Sublethal effects of chemical pollution in benthic fish from Marine Spanish waters | Efectos subletales de la contaminación química en especies de peces bentónicas de aguas españolas

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English. Thousands of potentially toxic chemical substances are introduced into the oceans every day. Even following decades of research and monitoring there is still a limited understanding of their effects on marine organisms and ecosystems. A complicating factor is the general lack of a direct relationship between elevated concentrations of contaminants in the tissues of marine organisms and effects. In addition, some biological responses have been shown to occur even at very low contaminant concentrations. A development and deployment of biological effects measurements (biomarkers and bioassays) along with analytical chemistry has increasingly been used as early warning signals of adverse environmental change in marine ecosystems. Contaminant-related biomarker responses include physiological or biochemical responses in organisms exposed to certain class of contaminants. Biomarker responses in fish from natural populations appear to be more sensitive than other methods, as they provide integrated information about contaminant exposure and effects as well as indicate acute pollution incidences. In vivo and in vitro bioassays contribute to our understanding of how pollutants affect living organisms but can also aid in deducing general mechanisms of action, to aid in categorization and overall assessment of effects. Fish is the most diverse class of vertebrates and can bridge human and ecological health in ecotoxicology studies. Benthic fish are suitable organisms for marine chemical pollution studies and the assessment of its health is it is not only an issue of conservation ecology but has clear links to utilisation of marine resources and human health. The main aims of this thesis were to clarify sublethal effects of environmental chemical contaminants on biological responses in three benthic fish species (Lepidorhombus boscii, Callionymus lyra and Mullus barbatus) from Spanish marine areas as well as to develop the use of contaminant-related biological responses in fish into an integrated framework for future Spanish pollution environmental monitoring programmes. This thesis comprises five scientific papers [I-V], all of which have been published in peer-reviewed international journals. Benthic fish were sampled under different field scenarios: i) from the northern Spanish shelf shortly after a medium oil spill ii) and repeatedly in the same area over a period of three years, iii) from chronically polluted areas on the Spanish Mediterranean inner shelf iv) and from the Mar Menor coastal lagoon (SE Spain). In all scenarios, fish were also simultaneously sampled from reference areas to allow a direct comparison. Standardised protocols were developed and implemented to minimise variability due to environmental and biological factors. In addition to direct use, the methods were evaluated and data from both monitoring and laboratory studies used to develop a framework for monitoring contaminants in marine ecosystems. The biological measurements investigated as part of this thesis comprised validated contaminant-related biomarkers, such as detoxification enzymes related to biotransformation phase I, e.g. ethoxyresorufin-O-deethylase activity (EROD) and biliary concentrations of hydroxylated metabolites of polyaromatic hydrocarbons (PAHs). Other contaminant-related biomarkers, whose applicability in marine fish is still under discussion and need further research, such as enzymes related to the organic xenobiotic biotransformation phase II (gluthatione-S-transferase activity (GST)), antioxidant enzymes (glutathione reductase (GR) and catalase (CAT)), metallothionein (MT) and DNA integrity were also studied. In addition, the in vitro ER-LUC reporter gene assay was used as a screening tool to estimate the total exposure to estrogenic active compounds in bile from fish collected in different areas as well as the estrogenic potency of hydroxylated PAHs and alkylphenols. Chapter 2 presents results from analyses of a range of hepatic biomarker responses five months after the Prestige oil spill using the two benthic fish species four-spot megrim, L. boscii, and the dragonet, C. lyra. The fish were collected from different areas along the northern Iberian shelf, to clarify the spatial extent of effects from the spill, as well as species-dependent responses [paper I]. No data were available for the concentration of chemical contaminants in the fish at the time of the study. Biomarker responses did however suggest that fish from some areas were affected by existing chronic pollution before the Prestige oil spill happened. Due to the lack of pre-spill biomarker data from the available species and this region, it was not possible to directly attribute biological effects observed in fish to the petroleum hydrocarbon components of the spilled oil. As a follow-up, Chapter 3 is a study to determine whether biomarker responses, in the same fish species, would vary over the immediate years following the Prestige oil spill [paper II]. The results indicated significantly decreasing hydrocarbon-associated biological responses over time and a subsequent recovery to presumed baseline levels. Overall, biomarker responses were in agreement with oil slick trajectories and the spatial distribution of tar aggregates found on the bottom shelf after the accident. The results of these studies showed that sublethal effects in fish were lasting for at least 2-3 years following the spill and they have been the first to show that an oil spill in open waters may affect benthic fish species hundreds of kilometres away.Chapter 4 presents the guideline document that was prepared to formalise the sampling design, species selection and the selection of appropriate biological effects techniques. The guideline focused particularly on toxicity assessment and strategies for monitoring biological effects associated with oil spills, with particular reference to European coastal waters [paper III]. The guideline document concluded that it is critical to have ongoing effects-based biomonitoring programmes in areas with high risk of oil-spill accidents.In Chapter 5, an integrated study was used to clarify whether contaminant concentrations in chronically polluted coastal areas in the Iberian Mediterranean inner shelf were at levels that cause adverse health effects in the benthic fish Mullus barbatus [paper IV]. The study combined chemistry in biota and surface sediments, biomarker responses as well as reproductive and nutritional physiological indicators. The study included a coastal area dominated by metal contamination (Portmán). The results showed alterations of some biomarkers, i.e. EROD, DNA integrity, muscle lipid content and gonadosomatic index, in fish from the more contaminated areas, although responses were not dramatic. The contaminant body burden in fish only partly reflected the presence of the same contaminants in the sediments of the study areas, presumably due to varying bioavailability and biotransformation capacity of the fish.Finally, Chapter 6 presents an integrated chemical and biological study to assess exposure to estrogenic contaminants to male fish [paper V]. The use of the in vitro ER-LUC reporter gene assay combined with chemical analyses in bile extracts highlighted some of the challenges using chemical analyses in that only a minimal fraction of the total estrogenic activity observed in male fish could be explained by the observed concentrations of estrogenic substances included in the chemical analyses.The results reported in chapter 2 and 3 have shown that L. boscii and C. lyra are appropriate species for monitoring programmes along much of the northern Iberian shelf. Similarly, the results reported in chapter 5 and 6 support the use of M. barbatus as a target species for monitoring programmes in Spanish Mediterranean waters. Interspecies differences in constitutive levels of biomarker responses were found for the three species, particularly for EROD activity and catalase activity. Results obtained in this thesis made it possible to establish preliminary assessment criteria (AC) for EROD activity in the three studied species and preliminary baseline levels of PAHs bile metabolites and bile estrogenicity in M. barbatus. A general discussion of the studies included in the thesis as well as recommendations for future biomonitoring programmes of chemical pollution in marine Spanish waters can be found in Chapter 7. Final concluding remarks and future perspectives are presented in Chapter 8.