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Trabajo presentado en la SETAC Europe 27th Annual Meeting (Environmental Quality Through Transdisciplinary Collaboration), celebrada en Bruselas del 7 al 11 de mayo de 2017., Detection of pharmaceuticals in the aquatic environment is an emerging issue of concern. Although the majority of drugs occur in the aquatic environment at trace level (ng L-1 to low μg L-1), most of them have been specifically designed to be biologically active at low concentrations in humans and animals and therefore they can represent a risk for aquatic organisms particularly under chronic exposure. The knowledge on the biological effects of pharmaceuticals on aquatic organisms is still scarce, moreover since these compounds occur in aquatic environment as complex mixtures. Ibuprofen (IBU) is one of the most used non-steroidal anti-inflammatory drugs: its ability to induce toxic effects in aquatic organisms at environmentally relevant concentrations has been widely proven. Ciprofloxacin (CIP) and flumequine (FL) are broad -spectrum antibiotics of the fluoroquinolones class. Fluoroquinolones toxicity was observed in rodents producing among others oxidative stress, cyto and neurotoxicity. However, ecotoxicological effects on aquatic organisms of CIP and FL are practically unknown. In our work biochemical responses related to oxidative stress (CAT, SOD, GR, T-GPx activities and LPO levels), detoxification (GST activity) and neurotoxicity (AchE activity) were studied in the red swamp crayfish Procamabarus clarkii after sub-chronic exposure to 10 and 100 μg L-1 of a mixture of IBU, CIP and FL. Recovery of organisms was also evaluated after 1 week of post-exposure depuration. Additionally transcriptional responses related to antioxidant and stress (CAT, GST, heat shock protein 90HSP90 and metallothioneinsMT), apoptotic (cathepsin-L CatL, 14-3-3 zeta protein14 -3-3ζ), immune (thymosin Thy, toll-like receptorsPcToll) and inflammatory (collagen alpha chain -like proteinColA) processes and neurotransmission modulation (neuropeptide precursor proteinNP, myosuppressin -like neuropeptide precursor MSNP, synaptome-associated proteinSNAP) were evaluated. Results obtained for biomarkers and gene expression indicate a general activation of different oxidative stress related features in both enzymatic and transcriptional responses, particularly at the highest concentration of drugs. Therefore, exposure of pharmaceuticals mixtures to P. clarkii can produce oxidative stress as indicated by enzymatic and transcriptional responses. It has still to be evaluated if these effects can have long term physiological effects on aquatic ecosystems and human health.

Trabajo presentado en el 6th Congress of the Spanish Proteomic Society, celebrado en Cádiz del 15 al 18 de noviembre de 2016., Over the last decades engineered gold nanoparticles (AuNPs) have been developed for and introduced into a growing number of commercial and industrial products. Up to date limited information is available on the ecotoxicological responses in non-target organisms, in par-ticular under environmental relevant concentrations. Notwithstanding their promising applica-tions, increased understanding of their behaviour and effects is required to manage potential risk for non-target organisms.We studied differential protein expression in response to citrate capped AuNPs (21.5 ± 2.9 nm) in the digestive gland of the bivalve Ruditapes philippinarum after 1 and 7 days of expo-sure at an environmental relevant concentration (750 ng L-1), using a 2nd-generation (iTRAQ-8plex) proteomic approach.2200 proteins were identified to be simultaneously expressed in control and treatment and a regularized regression approach (Elastic Net) identified 105 of those to be expressed dif-ferentially. Homology-based functional annotation could be successfully performed for 77% of all sequences and 75% of the differentially expressed sequences and subsequent analysis identified potential enrichment in various stress and injury related functions. Identified pro-teins may be useful as biomarkers of environmental nanoparticle pollution and provide insight about the mode of action of engineered gold nanoparticles.

Trabajo presentado en el 30th ESCPB Congress (New European Society for Comparative Physiology and Biochemistry, "Unraveling complexity: from molecules to ecosystems"), celebrado en Barcelona del 4 al 7 de septiembre de 2016., Copper oxide nanoparticles (CuO NPs) are increasingly investigated, developed and manufactured for a wide and promising array of commercial and industrial products. Their increased production use and disposal will eventually lead to their increased release into the aquatic environment and potentially affect non-target organisms within. Although CuO NPs have been identified to be potentially cytotoxic, little is known about their behaviour in the marine environment and its potential influence on their cytotoxicity. We studied aggregation and dissolution kinetics of two commercially available bare copper oxide nanoparticles with nominal similar mean sizes (~ 40 nm), but distinct synthesis processes (wet chemistry and combustion synthesis) in various environmental and experimental relevant conditions. In addition cytotoxicity and DNA damage, as well as gene expression of oxidative stress, inflammatory response, DNA damage repair and cell death mediator markers were studied in Ruditapes philippinarum haemocytes after 24 h in vitro exposure and compared with sub-micron CuO (~ 500 nm) and water soluble Cu treated cells. Our results indicate that aggregating behaviour largely influences the toxicity of CuO NPs by influencing their susceptibility to ion leaching from the particle/aggregate surface. Gene expression analysis identified highly similar modes of action for all tested particulate and ionic Cu forms, further substantiating that nano and bulk CuO toxicity might largely be driven by ionic Cu. In addition our work highlights various differences in the behaviour of CuO NPs in environmental and culture conditions that need consideration when extrapolating results from in vitro to natural (or environmental) conditions.

Trabajo presentado en el XVIII Seminario Ibérico de Química Marina, celebrado en Alicante (España) de 20 al 22 de julio de 2016., Pharmaceuticals are pollutants of potential concern in the aquatic environment where they are commonly introduced as a complex mixture. In this study we have evaluated the toxic effects produced in clams Scobicularia plana by a mixture of ciprofloxacine, flumequine and ibuprofen, three pharmaceuticals that are produced and used in large quantity and have a wide spread occurrence in aquatic environment. Clams were exposed to two mixture concentrations (at 10 and 100 μg·L-1 levels) during 28 days (21 days of exposure and 7 of depuration) and oxidative and neurotoxic effects were assessed using a multi-biomarker approach: CAT, GR, GPx, GST, SOD, LPO levels and AchE activity were evaluated in gills and digestive gland over time. The present study demonstrated that the combination of three pharmaceutical compounds, particularly at the highest concentration tested, have a considerable effect on the activities of antioxidant and detoxifying enzymes and therefore on the oxidative status of S. plana., This work has been funded by the Ministerio de Economia y Competitividad of the Spanish Government under Project CTM2012-38720-C03-03, and ERF funds.

Trabajo presentado en el Scotland and Valencia workshop on Bayesian Statistics (ScoVa16 Workshop, VABAR), celebrado en Valencia el 28 y 29 de enero de 2016., Among others, proteomic research aims to identify and quantify relative changes in pro-tein abundance between individuals under different circumstances. In ecotoxicology, pro-teomics is applied to test organisms exposed to environmental contaminants, and is es-pecially useful for the evaluation of chronic low-level exposure effects that manifest atmolecular levels of organization. Obtained information can be used to better understandmetabolic pathways and networks the contaminant interferes with, providing a betterknowledge of the mode of action of the contaminant, as well as for contaminant specificbiomarker development for environmental monitoring purposes. In a laboratory-basedexperiment, we exposed the non-target marine bivalve, the Manila clamRuditapes philip-pinarumto an environmentally relevant concentration (0.75μg L−1) of weakly agglomer-ating citrate AuNPs (∼20 nm). An 8-plex iTRAQ-based bottom-up proteomic approachfollowed by tandem mass spectrometry led to the identification and quantitation of 2200expressed proteins. In order to identify statistically significant features, the obtained datawas analysed using multiple contrast analysis corrected by FDR aimed at the control ofthe proportion of false positives among rejected hypotheses. However, the experimentaldesign applied resulted in a higher number of variables than samples or observations lead-ing to an over-conservative approach. Alternatively, the restricted model regressions Lassoand Elastic-Net were applied to provide better information on what proteins differentiatebetween control and treated clams through penalization of the coefficients of the variablesthat are not associated with the response variable zero. Elastic-Net revealed 105 iden-tified proteins of which many were the same as those obtained using multiple contrasts.Ultimately, samples were classified after dimension reduction by PLS-DA. This provideda good classification of the samples, in which all variables are projected along the com-ponents creating the model. Several obtained proteins were related to oxidative stress,inflammatory response and cytoskeleton and correlated with several features identified inq-PCR gene expression analysis carried out on the same samples previously.

Trabajo presentado en el 10th Iberian Congress on Environmental Contamination and Toxicology y el 7th Iberoamerican Congress on Environmental Contamination and Toxicology (CICTA 2015), celebrados en Vila Real (Portugal) del 14 al 17 de julio de 2015., Engineered Gold nanoparticles (AuNPs) are introduced into a growing number of commercial products. Notwithstanding their promising applications, the increased likelihood of their release into environmental compartments and the resulting exposure on the species within needs to be met by an increased understanding of their behaviour and effects. Up to date limited information is available on the ecotoxicological risk for non-target organisms, with the majority of research focusing on magnified expectable environmental concentrations. In our laboratory-based study the bivalve Ruditapes philippinarum was chosen as a model to evaluate differential protein expression of citrate capped AuNPs (21.5 ± 2.9 nm) at an environmental relevant concentration (0.75 μg L-1). We used a 2nd-generation (iTRAQ-8plex) proteomic approach to identify and quantify differently expressed proteins in the digestive gland after 1 and 7 days of exposure in comparison to control organisms.The iTRAQ results demonstrate that the digestive gland is affected by environmentally relevant concentrations of citrate capped gold nanoparticles. Over 2500 proteins involved in a wide range of metabolic pathways and/or physiological processes were identified. Altered proteins may be useful as biomarkers of environmental nanoparticle pollution and provide insight about the mode of action of engineered gold nanoparticles.

Trabajo presentado en el 10th Iberian Congress on Environmental Contamination and Toxicology y el 7th Iberoamerican Congress on Environmental Contamination and Toxicology (CICTA 2015), celebrados en Vila Real (Portugal) del 14 al 17 de julio de 2015., The development of advanced analytical techniques have allowed to detect the occurrence and fate of pharmaceutical compounds in different matrixes such as ground water and sediment, either in their parent compound or in the form of metabolites. Many of these compounds have at least one asymmetrically substituted carbon atom and are, therefore chiral. There are very few studies about the chiral behavior of chemicals in the aquatic environment and the potential negative effects in the organisms. An example of these compounds is Ibuprofen (IB); a widely used nonsteriodal anti-inflammatory drug (NSAIDs). IB is formed by two enantiomer forms: R and S. In order to improve the knowledge about the different ecotoxicological effects that different enantiomeric forms can have in aquatic organisms, we performed a series of toxicity tests on the freshwater shrimp Atyaephyra desmarestii as model organism. A. desmarestii is widely distributed in clean freshwater bodies and has been successfully employed in pharmaceuticals ecotoxicity tests. Selected sublethal endpoints were osmoregulatory capacity and ingestion- and respiration rate as indicators of physiological changes. The shrimps were exposed at IB in its composite form and both enantiomeric forms at environmental relevant concentrations.Given that different enentiomers of a chiral compound can cause different biological effects or the presence of one enantiomer may inhibit the metabolism of the other, it is important know not only the fate but also the effect of these substances in non-target species to improve the environmental risk assessment of chiral compounds.

Trabajo presentado en el 10th Iberian Congress on Environmental Contamination and Toxicology y el 7th Iberoamerican Congress on Environmental Contamination and Toxicology (CICTA 2015), celebrados en Vila Real (Portugal) del 14 al 17 de julio de 2015., Arsenic is a well known toxicant released into the environment through different industrial processes and agricultural usage which environmental concentrations normally between 1 and 10 μg/L reaching milligram concentrations in polluted areas. In the aquatic environment arsenic exists in different chemical forms being arsenate (Asv) is the most common form under oxic conditions. In the present study marine clam Scrobicularia plana was used as a model organism to investigate oxidative stress and bioaccumulation related to Asv exposure. Clams were exposed to two concentrations of Asv (10 and 100 μgL-1) for 14 days and samples were collected after a 1, 7 and 14 days of exposure. Antioxidant biomarker including glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPX), aceticholinesterase (AchE) and superoxide dismutase (SOD) as well as lipid peroxidation (LPO) in gills and digestive glands were analyzed. .SOD activity increased in both tissues after one day of exposure to the highest concentration of Asv . A significant decrease in AchE and GR activities was observed in gills after seven days at both concentrations. Also GR activity decreased in digestive gland at the day 14. On the other hand, GST, GX and LPO didn’t show any significant alteration over time. The results indicate that exposure to environmental concentrations of Asv can induce oxidative stress. The concentration of Asv was determined in clam digestive gland: S. Plana showed a moderate bioaccumulation ability when exposed to both concentrations of Asv. .

Trabajo presentado en la SETAC Europe 25th Annual Meeting (Environmental protection in a multi-stressed world: challenges for science, industry and regulators), celebrada en Barcelona del 3 al 7 de mayo de 2015., According to the European Water Framework Directive, the assessment of the ecological status of freshwater systems is based on biological, physicochemical and hydromorphological quality. Some studies have observed that biological status is not always in coherence with chemical status, maybe due to the adaptation mechanisms of aquatic organisms under chronic chemical exposure. In these situations, ecotoxicity tests could be useful to resolve these divergences. The use of effect-based monitoring tools has been mentioned in the context on the Common Implementation Strategy (CSI) focused on regulated substances, which involves that many substances are not considered and their contributions to toxicity are lacking. The general aim of this study is to design a methodology to categorize the ecotoxicological status of rivers. The specific aims are: to compare the effectiveness of different ecotoxicity tests performed with freshwater sediments; to evaluate the relation between ecological status, pollutant concentent, their bioavailability, and water and sediment ecotoxicit y; and to develop a general methodology to evaluate the relative contribution of the main pollutants to the toxicity in order to improvemanagement tools. Two sampling campaings were carried out in 2013 and 2014 within the Ebro River (Spain) watershed. Therteen composite samples of sediments were collected and data on the chemical and ecologycal status has been achieved. The ecotoxicity of pore water and whole sediments was evaluated by Vibrio fischeri, Nitzschia palea, Pseudokirshneriella subcapitata, Daphnia magna and Chironomus riparius. In addition, the trace metals bioavailability was calculated by a sequential extraction according to the BCR method. To distinguish the potentially toxic fraction associated to trace metals burden of sediments, an analysis of acid-volatile sulphide and simultaneously extracted metals was performed. The results indicate that the ecotoxicological approach in most of the analyzed sediments is in agreement with their ecological status. This study demonstrates that the integration of chemical, biological and ecotoxicological analyses could be crucial to understand the hazard of pollutants in aquatic ecosystems, especially, in freshwater sediments. Moreover, it corroborates that cost-effective and rapid screening short-term bioas says could be useful to determine the surface water ecotoxicological status.

Trabajo presentado en el ImageNano 2015 (Bringing together Nanoscience and Nanotechnology), celebrado en Bilbao del 10 al 13 de marzo de 2015., The increasing use of nanomaterials in industrial applications for the last decade implies an increase of the concern about potential toxic effects of released engineered nanoparticles to the environment. In fact, aquatic ecosystems are the main receptors of chemical pollution, including nanoparticles. Surface waters and, to a greater extent, oceans, will be the final receptors for all wasted NPs (Fabrega et al., 2011; Gong et al., 2011; Whiteley et al., 2013). The transition from freshwater to estuarine and marine ecosystems represents drastic changes in ionic strength, organic matter and other physicochemical properties, which affect to particle propertiesand their fate and behavior. Nevertheless, knowledge about the effects and the mechanisms of toxicity of the different NPs on the aquatic and, over all, marine biota is quite far away to be complete (Boxall et al.; 2007; Baun et al., 2008; Chinnapongse etal., 2011; Walters et al., 2014). Toxicity of metallic nanoparticles to aquatic organisms seems to be related to the physical and chemical properties of those nanoparticles as well as their behavior in aquatic media where dissolution, aggregation and agglomeration processes occur. These ones affect to metal bioavailability. In this study a review of fate, behavior and toxicity of metallic nanoparticles is carried out taken in account the differences between fresh and marine environment with special emphasis on phytoplankton., This work has been funded by the projects PE2011-RNM-7812 Junta Andalucía and CTM2012-38720-C03-03 Plan Nacional Investigación (MINECO).

Trabajo presentado en el 2nd Marine NanoEcoSafety Workshop (MANET), celebrado en Palermo el 17 y 18 de noviembre de 2014., Gold nanopar ticles (AuNPs) h ave unique physical and chemical properties and are therefore used and developed for a wide r ange of novel applications in medicine, biology and chem istry. However in recent years concern has been raised over their ability to enter cells, organelles and n uclei, and provoke oxidative stress. In a laboratory-based experiment the non -t arget marine bivalve Ruditapes philippinarum was used as a model organismo Uptake, elimination and molec ular effects of ~20 nm citrate AuNPs at 0.75 Ilg 1-1 were studied over 7 days exposure and equal length elimination periodo Our results demonstrate that the AuNPs were readily taken up into the digestive glan d > gilIs. We further observed limited oxidative stress and inflammatory responses. The activity of several antioxidant en zy mes was increased in an organ sp ecific manner, with the digestive gland ex.hibiting the majority of its enzyme activity within 2,1 hours of exposure. Gene expression analysis highlighted the involvement of inflammatory re sponse genes in digestive gland tissue, as measured by changes in T NF-a and Gadd45 expression. Suggesting that one m echanism of citrate AuNP toxicity i5 well described by the oxidative stress par adigm. Further a significant elimination of Au from the digestive tract within t he purification period was observed, with excretion being an important pathway. In conclusiol1, wh ile not producing significant oxidative dam ag e, concern is rajsed regardi ng the effects of ch ronic AuNP exposure.

Trabajo presentado en la Final SCARCE International Conference (River Conservation Under Water Scarcity: Integration of water quantity and quality in Iberian Rivers under global change), celebrada en Tarragona el 20 y 21 de octubre de 2014., According to the European Water Framework Directive (WFD), assessment of surface water status is based on ecological and chemical status that not always are in coherence. In these situations, ecotoxicity tests could help to obtain a better characterization of the ecosystems. The general aim of this work was to design a cost-effective methodology to study the ecotoxicological status of freshwater systems that could be useful and complementary to ecological status defined by the WFD by applying a quality triad integrating chemical, physicochemical, biological and ecotoxicological data. The specific aims of this work have been: (1) to compare the effectiveness and viability of different ecotoxicity tests performed with freshwater sedimen ts (whole sediment and pore water) taking as target organisms different aquatic species, and (2) to evaluate the relationship between ecological status, pollutant concentrations (paying special attention to metals availability), and pore water and sediment ecotoxicity. For this purpose, sediments from thirteen sampling sites within the Ebro river watershed (NE Spain) were collected for ecotoxicity characterization. The ecotoxicity of pore water has been evaluated employing the test organisms Vibrio fischeri, Pseudokirschneriella subcapitata and Daphnia magna, while whole sediment ecotoxicity was evaluated using Vibrio fischeri, Daphnia magna, Nitzschia palea and Chironomus riparius. An analysis of acid-volatile sulfide (AVS) and simultaneously extracted metals (SEM) was performed to evaluate the sediment toxicity associated to bioavailable metals. Moreover, data about priority pollutants defined by the WFD in wate r, sediment and fish as well as data of surface water status of each sampling point were provided by the Monitoring and Control Program of the Ebro Waterbodies. In general terms, whole sediment bioassay s have shown more toxicity than pore water tests. Among the different organisms used, P.subcapitata and C. riparius were the most sensitive in pore water and whole sediment, respectively. Our evaluation of th e ecotoxicological status showed high coincidences with the ecological status, established according to the WFD, especially when ecosystem disruption due to numerous stressors (presence of metals and organi c pollution) was observed. These results allow us to confirm that, when chemical stressors affect the ec osystem functioning negatively, an ecotoxicological approach, provided by suitable bioassays in pore water and whole sediment, could be able to detect these changes with accurate sensitivity., Authors would like to thank the Spanish Ministry of Economy and Competitiveness for its financial support through the project SCARCE (Consolider-Ingenio 2010 CSD2009-00065).

Concerns are being expressed recently over possible environmental effects of human pharmaceuticals. Although the likelihood of acute toxicity is low, the continuous discharge of pharmaceuticals into the aquatic environment means that sublethal effects on non-target organisms need to be seriously considered. One-year-old Atlantic salmon parr were exposed to 7.85 ± 0.13 μg L-1 of the antidepressant drug Carbamazepine (CBZ) for five days to investigate changes of mRNA expression in the brain by means of a custom 17k Atlantic salmon cDNA microarray. The selected concentration is similar to upper levels that can be found in hospital and sewage treatment plant effluents. After treatment, 373 features were differently expressed with 26 showing up- or down-regulation of ≥2-fold (p ≤ 0.05). Among the mRNAs showing the highest change were the pituitary hormones encoding features somatolactin, prolactin and somatotropin, or growth hormone. Functional enrichment and network analyses of up- and down-regulated genes showed that CBZ induced a highly different gene expression profile in comparison to untreated organisms. CBZ induced expression of essential genes of the focal adhesion and extracellular matrix - receptor interaction pathways most likely through integrin alpha-6 (itga6) activation. Negative regulation of apoptotic process, extracellular matrix organization and heme biosynthesis were the most enriched biological process related GO-terms, with the simultaneous enrichment of collagen and extracellular region related cellular component GO-terms, and extracellular matrix structural constituent, hormone activity and chromatin binding molecular function related GO-terms. These results show that relatively low doses of CBZ may affect brain physiology in exposed salmon parr, targeting similar processes as in human, indicating a high degree of conservation of targets of CBZ action. However, and since the mRNAs showing most changes in expression are critical for adaptation to different stressors and life history transitions in Atlantic salmon, more research should be undertaken to assess CBZ effects to avoid impairment of normal development and maintenance of natural populations.

Trabajo presentado en la SETAC Europe 24th Annual Meeting (Science Across Bridges, Borders and Boundaries), celebrada en Basilea del 11 al 15 de mayo de 2014., According to the European WFD, ecological status is based on three quality elements: biological, physicochemical and hydromorphological, but ecotoxicological status is still not included. Biological status is not always in coherence with chemical status, maybe due to the adaptation mechanisms of aquatic organisms under chronic chemical exposure. In these situations, ecotoxicity tests could be useful to resolve these divergences. Moreover, the evaluation of the freshwater ecological status often supposes time, workforce expertise and costs, that sometimes the water management organisms can not assume.