Biomarcadores bioquímicos de exposición en bivalvos como herramientas para la evaluación de la salud ambiental frente a la contaminación de plaguicidas y metales pesados, en ecosistemas marino-costeros del Caribe Colombiano
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Abstract
El estudio de biomarcadores, definidos como el “cambio en diferentes tipos de respuestas biológicas, relacionadas con diversos niveles de exposición o efectos tóxicos de sustancias químicas, principalmente aquellas de tipo xenobiótico” son un importante componente en la evaluación de la salud ambiental de ecosistemas. Sin embargo, en Colombia, este campo de investigación hasta ahora ha tomado relevancia y son pocas las investigaciones que se han desarrollado sobre su uso en programas de monitoreo.
El objetivo principal de la presente disertación fue el de evaluar la utilidad de cuatro biomarcadores bioquímicos (Metalotioneína- MT, y la actividad de tres enzimas, Colinesterasa- ChE, Catalasa- CAT y Anhidrasa Carbónica- AC) como una técnica alternativa para estimar la salud ambiental de ecosistemas costeros en el Caribe Colombiano, con énfasis en exposición a metales pesados y plaguicidas. Para ello, se utilizaron ostras (Crassostrea sp. y Saccostrea sp.) como especie indicadora, escogiendo tres tejidos (branquias, glándula digestiva y músculo aductor). La investigación se desarrolló en dos fases: una de laboratorio, para medir en condiciones controladas la respuesta concentración-dependiente sub-letal de los biomarcadores a la exposición de dos metales (cadmio y cobre) y dos plaguicidas (clorpirifós- Clo e imidacloprid- Im), en Saccostrea sp. La segunda fase midió las respuestas de MT y ChEs en ostras colectadas en campo (poblaciones mixtas de Crassostrea sp. y Saccostrea sp.), en 5 sitios intermareales del Caribe Colombiano (Taganga, la Marina de Santa Marta, Bahía de Cartagena (Ecopetrol y Contecar) y la isla de Barú, en la Bahía de Barbacoas). Los análisis de los biomarcadores se acompañaron de análisis de metales en sedimento y en tejidos de ostras (As, Cd, Cr, Cu, Hg, Ni, Pb, Sn y Zn), así como de un barrido de 42 plaguicidas (22 organofosforados -OP y 20 organoclorados- OCP).
Los resultados de los bioensayos mostraron respuestas
Summary in foreign language
The study of biomarkers, defined as the "change in different types of biological responses, related to various levels of exposure or toxic effects of chemical substances, mainly those of xenobiotic type" is a valuable component in the diagnosis of environmental health of ecosystems. However, in Colombia, this field of research is still in development and few investigations have been conducted employing biomarkers in field-based monitoring programs.
The main objective of the present dissertation was to evaluate the utility of four biochemical biomarkers (metallothionein-MT, cholinesterase activity-ChE, catalase activity-CAT and carbonic anhydrase activity-AC), as complements to estimating the environmental health of coastal ecosystems in the Colombian Caribbean, with a specific focus on exposure to heavy metals and pesticides. For this, cup oysters (Crassostrea sp. and Saccostrea sp.) were used as indicator species, with biomarkers analyzed in three target tissues (gills, digestive gland and adductor muscle). The research was carried out in two stages: first, in the laboratory, to measure under controlled conditions, the sub-lethal concentration response of biomarkers to the exposure of two metals (cadmium and copper) and two pesticides (chlorpyrifos, Clo, and imidacloprid, Im), in individuals of Saccostrea sp. The second stage measured the responses of MT and ChE activity in field-collected oysters (mixed populations of Crassostrea and Saccostrea), from five intertidal sites in the Colombian Caribbean coast (Taganga Bay, Santa Marta Marina, Cartagena Bay (Ecopetrol and Contecar station) and Barbacoas Bay (peninsula of Barú). The analysis of the biomarkers was accompanied by analysis of metals (As, Cd, Cr, Cu, Hg, Ni, Pb, Sn and Zn), as well as a suite of 42 pesticides (22 organophosphates - OP and 20 organochlorines- OCP) in sediment and in oyster tissues. To test for correlation between biomarker responses and environmental covariables (temperature, salinity, pH and dissolved oxygen concentration, as well as metal concentrations in tissues and sediments), a suite of multivariate techniques (principal component analysis and stepwise multiple regression) was used to partition variance contributions and to identify explanatory variables.
The results of the laboratory bioassays showed sensitive and differential responses of the four biomarkers in oysters exposed to the four pollutants. For MT, an increase in MT concentrations was observed at Cd concentrations > 100 μg / L (correlating with Cd bioaccumulation in tissues), while for Cu, Clo and Im, no significant changes in MT concentrations were apparent. In the case of cholinesterase activity (ChEs), it was possible to discriminate three different functional sub-fractions showing differential responses to the four pollutants, termed total cholinesterase activity (T-ChE), cholinesterase activity sensitive to the inhibitor eserine (Es-ChE) and cholinesterase activity resistant to eserine (Er- ChE). T-ChE and Es-ChE activity were diminished at high concentrations of Clo (100 mg / L). Similarly, exposure to Cd and Cu (> 100 μg / L) as well as Im (100 mg / L) resulted in significant, albeit differential, changes, in the activity of the three ChE fractions. For catalase (CAT) and carbonic anhydrase (CA), large variability in enzyme activity was noted and no marked linear dose-dependent responses were identified, except for a significantly higher CAT activity in the digestive gland at higher Cu concentrations. On the other hand, the responses of CAT activity in digestive gland (exposed to Cu) and of AC activity in digestive gland (Cu) and gills (Cu, Clo, Im) appeared to conform to non-linear Michaelis-Menten kinetics, with marked increases in enzyme activity at lower contaminant concentrations and apparent saturation at elevated concentrations. The tissues that responded with greater sensitivity to the four xenobiotics were digestive gland and gills, while adductor muscle typically showed lower activities and concentration-invariant responses. The results of the laboratory experiments allowed exploring the behavior and sensitivity of the four biomarkers and helping define likely response ranges and patterns for the four biomarkers in a Colombian bivalve species.
The field results showed that the concentrations of OP and OCP in tissue and sediment were below the limits of detection in all the sampling sites. As for metals, the concentrations of As, Cd, Cu and Hg in sediment exceeded the TEL (threshold effects level, according to Buchman, 2008) in several locations, whereas the concentrations of Cd, Cu and Pb in tissues were in the ¨mid” concentration range of the NOAA Mussel Watch program (reference species C. virginica). As a notable exception, Cd tissue concentrations exceeded the NOAA "high" range, with 15 μg / g dw at Barú Island and Contecar, in addition to exceeding the maximum limit for human consumption established by the Colombian National Resolution 122 of 2012.
MT concentrations and ChE activities in the gills and digestive glands were within the range previously reported for other species of bivalves from other regions. For MT, the concentrations in both tissues varied between sites by a factor of 5 to 6, with the highest averages at Barú Island and the Bay of Cartagena. For ChE activity (measured in three sub-fraction), high spatial and temporal variation was observed, with overall greater activities for Contecar, Taganga and Marina, and lowest activities for Barú.
Statistical analysis (principal component analysis - PCA) was used to group environmental covariables into two general types of environmental gradients: two physicochemical (“natural”) gradients, represented by pH, temperature, dissolved oxygen and salinity, and three metal (“pollution”) gradients, represented by metal concentrations in tissues and sediments. Stepwise multiple regression analysis, using individual environmental variables as well as the consolidated PCA gradients, identified different sets of predictor variables for the different biomarker responses, with metal variables and the three metal PCA gradients prevailing as predictor variables over the physical-chemical variables or consolidated PCA axes. For example, concentrations of MT in digestive gland and gills correlated positively with the concentrations of Cd and Ni in whole oyster tissues as best predictor variables, whereas total ChE activity in digestive gland correlated positively with tissue Pb, and T-ChE activity in gills correlated positively with tissue Zn. Analogously, Es-ChE activity in digestive gland correlated positively with tissue Pb and Hg and negatively with tissue Cd and Sn as significant predictor variables, whereas Es-ChE activity in gills was best predicted by a positive correlation with tissue Zn. Nevertheless, statistical and ecologically significant covariance was also observed between the concentration of MT in oyster tissues (digestive gland and gills) and water pH (positive), and between the total ChE activity and salinity (negative correlation) and temperature (positive correlation), indicating that both metal contaminants as well as water physicochemical variables seem to be involved in generating the spatial-temporal variations observed in the biomarker patterns.
The combined results of the laboratory bioassays and field study allow to benchmark the behavior and sensitivity of four biomarkers in Colombian bivalve species, by establishing expected response ranges of these biomarkers in intertidal oysters exposed to different types of environmental contaminants. The observed correlations between biomarkers (MT and ChE) with a multitude of environmental variables, both natural (physicochemical) as well as anthropogenic (metals), and the highly variable and often non-linear dose-effect relationships observed in laboratory bioassays illustrate the promise as well as the complexity of using biochemical biomarker responses as diagnostic tools for environmental monitoring.
Finally, as a non-expected finding, molecular analyses of the oyster communities of the Colombian Caribbean showed that these correspond to mixed populations of species of the genus Crassostrea sp. and Saccostrea sp.
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