Thesis Ane 29-11-2016
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DEPARTMENT OF ZOOLOGY AND ANIMAL CELL BIOLOGY (UPV/EHU) UMR CNRS EPOC 5805 (Université de Bordeaux) Understanding the impact of silver as an emerging contaminant in the Ibaizabal and Gironde Estuaries International Ph. D Thesis submitted by Ane Rementeria Ugalde For the degree of Philosophiae Doctor November, 2016 (cc)2016 ANE REMENTERIA UGALDE (cc by-nc-sa 4.0) FUNDING The University of the Basque Country (UPV/EHU) through the grant for performing the PhD thesis in cotutelle between the University of the Basque Country (UPV/EHU) and the University of Bordeaux. Basque Government through a grant to the Consolidated Research Group “Cell Biology in Environmental Toxicology” (GIC07/26-IT-393-07, 2007-2012 and GIC12/149-IT-810-13, 2013-2018). University of the Basque Country by means of a grant to the Unit Formation and Research “Ecosystem Health Protection” (UFI11/37, 2011-2014). Spanish Government through a research project CTM2012-40203-C02-01 (MINECO). ACKNOWLEDGMENTS I wish to thank, ° Prof. Jörg Schäfer and Dr. Beñat Zaldibar my PhD supervisors for giving me the chance to carry out this thesis work. For their effort, advices and help. ° Prof. Ionan Marigómez and Prof. Manu Soto director and deputy directors of PiE for gently admitting me in the Plentzia Marine Station (PiE) and allowed me to perform the experimentations. ° Prof. Miren Cajaraville, head of Cell Biology in Environmental Toxicology (CBET) research group, UPV/EHU, for giving the opportunity of joining her team. EXTENDED ABSTRACT Estuarine environments have provided humans food sources and the capacity to exchange goods with the rest of the world through navigation since ancient times. In consequence, many historical main human settlements have occurred around estuaries, leading to their degradation by the modification of their natural functioning and also by increased pollutant inputs due to various anthropogenic sources. In the last centuries, estuaries have become major sites for waste disposal and European estuaries are not an exception. Nowadays, many European estuaries show recovery processes mainly as a result of the general decline on industrial activities and the increased environmental awareness of the society, which has led to the implementation of new policies and regulations. Within the Bay of Biscay two estuaries could be considered as good examples of these recovery processes: the Ibaizabal (Bilbao, Basque Country, Spain) and the Gironde (South-Western France) Estuaries. Despite all the improvements that both estuaries have undergone, pollutant concentrations are still relevant in their waters and sediments. Among the pollutants present in the mentioned compartments (water and sediments), metallic pollution is of special relevance for the scientific community and environmental managers. In fact, metals can exert toxicity effects on aquatic biota at very low exposure levels and they can bioaccumulate or biomagnify through the food chain, potentially affecting both aquatic environmental and human health. Among the different metals showing anomalies in estuaries silver (Ag) and copper (Cu) are of special interest. Silver is regarded as a very toxic element for aquatic biota, particularly in its ionic form Ag +, whereas Cu, is an essential element for living organisms but also toxic once certain exposure levels have been surpassed. These two elements have been extensively used by humans since ancient periods and nowadays they share to some extent similar pollution sources. Mining and metallurgy have been the classical pollution sources for Ag whereas more recently (during the last decades of the XX th century) main Ag inputs have been related to photographic and electronic industries and also to Ag uses in hospitals. In fact, Ag has been considered as a good tracer for urban pollution during several decades. Nowadays, additional Ag inputs into the environment also originate from domestic and urban wastewaters, being increasingly related with the use of Ag nanoparticles in textiles and personal care products due to the bactericidal properties of Ag. Furthermore, Ag-iodide (AgI) is also used in agriculture to prevent hail storms through networks of cloud seeding devices. In the case of Cu, classical sources for this element have also been related to mining and metallurgy industry. Other Cu sources are fungicides used in agriculture (where Cu appears as CuSO 4) and, more recently, navigation. In fact since the ban of organo-tin compounds, Cu has become a major agent of the antifouling paints avoiding the attachment of biological organisms to the hulls of vessels. Environmental water monitoring programs have relied on the use of sentinel species, being bivalves such as mussels and oysters widely employed for this purposes since 1976, when “The Mussel Watch” biomonitoring program was first implemented in the USA. Bivalves accumulate pollutants in their tissues with little metabolic transformation and on the other hand, their response in terms of toxicology can reflect the effects caused in the environment by pollutants. Additionally, as they are sedentary organisms, bivalves can record spatial and temporal changes in pollution. In this context, environmental biomonitoring programs rely on the collection of sentinel species for subsequent chemical measurement of pollutants in combination with biological measurements, which include alterations at cell and tissue levels. The latter, known as biomarkers, provide important information not only about individuals´ health status, but also about the surrounding environmental quality offering the perspective of predicting future changes that may occur at higher biological organisation levels, such as population or ecosystem. However, for a proper understanding of the information obtained by biomarkers, a battery of them should be applied, otherwise proper understanding of the organisms’ response to pollutant stress would not be possible. The battery of biomarkers applied In the present work included the measurements of: Metallothionein (MT) induction, intralysosomal metal accumulation (through autometallography), lipofuscin content determination, neutral lipid content determination, digestive gland atrophy, connective to digestive diverticula ratio, histopathological alterations and measurements of condition index (CI). Recently, different indices have been proposed for biomarker integration in order to obtain an integrated and simplified view of animals’ response, For example, the Integrative Biological Response (IBR) index has been successfully applied in different sentinel organisms under both experimental and field conditions. In fact, biomonitoring studies that have included this index have been able to discriminate between spatial and temporal tendencies. Since previous works have reported possible interactions between Cu and Ag, affecting their bioavailability and accumulation in oysters, one objective of the present thesis was to acquire a better understanding of such interactions between these metals as well as on the potential of using oysters to understand Ag and Cu behaviour and effects in estuaries. Accordingly, the present work relies on four main approaches: one field study and three laboratory experiments carried out at the Plentzia Marine Station (PiE, UPV/EHU). All biological measurements were carried out at the Cell Biology in Environmental Toxicology (CBET research group, UPV/EHU), whereas chemical measurements were performed in the “Transferts Géochimiques des Métaux à l'interface continent ocean” research group (TGM, UMR CNRS EPOC 5805, Université de Bordeaux). The first activity is based on a field study aiming to assess the environmental health status of three estuaries (Ibaizabal, Oka and Gironde) of the Bay of Biscay using mussels and oysters as sentinel organisms. Two of the analysed estuaries (Ibaizabal and Gironde) are well known for chronical metal pollution whereas the Oka Estuary, located within the UNESCO Reserve of the Biosphere of Urdaibai is regarded as a control site. Collected animals were processed for chemical analysis of Ag and Cu accumulation in tissues and also for biological measurements. Obtained chemical results revealed higher metal concentrations in individuals inhabiting the Gironde Estuary. Moreover, clearly higher metal accumulation values occurred in oysters than in mussels from the same sites. These results were in clear concordance with the obtained values for intralysosomal metal accumulation with higher metal levels in bivalves from the Gironde Estuary. In agreement, the applied battery of biomarkers was able to reflect in both species the higher pollutant pressure in the Gironde Estuary with mussels and oysters showing higher levels of digestive epithelium atrophy and disturbed digestive tissue integrity. Consequently, the IBR index also indicated a more altered health status of oysters and mussels from the Gironde Estuary compared to Ibaizabal and Oka Estuaries. The second approach of the present work was focused on a laboratory exposure experiment in which oysters Crassostrea gigas were exposed through direct pathway to a range of environmentally relevant concentrations of Ag and Cu alone and in combination. In this experiment, stable isotopes ( 107 Ag and 63 Cu) were used, allowing using relatively low exposure concentrations (close to environmental levels) of metals and their subsequent quantification in oysters’ tissues. The experiment lasted 28 days and the measured biomarkers integrated into the IBR index were able to indicate that even at very