Common biomarkers for the assessment of marine pollution
One way to assess the health of an ecosystem is to use of biomarkers to assess marine pollution. This article looks at a number of suitable biomarkers that can be used on bivalves or fish to assess the level of marine pollution.
- 1 Introduction
- 2 Biomarkers of exposure
- 3 Biomarkers of morphological damage
- 4 Biomarkers of stress
- 5 Biomarkers of genotoxicity
- 6 Biomarkers of reproductive impairment.
- 7 References
The use of biomarkers stands for a fundamental approach in the assessment of ecosystem health. It allows the early detection of biological changes due to exposure to chemical pollutants, which may result in long-term physiological disturbances. Mussels, such as Mytilus edulis and other marine bivalves, as well as fish (e.g. Mullus sp., Platichthys flesus L., Zoarces viviparus, Perca sp.) are widely used in monitoring programs as sensitive indicators, so-called sentinel organisms, of the exposure to, and the biological effects of metals and organic pollutants.        The choice of molluscs in monitoring programs is based on their wide geographic distribution, their abundance and accessability in the field as well as in aquaculture. In the case of fish, even though the sampling is expensive, the importance of their use is linked to their position in the trophic chain and their high commercial value. Both bivalves and fish strongly accumulate organic and anorganic pollutants.
Contrary to chemical monitoring of organisms, which principally evaluates the presence of pollutants in tissues by chemical analysis, biomonitoring methods evaluate not only the presence, but what is more significant, the response of the organisms to these pollutants by the assessment of biomarkers, i.e. parameters that reflect their effects at the molecular, cellular, organ, and organism level. The use of these biomarkers in monitoring does not replace chemical monitoring or population studies, but it integrates them in determining the toxic effects of pollutants, also when they are present at low, sub-lethal concentrations.
For example, initial studies concerning the toxic effects of pollutants on the cell structure and function showed morphological aberrations (Fig. 1) and changes on cell organelles, such as lipid inclusions (Fig. 2). Thes parameters are now used in monitoring programs. Some biomonitoring strategies involve the assessment of biomarkers in sentinel organisms. Two main features characterize the suitability of biomarkers for use in large research and monitoring projects (for example MED POL of United Nations, BEEP project of EU, etc.). These are a low cost combined with non-complicated and non-expensive research equipment and most important, the availabitity of the techniques in routine laboratories.
Suitable biomarker parameters that are measured in bivalves and fish as sentinel organisms are presented below. There are two main groups: biomarkers that reflect exposure and effect related biomarkers. The latter can be subdevided in biomarkers of physiological stress, of morphological damage, of genotoxicity, and of reproductive toxicity.
Biomarkers of exposure
Biomarkers of exposure include parameters that reflect exposure to a specific class of pollutants, i.e. the biomarkers of this class are specific. Tissue levels of metallothioneins,   inhibition of cholinesterase activity,   peroxisomal proliferation   mixed function oxygenases,     and EROD activity  are the main biomarkers of this category.
For example, metallothioneins (MTs) are cytosolic proteins with a high affinity for IB and IIB metal ions. They are normally expressed in animal tissues and are involved in heavy metal homeostasis. In an environment with high metal concentrations MTs are over-expressed  . This has been observed, among others, in fish and mussels.    Therefore, MTs are indicators of metal contamination and are widely used as a tool for coastal biomonitoring programs, such as the 5 year monitoring program in Thermaic Gulf.
Biomarkers of morphological damage
A number of histological biomarkers reflect morphological alterations due to contaminants,     such as thinning of the epithelium, increase in the number of epithelial basophil cells, changes in the ratio of basophil to digestive cells, the ratio between the lysosome/cytoplasm volumes.
A reduction of the digestive epithelium thickness was the main difference between exposed and reference groups. A reduction or loss of digestive synchrony was also noted after exposure to pollutants.  Changes in the proportion of digestive tubule phases, thinning of the digestive epithelium and increased volume density of basophilic cells was noted in mussels exposed to the water accommodated fraction of different oils.   Similarly, an increase in the number of epithelial basophil (secretory) cells was observed and the appearance of lipid vacuoles in both digestive and basophil cells.
Biomarkers of stress
Biomarkers related to physiological stress indicate the effects of a wide range of pollutants; they are not substance specific. Parameters are : response to other stressors, and histological alterations.
Response to chemical and non-chemical stress
This category refers to parameters able to indicate whether an there is an effect at organism level. The biomarkers stress on stress   and the scope for growth   are the main representative of this class.
The simple biomarker stress on stress provides information about the effects of environmental stressors to the organism. The parameter indicates whether contaminants have affected the capacity of molluscs to survive under stressful condition, such as during exposure to air. Its application is very simple and is calculated by the survival time when animals are exposed to air.  
Histological stress parameters
Lysosome membrane stability in cryosections, .  lysosome membrane stability-neutral red retention time, . oxidative stress / lipofuscin lysosomal content    and neutral lipid accumulation are the main biomarkers of this category.
The assessment of histological biomarkers are assessed using histochemical / cytochemical techniques, which offers the advantage of the the possibility to analyse a number of other parameters in parallel, such as lipofuscin and unsaturated neutral lipid content (Figures 1 and 2), as well as changes in basic proteins in situ.
A characteristic example of this class is lysosomal membrane stability in cryosections (LMS).   The biomarker is related to changes due to pollution in the membranes of the lysosomes, which constitute main sites of toxic metal and organic pollutant’s sequestration and detoxification.  This biomarker is mainly applied on digestive gland cryosections of mussels and is based on the cytochemical detection of the lysosomal enzyme N-acetyl-β-hexosaminidase and on the time of acid labilization treatment required to produce lysosomal maximum staining intensity (see figure 3). Minor values of the technique characterize relatively polluted marine areas, while higher ones less polluted areas.
Biomarkers of genotoxicity
Usually includes biomarkers related to the single and double strand breakages, modified bases, DNA-DNA crosslinks, and DNA-protein crosslinks. Alkaline elution and COMET assay are two of the well known biomarkers of this category.      
The micronucleus(MN) test is a cytogenetic technique commonly used for the evaluation of genotoxic effects caused by chemical stressors. Micronuclei are formed during cell division when complete or fragment chromosomes fail to be incorporated in the daughter nuclei, forming small additional nuclei. The test consists in the scoring of cells containing one or more cytosolic micronuclei in addition to the main nucleus. as well as in in different cell types of fishes.   
Biomarkers of reproductive impairment.
A variety of compounds may affect reproduction through interference with the hormonal system: the endocrine disruptors. Histological and biochemical biomarkers reflect the effects of these substances: .
Histological parameters for assesment of changes in gonad development are often used in both molluscs and fish studies. Biomarkers assessing gonad development (such as ‘simple gonad index’ or ‘changes in structure of gonad tissue’) are helpful, giving indications of the effects of pollution on the reproductive performance of animals.     
Biochemicalbiomarkers for endocrine disruption are ‘vitellogenin levels’, ‘zona radiata proteins’   and ‘steroid hormones balance’  have been regarded as useful tools for an assessment of endocrine disruption caused by chemicals in fish.  
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