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.
The use of biomarkers stands for a fundamental approach in the assessment of ecosystem health. It might allow the detection of early biological changes, which may result in long-term physiological disturbances. Mussels, such as Mytilus spp and other marine bivalves, as well as fishes (Mullus sp., Platichthys flesus L., Zoarces viviparus, Perca sp.) are widely used in both, field and laboratory experiments as sensitive bioindicators of trace metal or organic substances contamination.        The choice of molluscs in monitoring programs is taken on the basis of their wide geographic distribution, their non-complicated availability in the field and aquacultures, while in the case of fishes, even though there are logistic problems related to the cost of sampling, caging, transportation, etc, the importance of their use is linked to their key position in the trophic chain and their high commercial value.
Contrary to chemical monitoring, which principally evaluates the presence of pollutants in cells and tissues using chemical analyses, biomonitoring methods evaluate not only the presence, but what is more significant, the response of the organisms to these pollutants assessing their effects at the molecular, cellular, tissue/organ, and organism level. The use of biomarkers does not replace chemical monitoring or strategies using population studies, but it integrates them providing an unique contribution in determining the toxicity of pollutants, even when they are present at low, sub-lethal concentrations.
Initial studies concerning the toxic effects of pollutants on the cell structure and function showed changes on cell compounds, such as lysosomes, lipids inclusion, peroxisomes etc., as well as on physiological responses, for example the reproductive performance of the animals. The progression and the advances of aquatic toxicology in the recent years allowed the development of biomonitoring strategies that employed sentinel organisms and biomarkers. Two main features characterize the biomonitoring techniques regarding their suitability for usage by large research projects (for example MED POL of United Nations, BEEP project of EU, etc.). The first is their low coast combined to relatively non-complicated and non-expensive research equipments and the second and most important, their proposal and use by many researchers and laboratories.
Below are listed in some detail examples of suitable biomarkers to be utilized on bivalves or fish as sentinel organisms:
Biomarkers of stress
Usually this class includes biomarkers related to the general physiological stress that the organisms undergo due to a wide range of pollutants, i.e., the biomarkers of this class are not specific. ‘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 application of these biomarker needs histochemical / cytochemical approach and frozen sections, giving an advantage related to the possibility to analyse a number of other parameters in parallel, such as lipofuscin and unsaturated neutral lipids contents (see 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 figures 3 and 4). 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 test’ (MN) is a well known cytogenetic technique commonly used for the evaluation of genotoxic effects caused by environmental stressors. Micronuclei are formed during cell division when complete or fragment chromosomes fail to be incorporated in the daughter nuclei, forming so small or more additional nuclei. The test consists in the scoring of cells containing one or more cytosolic micronuclei in addition to the main nucleus and has been applied in wild and caged marine invertebrates,  as well as in in different cell types of fishes.   
Biomarkers of exposure
Usually includes biomarkers whose changes can be related to the organism 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.
As an example, ‘levels of metallothioneins’ (MTs) refer to low-molecular-weight cytosolic proteins rich in sulphyhydryl (-SH) residues, with a high affinity for IB and IIB metal ions. These metalloproteins are normally expressed in animal tissues and are involved in heavy metal homeostasis, while in environments with high metal concentrations are over-expressed   both in fish, and mussels.    Therefore, MTs have been regarded as an indicator of metal contamination and widely used as a tool for biomonitoring programs (Fig. 5). 
This class usually includes biomarkers whose changes can be related to morphological alterations suggestive of harmful changes 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 at the organism level
This category refers to parameters able to indicate whether an effect at organism/population level is produced. The biomarkers ’stress on stress’   and the ‘scope for growth’   are the main representative of this class.
The effects of environmental stressors at the organism level can be evaluated also by the use of the simple biomarker ’stress on stress’, assessing whether contaminants have affected the capacity of molluscs to survive to environmental change, such as the air exposure. Its application is very simple and is calculated by the survival time when animals are exposed to air.  
Reproductive performance and endocrine disruptors
This class refers to fundamental aspects of research related to the biological effects of pollutants, such as the gonad development related to the reproductive performance of the animals and endocrine disruptors, related to the interference of the hormonal pathway inside the cells.
Parameters assessing changes in gonad development are often used for 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.     
Concerning endocrine disruption, biomarkers such as ‘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.  
Recent studies enhance the use of caged organisms in biomonitoring studies. Using caged organisms for 3-4 weeks (mainly mussels or fishes), the standardization of the results and the comparison of control organisms to animals collected from polluted sites is easier. Nowadays, a ‘2-tier approach’ has been proposed aimed at developing a cost-effective strategy which environmental agencies can afford.  The application of this approach comprises an initial screening of the various sampling station using a highly sensitive low-cost biomarker, such as lysosomal membrane stability (Tier 1). Only in the areas that an alteration in lysosomal stability has been established, the general stress of the animals is then to be assessed by utilizing the full battery of biomarkers (Tier 2). The data collected should be then run through an “expert system”   software to obtain an objective assessment of the pollutant induced stress syndrome.
- Krishnakumar, P.K., Casillas, E., Varanasi, U., 1994. Effect of environmental contamination on the health of Mytilus edulis from Puget Sound, Washington: cytochemical measures of lysosomal responses in the digestive cells using automatic image analysis. Marine Ecology Progress Series 106, 249–261.
- Regoli, F., Orlando, E., 1994a. Seasonal variation of trace metal concentrations in the digestive gland of the Mediterranean mussel Mytilus galloprovincialis: comparison between a polluted and a non-polluted site. Archives of Environmental Contamination andToxicology 27, 36–43.
- Regoli, F., Orlando, E., 1994b. Accumulation and subcellular distribution of metals (Cu, Fe, Mn, Pb and Zn) in the Mediterranean mussel Mytilus galloprovincialis during a field transplant experiment. Marin Pollution Bulletin 28 (10), 592–600.
- Walsh, K., Dunstan, R.H., Murdoch, R.N., 1995. Differential bioaccumulation of heavy metals and organopollutants in the soft tissue and shell of the marine gastropod,Austrocochlea constricta. Archives of Environmental Contamination and Toxicology 28, 1095 35–39.
- Viarengo, A., Ponzano, E., Dondero, F., Fabbri, R., 1997a. A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs. Marine Environmental Research 44 (1), 69–84.
- Regoli, F., 1998. Trace metals and antioxidant enzymes in gills and digestive gland of the Mediterranean mussel Mytilus galloprovincialis. Archives of Environmental Contamination and Toxicology 991 34, 48–63.
- Lafontaine, de Y., Gagne, F., Blaise, C., Costan, G., Gagnon, P., Chan, H.M., 2000. Biomarkers in zebra mussels (Dreissena polymorpha) for the assessment and monitoring of water quality of the St Lawrence river (Canada). Aquatic Toxicology 50, 51–71.
- Moore, M.N., 1985. Cellular responses to pollutants. Marine Pollution Bulletin 16, 134–139.
- Viarengo, A., Moore, M.N., Mancinelli, G., Mazzucotelli, A., Pipe, R.K., Farrar, S.V., 1987. Metallothioneins and lysosomes in metal toxicity and accumulation in marine mussels: the effect of cadmium in the presence and absence of phenanthrene. Marine Biology 94, 251–257.
- Lowe, D.M., Pipe, R.K., 1994. Contaminant induced lysosomal membrane damage in marine mussel digestive cells: an in vitro study. Aquat. Toxicol. 30, 357-365.
- George, S.G., Viarengo, A., 1985. A model for heavy metal homeostasis and detoxication in mussel. Mar. Pollut. Physiol.: Recent advances, 125-143.
- Viarengo, A., 1989. Heavy metals in marine invertebrates: mechanisms of regulation and toxicity at the cellular level. CRC Critical Reviews in Aquatic Science 1, 295–317.
- Viarengo, A., Nott, J.A., 1993. Mechanisms of heavy metal cation homeostasis in marine invertebrates. Comparative Biochemistry and Physiology 104, 355–372.
- Koehler, A., Deisemann, H., Lauritzen, B., 1992. Histological and cytochemical indices of toxic injury in the liver of the dab Limanda limanda. Mar. Ecol. Prog. Ser. 91, 141-153.
- Koehler, A., 2004. Toxic injury and gender-specific hepatocellular carcinogenesis in flounder (Platichthys flesus (L.). Aquat. Toxicol. 70, 257-276.
- Moore, M.N., 1976. Cytochemical demonstration of latency of lysosomal hydrolases in digestive cells of the common mussel Mytilus edulis, and changes induced by thermal stress. Cell Tissue Research 175 (3), 279–287.
- UNEP/RAMOGE, 1999. Manual on the biomarkers recommended for the MED POL biomonitoring programme. UNEP, Athens, Greece.
- Moore, M.N., 1985. Cellular responses to pollutants. Marine Pollution Bulletin 16, 134–139.
- Batel, R., Vukmirovic, M., Jacsic, Z., Bihari, N., 1994. Impact of pollution on DNA fragmentation in marine invertebrates. Use of Aquatic Invertebrates as Tools for Monitoring the Environmental Hazards, 109-117.
- Bolognesi, C., Landini, E., Roggieri, P., Fabbri, R., Viarengo, A., 1999. Genotoxicity biomarkers in the assessment of heavy metal effects in mussels: experimental studies. Environ. Mol. Mutagen. 33, 287-292.
- Bolognesi, C., Frenzilli, G., Lasagna, C., Perrone, E., Roggieri, P., 2004. Genotoxicity biomarkers in Mytilus galloprovincialis: wild versus caged mussels. Mutat. Res. 552, 187-196.
- Bolognesi, C., Perrone, E., Roggieri, P., Pampanin, D., Sciutto, A., 2006. Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions. Aquat. Toxicol. 78, S93-98.
- Lee, R.F., Steinert, S., 2003. Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat. Res. 544, 43-64.
- Frenzilli, G., Scarcelli, V., Del Barga, I., Nigro, M., Forlin, L., Bolognesi, C., Sture, J., 2004. DNA damage in Eelpout (Zoarces viviparous) from Goteborg harbour. Mutat. Res. 552, 153-162.
- Majone, F., Brunetti, R., Fumagalli, O., Gabriele, M., Levis, A.G., 1990. Induction of micronuclei by mitomycin C and colchicine in the marine mussel Mytilus galloprovincialis. Mutat. Res. 244, 147-151.
- Scarpato, R., Migliore, L., Alfinito-Cognetti, G., Barale, R., 1990. Induction of micronuclei in gill tissue of Mytilus galloprovincialis exposed to polluted marine waters. Mar. Pollut. Bull. 21, 74-80.
- Al-Sabti, K., Metcalfe, C.D., 1995. Fish micronuclei for assessing genotoxicity in water. Mutat. Res. 343, 121-135.
- Arkhipchuk, V.V., Garanko, N.N., 2005. Using the nucleolar biomarker and the micronucleus test on in vivo fish fin cells. Ecotoxicol. Environ. Saf. 62, 42-52.
- Barsiene, J., Lehtonen, K. K., Koehler, A., Broeg, K., Vuorinen, P.J., Lang, T., Pempkowiak, J., Šyvokien_, J., Dedonyte, V., Rybakovas, A., Repeka, R., Vuontisjärvi, H., Kopecka,J., 2006. Biomarker responses in flounder (Platichthys flesus) and mussel (Mytilus edulis) in the Klaipeda-B_ting_ area (Baltic Sea). Mar. Poll. Bull. 53, 422-436.
- Viarengo, A., Burlando, B., Dondero, F., Marrò, A., Fabbri, R., 1999b. Metallothionein as a tool in biomonitoring programmes. Biomarkers 4, 455-466.
- Sturm, A., da Silva de Assis, H.C., Hansen, P.D., 1999. Cholinesterases of marine teleost fish: enzymological characterization and potential use in the monitoring of neurotoxic contamination. Mar. Environ. Res. 47, 389-398.
- Sanchez-Hernandez, J.C., Moreno-Sanchez, B., 2002. Lizard cholinesterases as biomarkers of pesticide exposure: enzymological characterization. Environ. Toxicol. Chem. 21, 2319-2325.
- Rodriguez-Fuentes, G., Gold-Bouchot, G., 2004. Characterization of cholinesterase activity from different tissues of Nile tilapia (Oreochromis niloticus). Mar. Environ. Res. 58, 505-509.
- Cajaraville, M.P., Cancio, I., Ibabe, A., Orbea, A., 2003. Peroxisome proliferation as a biomarker in environmental pollution assessment. Microsc. Res. Tech. 61, 191-202.
- Orbea, A., Cajaraville, M.P., 2006. Peroxisome proliferation and antioxidant enzymes in transplanted mussels of four basque estuaries with different levels of polycyclic aromatic hydrocarbon and polychlorinated biphenyl pollution. Environ. Toxicol. Chem. 25, 1616-1626.
- Payne, J.F., 1976. Field evaluation of benzopyrene hydroxylase induction as a monitor for marine petroleum pollution. Science 191, 945-946.
- Stegeman, J.J., Hahn, M.E., 1994. Biochemistry and molecular biology of monooxygenase: current perspective on forms, functions, and regulation of cytochrome P450 in aquatic species. In: Malins, D.C., Ostrander, G.K., (Eds.), Aquatic toxicology: molecular, biochemical and cellular perspectives. Lewis Publishers, Boca Raton, FL, pp. 87-206.
- Bucheli, T.D., Fent, K., 1995. Induction of cytocrome P450 as a biomarker for environmental contamination in aquatic ecosystem. Crit. Rev. Environ. Sci. Technol. 25, 201-268.
- Goeptar, A.R., Scheerens, H., Vermeulen, N.P.E., 1995. Oxygen reductase and substrate reductase activity of cytochrome P450. Crit. Rev. Toxicol. 25, 25-65.
- Omura, T., Sato, R., 1964. The carbon monoxide-binding pigment of liver microsomes, I and II. J. Biol. Chem. 239, 2370-2385.
- Viarengo, A., Bettella, E., Fabbri, R., Burlando, B., Lafaurie, M., 1997b. Heavy metal inhibition of EROD activity in liver microsomes from the bass Dicentrarchus labrax exposed to organic xenobiotics: role of GSH in the reduction of heavy metal effects. Mar. Environ. Res. 44, 1-11.
- Bremner, I., 1987. Interactions between metallothionein and trace elements. Prog. Food Nutr. Sci. 11, 1-37.
- Webb, M., 1987. Toxicological significance of metallothinein. EXS 52, 109-134.
- Lowe, D.M., Moore, M.N., Clarke, R.K., 1981. Effects of oil on digestive cells in mussels:quantitative alterations in cellular and lysosomal structure. Aquat. Toxicol. 1, 213-226.
- Lowe, D. M., 1988. Alteration in the cellular structure of Mytilus edulis resulting from exposure to environmental contaminants under field and experimental conditions. Mar. Ecol. Prog.Ser. 46, 91-100.
- Cajaraville, M.P., Marigomez, J.A., Angulo, E., 1990. Short-term toxic effects of 1-naphthol on the digestive gland-gonad complex of the marine prosobranch Littorina littorea (L): a light microscopic study. Arch. Environ. Contam. Toxicol. 19, 17-24. 37.
- Cajaraville, M.P., Marigomez, J.A., Diez, G., Angulo, E., 1992. Comparative effects of the water accommodated fraction of three oils on mussels. 2. Quantitative alterations in the structure of the digestive tubules. Comp. Biochem. Physiol. C 102, 113-123.
- Viarengo, A., Canesi, L., Pertica, M., Mancinelli, G., Accomando, R., Smaal, A.C., Orunesu, M., 1995. Stress on stress response: a simple monitoring tool in the assessment of general stress syndrome in mussels. Mar. Environ. Res. 39, 245–248.
- Pampanin, D.M., Volpato, E., Marangon, I., Nasci, C., 2005. Physiological measurements from native and transplanted mussel (Mytilus galloprovincialis) in the canals of Venice. Survival in air and condition index. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 140, 41-52.
- Widdows, J., Donkin, P., 1992. Mussels and environmental contaminants: bioaccumulation and physiological aspects. In: Gosling, E., (Ed.), The mussel Mytilus: ecology,physiology, genetics and culture. Elsevier, Amsterdam, pp. 383-424.
- Widdows, J., Donkin, P., Staff, F. J., Matthiessen, P., Law, R. J., Allen, Y. T., Thain, J. E., Allchin, C. R., Jones, B. R., 2002. Measurement of stress effects (scope for growth) and contaminant levels in mussels (Mytilus edulis) collected from the Irish Sea. Mar. Environ. Res. 53, 327-356.
- Lowe, D., Pipe, R.K., 1985. Cellular responses in the mussel Mytilus edulis following exposure to diesel oil emulsions: reproductive and nutrient storage cells. Mar. Environ. Res. 17, 234-237.
- Kime, D.E., 1995. The effects of pollution on reproduction in fish. Rev. Fish Biol. Fish. 5, 52-96.
- Minier, C., Levy, F., Rabel, D., Bocquené, G., Godefroy, D., Burgeot, T., Leboulenger, F., 2000. Flounder health status in the Seine Bay. A multibiomarker study. Mar. Environ. Res. 50, 373-377.
- Van der Oost, R., Beyer, J., Vermeulen, N.P.E., 2003. Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ. Toxicol. Pharmacol. 13, 57-149.
- Aarab, N., Lemaire-Gony, S., Unruh, E., Hanson, P.D., Larsen, B.K., Andersen, O.K., Narbonne, J.F., 2006. Preliminary study of responses in mussels (Mytilus edulis)
- Haux, C., Björnsson, B.T., Förlin, L., Larsson, Å., Deftos, L.J., 1988. Influence of cadmium exposure on plasma calcium, vitellogenin and calcitonin in vitelogenic rainbow trout. Mar.Environ. Res. 24, 199-210.
- Spies, R.B., Stegeman, J.J., Rice, D.W., Jr., Woodlin, B., Thomas, P., Hose, J.E., Cross, J.N., Prieto, M., 1990. Sublethal responses of Platichtus stellatus to organic contamination in San Francisco Bay with emphasis on reproduction. In: McCarthy, J. F., Shugart, L. R.(Eds.), Biomarkers of Environmental Contamination. CRC Press, Boca Raton, FL, pp. 87-122.
- Karels, A., Soimasuo, M., Lappivaara, J., Leppänen, H., Aaltonen, T., Mellanen, P., Oikari, A.,1998. Effects of bleached kraft mill effluent on reproductive steroids and liver MFO activity in populations of perch and roach. Ecotoxicology 7, 123-132.
- Armstrong, D.T., 1990. Environ. stress and ovarian function. Biol. Reprod.34, 29-39.
- Martin-Skilton, R., Lavado, R., Thibaut, R., Minier, C., Porte, C., 2006. Evidence of endocrine alteration in the red mullet, Mullus barbatus from the NW Mediterranean. Environ. Pollut.141, 60-8.
- Viarengo, A., Lowe, D., Bolognesi, C., Fabbri, E., Koehler, A., (in press). The use of biomarkers in biomonitoring: A 2-tier approach assessing the level of pollutant-induced stress syndrome in sentinel organisms. Comp.Biochem. & Physiol.
- Viarengo, A., Burlando, B., Giordana, A., Bolognesi, C., Gabrielides, G.P., 2000. Networking and expert-system analysis: next frontier in biomonitoring. Mar. Environ. Res. 49, 483-486.
- Dagnino, A., Allen, J.I., Moore, M.N., Broeg, K., Canesi, L., Viarengo, A., 2007. Development of an expert system for the integration of biomarker responses in mussels into an organism animal health index. Biomarkers 12, 155-172.
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