- Email: [email protected]
Relationships between biochemical and physiological biomarkers responses measured on juvenile marine fish under environmental chemical contamination
S22
Abstracts / Comparative Biochemistry and Physiology, Part A 163 (2012) S10–S27
whereas BBP and DMP showed no toxicity in the fish cell line PLHC-1. A different toxic patter was observed in JEG-3, viz. DEHP was not toxic at the highest concentration tested (500 μM), while DMP showed significant cytotoxicity (1% at 500 μM). Finally, P450 aromatase activity has been determined in the S-9 fraction isolated from JEG-3 cells by using [1b-3H(N)]-androst-4-ene-3,17-dione as a substrate and measuring the formation of tritiated water. This assay allowed the detection of BPA, NP and OP as the most active in-vitro inhibitors of P450 aromatase in JEG-3 cells.
doi:10.1016/j.cbpa.2012.05.068
37. Holistic assessment of the impact of a dump site on the health of surrounding soils aided by biomarkers measured in microbes, plants and annelids A. Irizar (CBET Research Group, University of the Basque Country UPV/ EHU, Basque Country, Spain); L. Epelde, M. Anza, I. Martin (NeikerTecnalia, Basque Institute for Agricultural Research and Development, Basque Country, Spain); U. Artetxe, O. Barrutia (Plant Physiology, UPV/ EHU, Basque Country, Spain); N. Garcia-Velasco, D. Buey (CBET Research Group, UPV/EHU, Basque Country, Spain); J. Etxebarria (GAIKER Tecnological Centre, IK4 Research Alliance, Basque Country, Spain); N. Etxebarria (Analytical Chemistry , UPV/EHU, Basque Country, Spain); C. Garbisu (Neiker-Tecnalia, Basque Institute for Agricultural Research and Development, Basque Country, Spain); J.M. Becerril (Plant Physiology, UPV/EHU, Basque Country, Spain); I. Marigómez, M. Soto (CBET Research Group, UPV/EHU, Basque Country, Spain) Dumpsites containing urban and industrial wastes have always been a matter of concern due to the impact they can cause in the surrounding areas. Although the modern dumping sites have containment walls and other structures to control lixiviates and landslides, many old abandoned sites lack those safety measures and they could affect enclosing soil and aquatic ecosystems. Health status of soil ecosystems can be assessed through biological indicators and biomarkers measured in distinct key species that develop relevant functions in the ecosystems, in order to get an integrated view of soil health. Soil bacterial community, plants and decomposer invertebrates like earthworms play an important role in soil ecosystems and due to their close contact with soil (nutrient source and habitat) and their sensitivity to different pollutants, become very useful organism for soil health assessment. Thus, in this work, the effect of an abandoned dumping site in the surrounding ecosystem has been assessed through measurements of different biomarkers in the bacterial community and after ex situ exposures of plant and earthworms to soils collected around the dumpsite. The biomarkers indicated that the metals present in soils were not available for plants and earthworms due to the high pH produced by inertization treatment with CO3Ca. However, the most polluted point caused a deleterious effect on bacterial community. The decrease of pH up to natural soil levels (pH 4.4) caused an increase in metal bioavailability, which severely affected different parameters in the bacterial community, root elongation and photosynthesis in plants, and earthworm immune system. These results indicate that although, nowadays, the dumping site does not affect hardly the health status of the surrounding ecosystems, a decrease of the pH due to the continuous lixiviation of CO3Ca can be a risk for the health of surrounding areas.
doi:10.1016/j.cbpa.2012.05.069
38. Effects of the exposure of the clam Ruditapes philippinarum to carbamazepine, diclofenac and ibuprofen. Evaluation of enzymatic and molecular endpoints C. Trombini, M. Hampel, J. Blasco (Institute for Marine Sciences of Andalusia, Department of Ecology and Coastal Management, CSIC, Spain) Pharmaceuticals are an emerging class of environmental contaminants that are extensively used in human and veterinary medicine. Due to continuous production and consumption, along with a limited elimination by conventional technologies of water treatment, pharmaceuticals have been found in the aquatic environment at concentrations in the μg/L to ng/L range. Only little is known about the adverse effects on aquatic species being continuously exposed to these compounds. Thus in this study, the marine bivalve, Ruditapes philippinarum, was chosen as ecotoxicological model to evaluate the effects of exposure to pharmaceutical compounds selected from the most commonly found within the aquatic environment. Carbamazepine (CBZ) is an anticonvulsant and mood stabilizing drug used primarily in the treatment of epilepsy and bipolar disorder. Ibuprofen (IBU) and Diclofenac (DCF) are the most commonly used non-steroidal antiinflammatory drugs (NSAIDs). The organisms were exposed individually under semi-static conditions to environmentally relevant concentrations (15 μg/L) of the three compounds for 22 days (14 days of exposure and 8 of depuration). Samples (digestive gland and gills) were taken throughout the exposure period in order to monitor the development of the expression of the chosen endpoints over time. For the evaluation of enzymatic endpoints, a battery of biomarkers: acetylcholinestherase (Ache), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), dibenzylfluorescein dealkylase (DBF) and lipid peroxidation (LPO) was determined in the cytosolic fraction (S12, 12,000 g for 20 min at 4 °C) extracted from both tissues to evaluate the probable alterations to the oxidative stress status in the organisms exposed to pharmaceuticals versus controls. For the evaluation of molecular endpoints, transcription of selected target genes (e.g. inflammatory-, MXR-, energy pathway related genes) was evaluated by quantitative RT-PCR. Exposed organisms showed time dependent induction of the chosen parameters depending on the applied treatment, indicating that environmentally relevant concentrations of CBZ, DCF and IBU may alter the natural condition of exposed clams.
doi:10.1016/j.cbpa.2012.05.070
39. Relationships between biochemical and physiological biomarkers responses measured on juvenile marine fish under environmental chemical contamination E. Kerambrun, F. Henry (Univ Lille Nord de France, ULCO, LOG, F-62930 Wimereux, France and CNRS, UMR 8187, F-62930 Wimereux, France); W. Sanchez (Unité d'écotoxicologie in vitro et in vivo, Institut National de l'Environnement Industriel et des Risques (INERIS), BP 2, F-60550 Verneuil en Halatte, France); R. Amara (Univ Lille Nord de France, ULCO, LOG, F-62930 Wimereux, France and CNRS, UMR 8187, F-62930 Wimereux, France) Several biomarkers have been developed and described in the scientific literature and some of them may be used to assess the quality of the aquatic environment. However, in most cases, a single biomarker can hardly be used as an early signal exposure to contaminants and report the health status of organisms. The aim of our study was to
Abstracts / Comparative Biochemistry and Physiology, Part A 163 (2012) S10–S27
develop a multibiomarker approach on juvenile marine fish (3– 4 months) in order to evaluate the biological effects of chemical pollution at different levels of biological organization. Biochemical detoxification parameters (EROD, GST) and an antioxidant enzyme (CAT) were used as early warning tools of toxicity. In parallel, juvenile fish growth and health were analyzed by the measurement of different physiological biomarkers (somatic and recent growth, RNA:DNA ratio, morphometric and lipid indices). Sensitivity and relevance of biochemical and physiological biomarkers were tested on juvenile: i) sea bass exposed to acute petroleum pollution, ii) sea bass and turbot exposed to harbour and estuarine sediments during controlled and semi-controlled (caging) conditions. Throughout all experiments, responses of biochemical biomarkers were recorded as soon as a few days of exposure which confirm their use as an early warning tool of toxicity. EROD and GST were found to be sensitive to short exposure (2 and 4 days) of organisms to petroleum. However, when fish were exposed to harbour and estuarine sediments, biochemical biomarker responses were poorly correlated with the level of metal and PAH concentrations. On the contrary, juvenile growth and condition indices were found to be more sensitive to the different levels of contaminants analysed suggesting their potentiality to reflect deleterious effects of chemical contaminants on juvenile fish health. Therefore, this study supports the use of both biochemical and physiological biomarkers in biomonitoring programs to display a more comprehensive and integrative assessment of environmental quality.
doi:10.1016/j.cbpa.2012.05.071
40. Responses to temperature in three non-differentiating bacteria I. Garaizabal, Z. Bravo, M. Orruño, I. Arana, I. Barcina (Inmunología, Microbiología y Parasitología, Universidad del País Vasco UPV/EHU, Spain) The formation of resistance structures by differentiating bacteria to confront adverse environmental conditions is well described. Some non-differentiating bacteria which do not develop such structures, can survive for a long time by adopting the viable but nonculturable phenotype (Barcina and Arana, 2009; Colwell and Grimes, 2000) which is a strategy of adaptation to adverse conditions in which cells conserve detectable metabolic function but become unculturable. We have studied the response of three non-differentiating bacteria to temperature which is probably due to its variation the most common stress factor affecting the survival of bacteria. We have used Vibrio harveyi (psychrophile, oligotroph, marine inhabitant), Acinetobacter baumannii (mesophile, copiotroph, nosocomial bacterium) and, as pattern, Escherichia coli (mesophile, copiotroph, indicator bacterium). Bacterial populations, harvested during the stationary phase of growth, were maintained in sterile saline solution (A. baumannii and E. coli) or seawater (V. harveyi) at 4, 20 or 37 °C. Periodically, intact, active and culturable cells were enumerated. In all cases, we observed that bacterial strains maintained their integrity and total number of cells did not change throughout experimentation time, but V. harveyi suffered a drastic morphological change with size reduction at both temperatures studied (4 °C and 20 °C). However, a common pattern of response to temperature was not observed for the three bacteria studied. The effect of the temperature on survival pattern was dependent on the type of bacteria studied. Moreover, E. coli adopted the VBNC phenotype at 20 °C and 37 °C and V. harveyi entered into the VBNC state at 4 °C. However, resistance of A. baumannii could not be attributed to the display of this response. So, VBNC cannot be considered a common response to stress.
S23
References: Barcina I., Arana I. (2009). Rev Environ Sci BioTechnol 8:245–255. Colwell R.R., Grimes D.J. (2000). Nonculturable microorganisms in the environment. ASM Press, Washington, DC. doi:10.1016/j.cbpa.2012.05.072
41. Influence of feeding regimes on cell and tissue level biomarkers in digestive gland of mussels. E. Blanco-Rayón, M. Soto, I. Marigómez, U. Izagirre (CBET Research Group, Plentzia Marine Station (PIE), University of the Basque Country UPV/EHU, Basque Country, Spain) The application of a wide variety of biomarkers in mussels has been used in pollution monitoring programs and in laboratory experiments to assess the biological effects of pollutants. Nevertheless, there is lack of concern about certain factors that could affect the significance of biomarkers when running in door exposures. Likely, there are great differences in the literature regarding feeding regimens and type of food. Taking into account that one of the target organs in which most of the biomarkers are measured is the digestive gland, and since is the organ in charge of digestion, it is of vital importance to state accurate experimental conditions related with feeding. Mussels (Mytilus galloprovincialis) were maintained during 14 days under three different feeding regimes in laboratory conditions: starvation (S), commercial food for marine invertebrates administrated in pulses (M), and algae (Isochrysis galbana) added continuously by a peristaltic pump (A). Cell (lysosomal biomarkers) and tissue-level biomarkers were assessed in digestive gland of mussels at 3, 7 and 14 days. Results showed clear differences in studied biomarkers between mussels maintained at different feeding regimes at different times. These differences are a confounding factor of great magnitude for the interpretation of the biomarkers results in the assessment of biological effects produced by pollutants. A standardized protocol for a better understanding, interpretation and comparison between experiments is deeply discussed. doi:10.1016/j.cbpa.2012.05.073
42. Down the rabbit hole: Subcellular localization and x-ray microanalysis of a set of metallic nanoparticles in mussels A. Jimeno-Romero (CBET Research Group, University of the Basque Country UPV/EHU, Basque Country, Spain); D. Berhanu (Natural History Museum of London, UK); P. Reip (Intrinsiq Materials, Ltd., UK); M. Oron (AHAVA-Dead Sea Laboratories, Israel); D. Gilliland (Joint Research Center, Ispra, Italy); E. Valsami-Jones (Natural History Museum of London and Univ. Birmingham, UK); M.P. Cajaraville (CBET Research Group, UPV/EHU, Basque Country, Spain); A. Warley (Kings College London, UK); I. Marigómez, M. Soto (CBET Research Group, UPV/EHU, Basque Country, Spain) Electron microscopy is an invaluable tool that allows us to better understand physiological processes occurring at tissue, cell and organelle levels. Energy-dispersive X-ray spectroscopy (EDX) analysis allows acquiring information about changes in elemental composition in different cell compartments, shedding light into the mechanisms of accumulation and regulation. In the present work the accumulation of different metal containing nanoparticles (NPs) has been studied by means of transmission electron microscopy (TEM) and EDX spectrum analysis in mussels (Mytilus galloprovincialis) exposed for 3 days to the
Abstracts / Comparative Biochemistry and Physiology, Part A 163 (2012) S10–S27
whereas BBP and DMP showed no toxicity in the fish cell line PLHC-1. A different toxic patter was observed in JEG-3, viz. DEHP was not toxic at the highest concentration tested (500 μM), while DMP showed significant cytotoxicity (1% at 500 μM). Finally, P450 aromatase activity has been determined in the S-9 fraction isolated from JEG-3 cells by using [1b-3H(N)]-androst-4-ene-3,17-dione as a substrate and measuring the formation of tritiated water. This assay allowed the detection of BPA, NP and OP as the most active in-vitro inhibitors of P450 aromatase in JEG-3 cells.
doi:10.1016/j.cbpa.2012.05.068
37. Holistic assessment of the impact of a dump site on the health of surrounding soils aided by biomarkers measured in microbes, plants and annelids A. Irizar (CBET Research Group, University of the Basque Country UPV/ EHU, Basque Country, Spain); L. Epelde, M. Anza, I. Martin (NeikerTecnalia, Basque Institute for Agricultural Research and Development, Basque Country, Spain); U. Artetxe, O. Barrutia (Plant Physiology, UPV/ EHU, Basque Country, Spain); N. Garcia-Velasco, D. Buey (CBET Research Group, UPV/EHU, Basque Country, Spain); J. Etxebarria (GAIKER Tecnological Centre, IK4 Research Alliance, Basque Country, Spain); N. Etxebarria (Analytical Chemistry , UPV/EHU, Basque Country, Spain); C. Garbisu (Neiker-Tecnalia, Basque Institute for Agricultural Research and Development, Basque Country, Spain); J.M. Becerril (Plant Physiology, UPV/EHU, Basque Country, Spain); I. Marigómez, M. Soto (CBET Research Group, UPV/EHU, Basque Country, Spain) Dumpsites containing urban and industrial wastes have always been a matter of concern due to the impact they can cause in the surrounding areas. Although the modern dumping sites have containment walls and other structures to control lixiviates and landslides, many old abandoned sites lack those safety measures and they could affect enclosing soil and aquatic ecosystems. Health status of soil ecosystems can be assessed through biological indicators and biomarkers measured in distinct key species that develop relevant functions in the ecosystems, in order to get an integrated view of soil health. Soil bacterial community, plants and decomposer invertebrates like earthworms play an important role in soil ecosystems and due to their close contact with soil (nutrient source and habitat) and their sensitivity to different pollutants, become very useful organism for soil health assessment. Thus, in this work, the effect of an abandoned dumping site in the surrounding ecosystem has been assessed through measurements of different biomarkers in the bacterial community and after ex situ exposures of plant and earthworms to soils collected around the dumpsite. The biomarkers indicated that the metals present in soils were not available for plants and earthworms due to the high pH produced by inertization treatment with CO3Ca. However, the most polluted point caused a deleterious effect on bacterial community. The decrease of pH up to natural soil levels (pH 4.4) caused an increase in metal bioavailability, which severely affected different parameters in the bacterial community, root elongation and photosynthesis in plants, and earthworm immune system. These results indicate that although, nowadays, the dumping site does not affect hardly the health status of the surrounding ecosystems, a decrease of the pH due to the continuous lixiviation of CO3Ca can be a risk for the health of surrounding areas.
doi:10.1016/j.cbpa.2012.05.069
38. Effects of the exposure of the clam Ruditapes philippinarum to carbamazepine, diclofenac and ibuprofen. Evaluation of enzymatic and molecular endpoints C. Trombini, M. Hampel, J. Blasco (Institute for Marine Sciences of Andalusia, Department of Ecology and Coastal Management, CSIC, Spain) Pharmaceuticals are an emerging class of environmental contaminants that are extensively used in human and veterinary medicine. Due to continuous production and consumption, along with a limited elimination by conventional technologies of water treatment, pharmaceuticals have been found in the aquatic environment at concentrations in the μg/L to ng/L range. Only little is known about the adverse effects on aquatic species being continuously exposed to these compounds. Thus in this study, the marine bivalve, Ruditapes philippinarum, was chosen as ecotoxicological model to evaluate the effects of exposure to pharmaceutical compounds selected from the most commonly found within the aquatic environment. Carbamazepine (CBZ) is an anticonvulsant and mood stabilizing drug used primarily in the treatment of epilepsy and bipolar disorder. Ibuprofen (IBU) and Diclofenac (DCF) are the most commonly used non-steroidal antiinflammatory drugs (NSAIDs). The organisms were exposed individually under semi-static conditions to environmentally relevant concentrations (15 μg/L) of the three compounds for 22 days (14 days of exposure and 8 of depuration). Samples (digestive gland and gills) were taken throughout the exposure period in order to monitor the development of the expression of the chosen endpoints over time. For the evaluation of enzymatic endpoints, a battery of biomarkers: acetylcholinestherase (Ache), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), dibenzylfluorescein dealkylase (DBF) and lipid peroxidation (LPO) was determined in the cytosolic fraction (S12, 12,000 g for 20 min at 4 °C) extracted from both tissues to evaluate the probable alterations to the oxidative stress status in the organisms exposed to pharmaceuticals versus controls. For the evaluation of molecular endpoints, transcription of selected target genes (e.g. inflammatory-, MXR-, energy pathway related genes) was evaluated by quantitative RT-PCR. Exposed organisms showed time dependent induction of the chosen parameters depending on the applied treatment, indicating that environmentally relevant concentrations of CBZ, DCF and IBU may alter the natural condition of exposed clams.
doi:10.1016/j.cbpa.2012.05.070
39. Relationships between biochemical and physiological biomarkers responses measured on juvenile marine fish under environmental chemical contamination E. Kerambrun, F. Henry (Univ Lille Nord de France, ULCO, LOG, F-62930 Wimereux, France and CNRS, UMR 8187, F-62930 Wimereux, France); W. Sanchez (Unité d'écotoxicologie in vitro et in vivo, Institut National de l'Environnement Industriel et des Risques (INERIS), BP 2, F-60550 Verneuil en Halatte, France); R. Amara (Univ Lille Nord de France, ULCO, LOG, F-62930 Wimereux, France and CNRS, UMR 8187, F-62930 Wimereux, France) Several biomarkers have been developed and described in the scientific literature and some of them may be used to assess the quality of the aquatic environment. However, in most cases, a single biomarker can hardly be used as an early signal exposure to contaminants and report the health status of organisms. The aim of our study was to
Abstracts / Comparative Biochemistry and Physiology, Part A 163 (2012) S10–S27
develop a multibiomarker approach on juvenile marine fish (3– 4 months) in order to evaluate the biological effects of chemical pollution at different levels of biological organization. Biochemical detoxification parameters (EROD, GST) and an antioxidant enzyme (CAT) were used as early warning tools of toxicity. In parallel, juvenile fish growth and health were analyzed by the measurement of different physiological biomarkers (somatic and recent growth, RNA:DNA ratio, morphometric and lipid indices). Sensitivity and relevance of biochemical and physiological biomarkers were tested on juvenile: i) sea bass exposed to acute petroleum pollution, ii) sea bass and turbot exposed to harbour and estuarine sediments during controlled and semi-controlled (caging) conditions. Throughout all experiments, responses of biochemical biomarkers were recorded as soon as a few days of exposure which confirm their use as an early warning tool of toxicity. EROD and GST were found to be sensitive to short exposure (2 and 4 days) of organisms to petroleum. However, when fish were exposed to harbour and estuarine sediments, biochemical biomarker responses were poorly correlated with the level of metal and PAH concentrations. On the contrary, juvenile growth and condition indices were found to be more sensitive to the different levels of contaminants analysed suggesting their potentiality to reflect deleterious effects of chemical contaminants on juvenile fish health. Therefore, this study supports the use of both biochemical and physiological biomarkers in biomonitoring programs to display a more comprehensive and integrative assessment of environmental quality.
doi:10.1016/j.cbpa.2012.05.071
40. Responses to temperature in three non-differentiating bacteria I. Garaizabal, Z. Bravo, M. Orruño, I. Arana, I. Barcina (Inmunología, Microbiología y Parasitología, Universidad del País Vasco UPV/EHU, Spain) The formation of resistance structures by differentiating bacteria to confront adverse environmental conditions is well described. Some non-differentiating bacteria which do not develop such structures, can survive for a long time by adopting the viable but nonculturable phenotype (Barcina and Arana, 2009; Colwell and Grimes, 2000) which is a strategy of adaptation to adverse conditions in which cells conserve detectable metabolic function but become unculturable. We have studied the response of three non-differentiating bacteria to temperature which is probably due to its variation the most common stress factor affecting the survival of bacteria. We have used Vibrio harveyi (psychrophile, oligotroph, marine inhabitant), Acinetobacter baumannii (mesophile, copiotroph, nosocomial bacterium) and, as pattern, Escherichia coli (mesophile, copiotroph, indicator bacterium). Bacterial populations, harvested during the stationary phase of growth, were maintained in sterile saline solution (A. baumannii and E. coli) or seawater (V. harveyi) at 4, 20 or 37 °C. Periodically, intact, active and culturable cells were enumerated. In all cases, we observed that bacterial strains maintained their integrity and total number of cells did not change throughout experimentation time, but V. harveyi suffered a drastic morphological change with size reduction at both temperatures studied (4 °C and 20 °C). However, a common pattern of response to temperature was not observed for the three bacteria studied. The effect of the temperature on survival pattern was dependent on the type of bacteria studied. Moreover, E. coli adopted the VBNC phenotype at 20 °C and 37 °C and V. harveyi entered into the VBNC state at 4 °C. However, resistance of A. baumannii could not be attributed to the display of this response. So, VBNC cannot be considered a common response to stress.
S23
References: Barcina I., Arana I. (2009). Rev Environ Sci BioTechnol 8:245–255. Colwell R.R., Grimes D.J. (2000). Nonculturable microorganisms in the environment. ASM Press, Washington, DC. doi:10.1016/j.cbpa.2012.05.072
41. Influence of feeding regimes on cell and tissue level biomarkers in digestive gland of mussels. E. Blanco-Rayón, M. Soto, I. Marigómez, U. Izagirre (CBET Research Group, Plentzia Marine Station (PIE), University of the Basque Country UPV/EHU, Basque Country, Spain) The application of a wide variety of biomarkers in mussels has been used in pollution monitoring programs and in laboratory experiments to assess the biological effects of pollutants. Nevertheless, there is lack of concern about certain factors that could affect the significance of biomarkers when running in door exposures. Likely, there are great differences in the literature regarding feeding regimens and type of food. Taking into account that one of the target organs in which most of the biomarkers are measured is the digestive gland, and since is the organ in charge of digestion, it is of vital importance to state accurate experimental conditions related with feeding. Mussels (Mytilus galloprovincialis) were maintained during 14 days under three different feeding regimes in laboratory conditions: starvation (S), commercial food for marine invertebrates administrated in pulses (M), and algae (Isochrysis galbana) added continuously by a peristaltic pump (A). Cell (lysosomal biomarkers) and tissue-level biomarkers were assessed in digestive gland of mussels at 3, 7 and 14 days. Results showed clear differences in studied biomarkers between mussels maintained at different feeding regimes at different times. These differences are a confounding factor of great magnitude for the interpretation of the biomarkers results in the assessment of biological effects produced by pollutants. A standardized protocol for a better understanding, interpretation and comparison between experiments is deeply discussed. doi:10.1016/j.cbpa.2012.05.073
42. Down the rabbit hole: Subcellular localization and x-ray microanalysis of a set of metallic nanoparticles in mussels A. Jimeno-Romero (CBET Research Group, University of the Basque Country UPV/EHU, Basque Country, Spain); D. Berhanu (Natural History Museum of London, UK); P. Reip (Intrinsiq Materials, Ltd., UK); M. Oron (AHAVA-Dead Sea Laboratories, Israel); D. Gilliland (Joint Research Center, Ispra, Italy); E. Valsami-Jones (Natural History Museum of London and Univ. Birmingham, UK); M.P. Cajaraville (CBET Research Group, UPV/EHU, Basque Country, Spain); A. Warley (Kings College London, UK); I. Marigómez, M. Soto (CBET Research Group, UPV/EHU, Basque Country, Spain) Electron microscopy is an invaluable tool that allows us to better understand physiological processes occurring at tissue, cell and organelle levels. Energy-dispersive X-ray spectroscopy (EDX) analysis allows acquiring information about changes in elemental composition in different cell compartments, shedding light into the mechanisms of accumulation and regulation. In the present work the accumulation of different metal containing nanoparticles (NPs) has been studied by means of transmission electron microscopy (TEM) and EDX spectrum analysis in mussels (Mytilus galloprovincialis) exposed for 3 days to the