In recent years the most important pollutants in the marine environment are the polychlorinated and polybrominated aromatics, which are highly toxic and environmentally persistent. These are: polychlorinated biphenyls (PCBs), polychlorinated dibenzo dioxins (PCDDs), polychlorinated dibenzo furans (PCDFs), polycyclic aromatic hydrocarbons (PAHs), Tetrabutyl

Daphnia magna

Artemia salina

Microscopic aquatic organisms (diatoms, algae) for toxicity tests

Bivalves for ecotoxicology tests

Midges as bioindicators of pollution

Toxicity testing

tin (TBT), nitroaromatic compounds, phenols, phthalate esters, organochlorine pesticides [such as dichloro diphenyl trichloroethane (DDT), hexachlorocyclohexane (HCH), aldrine, dieldrin, heptachlor etc], heavy metals [Cr (VI), Hg, Cu, Cd, Ni, Pb, Zn, etc] and other toxic substances and mixtures of waste from industry.33,34 These pollutants can become a major threat to the health of the marine ecosystem due to their accumulation in the tissues of various species. Also, the pollutants disperse through the biomagnification into higher trophic levels across the food chains reaching areas of significant human activity.35 In order to evaluate the environmental impact of these pollutants on the marine ecosystem it has become important for a rapid assessment of their toxic effects on the marine organisms. Biological markers or biomarkers measured at the molecular and cellular level is of immense importance as sensitive tools for biological adverse effects and environmental

quality assurance. The biomarkers used for this purpose have many advantages but also various limitations. Some of the most important biomarkers used for marine organisms and as subsequently for integrated environmental risk assessment are: a) cytochrome P450 (indicator of exposure to organic contaminants PAHs, PCBs, etc), b) DNA-damage (strand breaks) and bulky DNA-adducts due to exposure to mutagenic inorganic and organic xenobiotics, c) Inhibition of Acetylcholinesterase (AChE) activity (organo-phosphorous, carbamates, Cd, PB, Cu, etc), d) Metallothionein synthesis in hepatic and other tissues (exposure to metals Zn, Cu, Cd, Hg, Fe, etc), e) Antioxidant enzymes (superoxide dismuatse, catalase, glutathione transferase, exposure to ROS , free radicals, pollutants causing oxidative stress), f) Lipid peroxidation (oxidants, metals, etc), and g) Vitellogenin induction (oestrogenic substances). Some of these biomarkers have been used to identify polluted marine sites and to assess the impact of pollution in coastal marine environments.36,37 There are molecular, cellular and whole animal biomarkers which can be measured in samples of body fluids, cells or tissues. Some biomarkers are specific to a certain set of pollutants and others which change in response to both pollutants and natural factors causing oxidative stress or adverse effects to biological metabolism. Some biomarkers have prognostic value and can provide an early warning test, but also offer specificity, sensitivity and can be applied to a wide range of organisms.38,39 MOLECULAR BIOMARKERS Antioxidant enzyme defences. All biological systems generate endogenous reactive oxygen species (ROS) and other oxidants during their aerobic metabolism and energy production in the mitochondria. Pollutants with redox potential can produce increasing amounts of ROS in marine species in polluted sites. All biological system are detoxified from ROS by enzymatic and non enzymatic antioxidant defences which are ubiquitus in the tissues of most animal species. Antioxidant defences in aquatic organisms have been extensively reviewed.40-43 The modulation of enzymatic antioxidants incl;ude superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, reductase and S-transferase (GPx). Heavy metals, PAHs, TCDD, Arochlor and other pollutants can generate ROS. Measurements of enzyme activity by spectroscopic techniques. Measurement of antioxidant enzymes and lipid peroxidation in mussels can be used as sensitive biomarkers for the biomonitoring of polluted marine sites.44,45 Cytochrome P4501A (CYP1A) induction (mainly hepatic). Plays a key role in the biotransformation of pollutants, such as dioxins, PCBs and PAHs. The biomarker is widely use both in vertebrates and invertebrates for environmental biomonitoring, especially in marine bivalves and fish. The induction is triggered via the cytosolic Aryl hydrocarbon hydroxylase receptor (AHH) due to exposure to pollutants. Measurements of the induction of CYP1A in terms of 7-ethoxyresorufin O-deethylase (EROD, monooxygenase enzyme) activities is used as a biomarker for marine pollution. Measurement of CYP1A enzyme activity can be performed by fluorometric or spectrophotometric techniques, and enzyme amount can me quantified by ELISA kits.46-49 Acetylcholinesterase inhibition (AChE). AChE is an enzyme responsible for hydrolyzing the neurotransmitter acetylcholine in to choline and acetic acid. The enzyme is located in the membranes of vertebrates and invertebrates. The inhibition of the AChE is linked directly with the mechanism of toxic action of organophosphorous and carbamade insecticides, as well as metals cadmium, lead and copper. Marine bivalves (oysters and mussels) because they are filterfeeders are known to be good indicators of chemical pollution. Measurements are focused on the enzyme activity. Measurements are performed by spectrophotometric techniques and delta pH metric measurements. Studies showed seasonal variation of AChE activity in mussel gill and their changes were related to the periods of pesticide used in the polluted areas.50-52

Metallothioneins (MT) (hepatic and other tissues). Metallothieneins are peptides found mainly in the cytosol, lysosomes and in the nucleus. They are found in many aquatic invertebrates and many species of fish. They are low molecular weight peptides, high in the amino acid cysteine (which contains a thiol group (-SH). The thiol group enable MTs to bind heavy metals.53,55 Metallothioneins can be induced by essential and non-essential metals in aquatic organisms (mollusks, crustaceans). The MT induction is leading to changes in several biochemical processes that have the potential to be used as biomarkers of exposure and evaluation of pollution in the marine environment.56-58 Lysosomal stability. Lysosomes in the epithelial cells (for example in the mussels digestive gland) function as a central site for sequestration and accumulation of toixic metals and organic xenobiotics, but they also play a key role in detoxification processes and further excretion of these compounds. The accumulation of xenobiotics and their metabolic products, at cases of increasing environmental pollution, inside the lysosomes of the aquatic organisms weakens their lysosomal membrane stability and may induce the diffusion of hydrolytic lysosomal enzymes into the cytosol. Thus, environmental toxicologists proposed the measurement of lysosomal membrane stability as a rational biomarker of general interest in aquatic organisms, especially in marine bivalves.59,60 Measurements of lysosomal membrane stability (through the neutral red retention assay) has been investigated as a biomarker in blood cells of marine organisms and earthworms in pollution spills of crude petroleum and other environmental cases of organic and metal pollutants.61-65 DNAoxidative damage and DNA-adducts (DNA strand breaks, 8-OHdG, DNA-bulky adducts). Genotoxic chemicals can cause DNA strand breaks and formation of DNA adducts. Reactive intermediates of xenobiotics and genotoxic compounds can directly bind to DNA forming DNA adducts, while others, comprising free radicals and reactive oxygen species (ROS) can form alkali-labile sites on DNA. DNA strand breaks result following excision repair. It is therefore conceivable that there is a close relationship (dose-dependent and time-dependent) between DNA adduct formation and DNA strand breaks Increased oxidative damage through the generation of ROS has been established as an important mechanism for DNA damage by various environmental pollutants. PAHs, such as Benzo(a)pyrene (BaP) can be metabolized into the reactive dioloepoxide which subsequently reacts with DNA to form stable and unstable DNAadducts.66,67 In recent years there were many new developments in the formulation of analytical techniques and methodologies (such as the Comet assay) to detect DNA damage and DNAadducts (hypomethylation, FappyGua, DNA-protein crosslinks, the mutagenic DNA-adduct 8-OHdeoxyGuanosine or 8-oxo-7,8-dihydro-2-deoxyguanosine, Heavy metal binding, DNA strand breaks, etc).68-72 Lipid peroxidation (LP, oxidative stress, through free radical mechanisms). Lipid peroxidation has also been used as a bioindicator of oxidative damage in aquatic organisms exposed in polluted environmental conditions. Transition metals are a classic source of free radical generation and other reactive oxygen compounds which subsequently caused cellular damage. Metals produce hydroxyl radicals (HO) which react with membrane lipids (LH) abstracting a hydrogen and resulting in a highly reactive carbon-centered lipid radical (L) which in the propagation phase adds rapidly an oxygen to produce the lipid peroxyl radical (LOO). The peroxyl radical can abstract hydrogen atoms from a number of in vivo sources, such as DNA and proteins. Alternatively, antioxidants (such as -tocopherol) can act as protectors of cellular integrity.73-75

Direct analysis of lipid peroxidation products is complicated, so the extent of lipid peroxidation is typically assessed by measuring levels of secondary products which are formed, such as aldehydes and ketones. The most used assay for LP is the thiobarbituric acid reactive substances (TBARs) test. This test is most frequently used as a measure of malondialdehyde (MDA), a secondary lipid oxidation product. However, because thiobarbituric cid reacts with a number of other oxidation products including 4-hydroxynoneal, other unsaturated aldehydes and endoperoxides from enzymatic routes, this test is nonspecific. Therefore, the TBARs test can only a crude measure of LP. Another measure of LP is the determination of diene conjugation because LP typically involves the formation of conjugated dienes from PUFA (polyunsaturated fatty acids) substrate. These conjugated hydroperoxide products absorb UV light in the region of 230-235 nm.76,77 Vitellogenin induction (estrogenic substances): Endocrine disruption has recently emerged as a major environmental issue, in terms of both science and public policy. Various compounds, both natural and anthropogenic, when discarded in the aquatic environment cause alterations of the endocrine system of organisms. Endocrine effects, both in single organisms and at the population level, have documented after exposure to high concentrations of certain endocrine disrupters. All indications are that this problem will continue to evolve because of the controversial nature of the topic and insufficient data at present.78,79 There are numerous studies that endocrine-disrupting compounds (EDCs) are acting at every level of hormone synthesis, secretion, transport, site of action, and metabolism. Xenobiotics may act on the endocrine system by affecting transcription and signal transduction and can act through receptor-mediated or non receptor-mediated mechanisms.80, 81 The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) was formed in the USA in 1995 to develop strategies for evaluating that have the potential for endocrine disrupting activities. EDSTAC recommended a two-tiered approach, with the first tier concerned with detecting, through a battery of assays in mammalian and non mammalian organisms, endocrinological active, affecting the estrogen, androgen and thyroid hormone systems. The second tier is designed to characterize the dose-response relationship of endocrine-disrupting compounds in wildlife and humans. The in vitro tests include estrogen receptor (ER) binding/transcriptional activation, androgen receptor (AR) and steroid hormone synthesis using minced testis. In vivo tests include uterotropic assay in adult ovariectomized rat. Measures of feminization of male fish and reproductive impairment and reproductive interference are used as biomarkers for estrogenic substances. Vitellogenin induction (Vtg) biomarker system is a proposed test from OECD for male and juvenile fish.. For example, adult male zebrafish (Danio rerio) are used in flow-through system for 8 days exposed to 17-estradiol (E2) or 17-ethinylestradiol (EE2) and compared to the sensitivity of the estrogen inducible vitellogenin biomarker.82 Mollusks and other fish species can be used for testing effluents polluted with estrogenic compounds.83 Concluding remarks: Environmental Pollution, Ecotoxicology and Biomarkers Ecotoxicology studies include all aspects of environmental pollution in aquatic and terrestrial systems. Studies in aquatic and terrestrial toxicology rely heavily on interdisciplinary scientific exploration. Such research encompasses a variety of topics, including toxicity testing, sublethal responses of individual organisms, effects on populations and communities, and filed research.84 Ecotoxicology studies have established a plethora of measurement endpoints that can be used to determine exposure and effects in diferrent organisms or ecologic systems.85,86 These biological indicators of pollution may include individual-based measurements of some biochemical, physiologic or morphologic endpoint or higher-order endpoint measurements including perturbations at population or higher levels. Pollution may result in a cascade of events, beginning with effects on homeostasis in individuals and extending through population, communities, ecosystems and landscapes. The biomarkers have helped substantially to establish

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