Ecotoxicological Testing of the Marine and Freshwater Ecosystems: Emerging Techniques, Trends, and Strategies - Chapter 3

chapter three Biomarkers in environmental assessment R. van der Oost, C. Porte-Visa, and N.W. van den Brink Contents Abstract .................................................................................................................89 Introduction ..........................................................................................................91 Biomarkers overview ..........................................................................................94 Phase I biotransformation enzymes ........................................................95 Phase II biotransformation enzymes .......................................................96 Oxidative stress parameters ......................................................................97 Biotransformation products ......................................................................97 Stress proteins, metallothioneins, and multixenobiotic resistance .....98 Hematological parameters ........................................................................98 Immunological parameters .......................................................................99 Reproductive and endocrine parameters ...............................................99 Neuromuscular parameters ......................................................................99 Genotoxic parameters ..............................................................................100 Physiological and morphological parameters .....................................100 Proteomics and genomics ........................................................................101 Invertebrate biomarkers ...................................................................................101 Phase I enzymes ........................................................................................102 Oxidative stress .........................................................................................102 Stress proteins, metallothioneins, and multixenobiotic resistance ...103 Reproductive and endocrine parameters .............................................104 Neuromuscular parameters ....................................................................104 Genotoxic parameters ..............................................................................104 Physiological and morphological parameters .....................................104 Fish biomarkers ..................................................................................................105 Phase I enzymes ........................................................................................105 Phase II enzymes ......................................................................................106 87 © 2005 by Taylor & Francis Group, LLC 88 Ecotoxicological testing of marine and freshwater ecosystems Oxidative stress parameters ....................................................................106 Biotransformation products ....................................................................107 Stress proteins, metallothioneins, and MXR ........................................108 Reproductive and endocrine parameters .............................................108 Neuromuscular parameters ....................................................................109 Genotoxic parameters ..............................................................................109 Physiological and morphological parameters .....................................110 Amphibian biomarkers .....................................................................................110 Phase I enzymes ........................................................................................ 111 Phase II enzymes ...................................................................................... 111 Oxidative stress .........................................................................................112 Hematological parameters ......................................................................112 Stress proteins ...........................................................................................112 Reproduction and endocrine parameters .............................................112 Neuromuscular parameters ....................................................................113 Physiological and morphological parameters .....................................113 Mammalian and avian biomarkers ................................................................114 Phase I enzymes ........................................................................................115 Phase II enzymes ......................................................................................115 Hematological parameters ......................................................................116 Stress proteins and metallothioneins ....................................................116 Endocrine parameters ..............................................................................116 Neuromuscular parameters ....................................................................117 Morphological and histological parameters ........................................117 Summary, discussion, and conclusions ..........................................................118 Invertebrates ..............................................................................................119 Fish ..........................................................................................................119 Amphibians ...............................................................................................120 Birds and mammals .................................................................................120 Perspectives and recommendations ...............................................................121 References ...........................................................................................................123 Appendix 1 .........................................................................................................138 Overview of all biomarker assays ..................................................................138 Appendix 2 .........................................................................................................141 Standard operating procedures of biomarker assays ..................................141 SOP 1: Isolation of microsomes and cytosol from liver tissues ................141 Solutions .....................................................................................................141 Further requirements ...............................................................................142 Needed per sample ..................................................................................142 Procedure ...................................................................................................142 SOP 2: EROD activity in liver microsomes ...................................................142 Principle .....................................................................................................142 Solutions .....................................................................................................143 Further requirements ...............................................................................143 Needed per determination (duplo) .......................................................143 Procedure ...................................................................................................143 © 2005 by Taylor & Francis Group, LLC Chapter three: Biomarkers in environmental assessment 89 Calculations ...............................................................................................144 SOP 3: Cyt P-450 and Cyt b5 Contents of Liver Microsomes ...................144 Principle .....................................................................................................144 Solutions .....................................................................................................144 Further requirements ...............................................................................144 Needed per determination (duplo) .......................................................144 Procedure for cyt b5 .................................................................................145 Procedure for cyt P-450 ...........................................................................145 Calculations ...............................................................................................145 SOP 4: GST Activity of Liver Cytosol ............................................................146 Principle .....................................................................................................146 Solutions .....................................................................................................146 Further requirements ...............................................................................146 Needed per determination (duplo) .......................................................146 Procedure ...................................................................................................146 Calculations ...............................................................................................147 SOP 5: SOD Activity in Liver Cytosol ...........................................................147 Principle .....................................................................................................147 Solutions .....................................................................................................147 Further requirements ...............................................................................147 Needed per determination (duplo) .......................................................148 Procedure ...................................................................................................148 Calculations ...............................................................................................148 SOP 6: ACHE Activity in Tissue Homogenates ...........................................148 Principle .....................................................................................................148 Solutions .....................................................................................................149 Further requirements ...............................................................................149 Needed per determination (triplicates in microplate method) .........149 Procedure ...................................................................................................149 Calculations ...............................................................................................150 SOP 7: Lysosomal Membrane Stability in Cells ...........................................151 Principle .....................................................................................................151 Solutions and chemicals ..........................................................................151 Further requirements ...............................................................................151 Procedure ...................................................................................................151 Interpretation of results ...........................................................................152 Abstract It has been demonstrated that an environmental risk assessment based solely on pollutant levels in the environment is generally not considered to be reliable. There is a growing awareness that risk assessors have to focus on the effects of the total mixture of contaminants present in the environment, instead of on the pres-ence of some selected compounds. Biomarkers are defined as © 2005 by Taylor & Francis Group, LLC 90 Ecotoxicological testing of marine and freshwater ecosystems changes in biological responses (ranging from molecular through cellular, and from physiological responses to behavioral changes) that can be related to exposure to, or to the toxic effects of, envi-ronmental chemicals. The main objective of this chapter is to provide an overview of studies that use biomarkers for the as-sessment of exposure and toxic impact of environmental contam-inants to various organisms. The possibilities for biomarkers to detect, identify, and assess pollutant exposure, primary actions, and effects on invertebrates, fish, amphibians, birds, and mam-mals are discussed in separate sections and reviewed for site-spe-cific case studies. The use of biomonitoring methods in the control strategies for chemical pollution has advantages over chemical monitoring. First, these methods measure effects in which the bioavailability of the compounds of interest is integrated with the concentration of the compounds and their intrinsic toxicity. Second, most bio-logical measurements form the only way of integrating the effects on a large number of individual and interactive processes. Al-though it has been demonstrated that biomarkers are useful mon-itoring tools, it is clear that more information is needed about the relation between biomarker responses and the health and fitness of organisms, and even more so between biomarker responses and the risks for the ecosystem. A limitation of most of the bio-logical-effect measurements is that biomarker data interpretation must always be carefully controlled for false-negative and false-positive results, since the effects of non–pollution-related confounding factors may interfere with biomarker responses. With respect to future biomarker research, it is important to real-ize that different concepts are needed for the specific purposes of environmental monitoring programs. In conclusion, it can be stated that biomarkers are promising tools for environmental risk assessment (ERA). In view of the present chemically oriented pollution-abatement policies and the need to reveal specific chemical problems, it is most probable that biolog-ical-effect analysis will never totally replace chemical analyses. The biomarker approach, therefore, should not be considered as a replacement for conventional assessment techniques, but as an important supplementary approach of great ecological relevance. Several guidelines for ecotoxicological research leading to an ac-tual incorporation of biomarkers in ERA monitoring are proposed in this chapter. Much work has to be done in order to test and interpret biomarker responses and to develop acceptable quality assurance (QA) procedures. Only when both scientific and legal credibility of this information is established can the biomarker © 2005 by Taylor & Francis Group, LLC Chapter three: Biomarkers in environmental assessment 91 techniques be fully applied in routine monitoring programs. Since it seems obvious that chemical monitoring alone is insufficient for a reliable classification of water quality, the efforts to incorpo-rate biological compounds to the ERA research will eventually be worthwhile. Introduction Environmental risk assessment (ERA) in its classic form was mainly focused on the relationship between partitioning of toxic substances in the environ-ment and the potential hazards of these pollutants if they exceed certain threshold levels. In the last decades, however, it has been demonstrated that a risk assessment based solely on pollutant levels in the environment is not considered to be reliable, partly due to the ability of various pollutants (and their derivatives) to mutually effect their toxic actions. There is a growing awareness that risk assessors have to focus on the effects of the total mixture of contaminants present in the environment, instead of on the fate of some selected compounds. The main objective of this chapter is to provide an overview of studies that use biomarkers for the assessment of exposure and toxic impact of environmental contaminants to various organisms. The term biomarker is generally used in a broad sense to include almost any measurement that reflects an interaction between a biological system and a potential hazard, whether chemical, physical, or biological (WHO 1993). Many more biomarker definitions can be found in the literature. A biomarker has been defined as a change in a biological response (ranging from molecular through cellular and from physiological responses to behav-ioral changes) that can be related to exposure to, or the toxic effects of, environmental chemicals (Peakall 1994). Van Gestel and van Brummelen (1996) defined a biomarker as any biological response to an environmental chemical at the subindividual level, measured inside an organism or in its products (urine, feces, hair, feathers, and so on), indicating a deviation from the normal status that cannot be detected in the intact organism. When biological responses are measured using whole organisms, Van Gestel and van Brummelen (1996), as opposed to most others, refer to bioindicators instead of biomarkers. Although most biomarkers discussed in this chapter are measured at molecular to tissue levels, the definition by the World Health Organization (WHO 1993) is preferred since it is not focused on a specific integration level. A pollutant stress situation normally triggers a cascade of biological responses, each of which may, in theory, serve as a biomarker (McCarthy et al. 1991). Above a certain threshold (in pollutant dose or exposure time) the pollutant-responsive biomarker signals deviate from the normal range in an unstressed situation, finally leading to the manifestation of a multiple-effect situation at higher hierarchical levels of biological organization. When del-eterious effects on populations or ecosystems become clear, the destructive process has often gone too far to save the ecosystem by remedial actions or © 2005 by Taylor & Francis Group, LLC ... - tailieumienphi.vn