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Microorganisms play unique, essential and integral roles in the biosphere. This work aims to assess the utility of soil's metaomics for environmental diagnosis. Doñana National Park (DNP) was selected as a natural lab since it contains a strictly protected core that is surrounded by numerous threats of pollution. Culture-independent high-throughput molecular tools were used to evaluate the alterations of the global structure and metabolic activities of the microbiome. 16S rRNA sequencing shows lower bacterial abundance and diversity in areas historically exposed to contamination that surround DNP. For metaproteomics, an innovative post-alkaline protein extraction protocol was developed. After NaOH treatment, successive washing with Tris–HCl buffer supplemented with glycerol was essential to eliminate interferences. Starting from soils with different physicochemical characteristics, the method renders proteins with a remarkable resolution on SDS-PAGE gels. The proteins extracted were analysed by using an in-house database constructed from the rRNA data. LC–MS/MS analysis identified 2182 non-redundant proteins with 135 showing significant differences in relative abundance in the soils around DNP. Relevant global biological processes were altered in response to the environmental changes, such as protective and antioxidant mechanisms, translation, folding and homeostasis of proteins, membrane transport and aerobic respiratory metabolism., This study was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, CTM2016-75908-R), by the Spanish Ministry of Science and Innovation (MICINN, PID2019-110049RB-I00), by the Andalusian Plan of Research, Development, and Innovation (PAIDI) and by the University of Cordoba ‘Plan Propio’ program for group BIO187. A. M. Herruzo-Ruiz received a predoctoral contract of the University of Cordoba (‘Plan Propio’). Funding for open access charge: Universidad de Cordoba / CBUA.

The spread of pesticides in water bodies integrated into agricultural landscapes may prevent some areas from being colonized. In this study, the effects on the colonization responses of D. magna exerted by gradients of realistic environmental concentrations of the pesticides chlorpyrifos, terbuthylazine and their mixtures were tested in a novel multicompartment non-forced exposure system. Furthermore, the effects of both pesticides and their mixtures on the swimming behavior and the neurotransmission activity of D. magna were analyzed using a traditional forced exposure system. The synthesis and concentration of the main environmental metabolites of terbuthylazine were also analyzed. Results confirmed that D. magna exposed to mixture gradients were able to detect the pollutants and their colonization dynamics were drastically inhibited. The swimming behavior increased in D. magna exposed to the highest concentration of the mixture treatment. AChE activity was only significantly inhibited in the D. magna exposed to the highest concentration of chlorpyrifos. Changes in swimming behavior could not be directly related to the effects on AChE. Furthermore, the synthesis of the metabolite terbuthylazine 2-hydroxy during the course of the experiments was confirmed. These results demonstrate the importance of integrating pesticide mixtures in both non-forced and forced exposure systems during ecotoxicological assays., This research was funded by the Spanish Ministry of Science, Innovation and Universities and the European Regional Development Fund (ERDF) through the project WETANPACK (grant number RTI2018-097158-B-C31) and MCIN/AEI/10.13039/501100011033 through the project BrEStress (PID2019-105868RA-I00).

Scrobicularia plana is a coastal and estuarine bivalve widely used in ecotoxicological studies. However, the underlying molecular mechanisms for S. plana pollutant responses are hardly known due to the lack of molecular databases. Thus, in this study we present a holistic approach to assess a robust reference transcriptome and proteome of this clam. A mixture of control and metal-exposed individuals was used for mRNA isolation. Four sets of high quality filtered preprocessed reads were generated (two quality scores and two sequenced lengths) and assembled with Mira, Ray and Trinity algorithms. The sixty-four generated assemblies were refined, filtered and evaluated for their proteomic quality. Eight assemblies presented top Detonate scores but one was selected due to its compactness and biological representation, which was generated: (i) from the highest quality dataset (Q20L100), (ii) using Trinity algorithm with all k-mers (AtKa), (iii) removing redundancy by CD-HIT (RR80), and (iv) filtering out poor contigs (F), that was subsequently named Q20L100AtKaRR80F. S. plana proteomic analysis revealed 10,017 peptide groups that corresponded to 2066 proteins with a wide coverage of molecular functions and biological processes, confirming the strength of the database generated., This study was supported by the Ministry of Economy and Competitiveness, Spain (CTM2016-75908-R). We would like to thank Dr. Mercedes Cousinou and Ms. Laura Redondo (Genomic Unit, SCAI, UCO) for their technical help in transcriptome sequencing. A.M. Herruzo-Ruiz received a predoctoral contract of the University of Cordoba (“Plan Propio”).

Temperature variations and thermal extremes events caused by climate change can have profound implications for the toxicity of pesticides in aquatic organisms. Using an innovative system (Heterogeneous Multi-Habitat Test System - HeMHAS) that allows the simulation of different scenarios within a spatially heterogeneous landscape, the effects on the habitat selection of Danio rerio fish caused by the pesticides fipronil and 2,4-D were studied as single compounds and in mixture and integrated with air temperature variation (20, 24 and 28 °C). As a result, D. rerio detected and avoided both pesticides at air temperatures of 20 and 24 °C; however, at 28 °C no significant difference was observed in habitat choice by fish. Additionally, when pesticides were mixed in a heterogeneously contaminated landscape, it was observed that D. rerio detected contamination and preferred the clean zone at 20 and 24 °C; however, at 28 °C the potential to escape from the most contaminated areas was impaired. Thus, contamination by both pesticides made the habitat selection behavior of fish at 20 and 24 °C more noticeable. In addition, the association between pesticides and temperature showed negative effects on the response of fish to detect and escape from contaminated environments, suggesting the influence of temperature in altering the ability of the organism to provide an efficient response to stress., R.A. Moreira would like to thank the São Paulo Research Foundation “Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP) for the post doctorate grant to the first author (Process number: 2020/16131-0 and 2017/24126-4). Financial support was also provided by FAPESP (2015/18790-3). C.V.M. Araújo is grateful to the grant (Ramón y Cajal contract: RYC-2017-22324) funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. This research was funded by MCIN/AEI/10.13039/501100011033 (BrEStress project: PID2019-105868RA-I00) and i-COOP2019 program from CSIC (MultiCecotox project: #COOPB20444).

This chapter introduces the concept of the weight line of evidence applied to the ocean acidification process. First, the acidification process is situated in the context of climate change and how pH decrease affects the carbon chemical equilibrium in seawater. The pH changes have a direct effect on aquatic organisms, especially those that are calcifying organisms. However, ocean acidification indirect effects—as results of the joint action with other stressors—are presented too. Considering the key role of metals in many biogeochemical processes, changes induced by ocean acidification on speciation are summarized. The combined effect of new pollutants and ocean acidification in coastal ecosystems and sensitive regions is considered. In the final remarks section, some open questions and future research have been addressed with a sustainable growth to avoid the irreversible collapse of marine ecosystems.

Selenium (Se) is a vital trace element for many living organisms inclusive of aquatic species. Although the antagonistic action of this element against other pollutants has been previously described for mammals and birds, limited information on the join effects in bivalves is available. To this end, bivalves of the species Scrobicularia plana were exposed to Se and Cd individually and jointly. Digestive glands were analysed to determine dose-dependent effects, the potential influence of Se on Cd bioaccumulationas well as the possible recover of the oxidative stress and metabolic alterations induced by Cd. Selenium co-exposure decreased the accumulation of Cd at low concentrations. Cd exposure significantly altered the metabolome of clams such as aminoacyltRNA biosynthesis, glycerophospholipid and amino acid metabolism, while Se co-exposure ameliorated several altered metabolites such asLysoPC (14:0), LysoPE (20:4), LysoPE (22:6), PE (14:0/18:0), PE (20:3/18:4) andpropionyl-L-carnitine.Additionally, Se seems to be able to regulate the redox status of the digestive gland of clams preventing the induction of oxidativedamage in this organ. This study shows the potential Se antagonism against Cd toxicity in S. plana and the importance to study join effects of pollutants to understand the mechanism underlined the effects., This study has been carried out within the research projects (CTM2012-38720-C03-03 and CTM2016-75908-R) founded by the Spanish Ministry of Economy and Competitiveness, who also provided a pre-doc grant for Chiara Trombini (BES-2013-063426), and the project PGC-2018- 096608-B-C21 from the Spanish Ministry of Science and innovation (MCIN). (Generación del Conocimiento. MCIN/AEI/10.13039/501100011033/FEDER “Una manera de hacer Europa”). Authors are grateful to FEDER (European Community) for financial support, Grant UNHU13-1E-1611. Rodríguez-Moro, G. thanks to Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020) and European Union for a post-doctoral grant (DOC_01115). Funding for open access charge: Universidad de Huelva / CBUA.

The high levels of contamination in aquatic ecosystems caused by pesticides and the organisms' consequent continuous exposure to it has made them vulnerable to damage. However, mobile organisms can avoid this continued exposure to contaminants by moving to less disturbed habitats. Therefore, through the use of the Heterogenous Multi-Habitat Assay System (HeMHAS), our objective was to evaluate the ability of Daphnia magna to detect and avoid habitats contaminated by fipronil and 2,4-D, in a spatially connected landscape. Further, the role of contamination by these pesticides, isolated and in mixtures, concerning the colonization of habitats by daphnids was also evaluated. Given that not all organisms successfully escape contamination, the chronic toxicity of the same pesticides using different parameters for D. magna (maternal survival, fecundity and maternal body length) was also evaluated. When evaluating the avoidance response by D. magna exposed to pesticides, there was no preference for the less contaminated areas for both compounds. However, organisms did not move to contaminated zones in the colonization experiments, with no immigration of daphnids to the zones with intermediate and the highest levels of fipronil, nor to the highest concentration of 2,4-D. Finally, the colonization by daphnids was significantly prevented when exposed to a mixture of the pesticides, in which the areas with the highest combinations of pesticide concentrations were not colonized by D. magna. Regarding the long-term chronic effects, negative consequences were observed, particularly for maternal body length, fecundity and maternal survival, due to the exposure to fipronil. Considering that pesticides can limit the areas colonized by organisms by making them unattractive, the risk of local population extinction may be underestimated if only standard endpoints involving forced exposure are studied.

The use of non-forced multi-compartmented exposure systems has gained importance in the assessment of the contamination-driven spatial avoidance response. This new paradigm of exposure makes it possible to assess how contaminants fragment habitats, interfering in the spatial distribution and species¿ habitat selection processes. In this approach, organisms are exposed to a chemically heterogeneous scenario (a gradient or patches of contamination) and the response is focused on identifying the contamination levels considered aversive for organisms. Despite the interesting results that have been recently published, the use of this approach in ecotoxicological risk studies is still incipient. The current review aims to show the sensitivity of spatial avoidance in non-forced exposure systems in comparison with the traditional endpoints used in ecotoxicology under forced exposure. To do this, we have used the sensitivity profile by biological groups (SPBG) to offer an overview of the highly sensitive biological groups and the species sensitive distribution (SSD) to estimate the hazard concentration for 5% of the species (HC5). Three chemically different compounds were selected for this review: copper, glyphosate, and Ag-NPs. The results show that contamination-driven spatial avoidance is a very sensitive endpoint that could be integrated as a complementary tool to ecotoxicological studies in order to provide an overview of the level of repellence of contaminants. This repellence is a clear example of how contamination might fragment ecosystems, prevent connectivity among populations and condition the distribution of biodiversity., C.V.M. Araújo is grateful to the grant (Ramón y Cajal contract: RYC-2017-22324) funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”.

Microplastics (MPs) pollution is a threat to the marine environment worldwide (Andrady, 2011) from the tropics to the poles (Waller et al., 2017). The Mediterranean Sea, a semi-closed basin, is heavily affected by this pollution as well (Llorca et al., 2020). The aim of this special issue was to collect the latest studies on MPs pollution in the Mediterranean Sea. MPs are plastic particles smaller than 5 mm in size that derive from industrial products (primary microplastics) or from the fragmentation of large plastic pieces (secondary microplastics) (Auta et al., 2017). MPs are found floating on the surface, along with those in the water column, in sediments, and up to the deep sea (Van Cawenberghe et al., 2013). MPs can be ingested by aquatic biota and they can be a vehicle for other pollutants such as persistent organic pollutants (POPs), that can be adsorbed and concentrated from the surrounding seawater. As a result, MPs can be transfer several toxic pollutants via the food chain (Fytianos et al., 2020). This editorial introduces to the Research Topic “Microplastics in the Mediterranean Sea,” highlighting the pollution of MPs in this peculiar ecosystem and aiming at a better understanding of the occurrence, distribution, and toxicity presence and the study of MPs in the Mediterranean Sea.

Surgical face masks are more present than ever as personal protective equipment due to the COVID-19 pandemic. In this work, we show that the contents of regular surgical masks: i) polypropylene microfibres and ii) some added metals such as: Al, Fe, Cu, Mn, Zn and Ba, may be toxic to some marine life. This work has got two objectives: i) to study the release rate of the products from face masks in marine water and ii) to assess the toxicity in Phaeodactylum tricornutum of these by-products. To achieve these two objectives, we performed release kinetic experiments by adding masks in different stages of fragmentation to marine water (i.e. whole face masks and fragments of them 1.52 ± 0.86 mm). Released microfibres were found after one month in shaking marine water; 0.33 ± 0.24 and 21.13 ± 13.19 fibres·mL−1 were collected from the whole and fragmented face masks, respectively. Significant amounts of dissolved metals such as Mn, Zn and Ni, as well as functional groups only in the water containing the face mask fragments were detected. Water from both treatments was employed to study its toxicity on the marine diatom. Only the water from the face mask fragments showed a significant, dose-dependent, decrease in cell density in P. tricornutum; 53.09 % lower than in the controls. Although the water from the face mask fragments showed greater effects on the microalgae population than the water from the whole face mask, the latter treatment did show significant changes in the photosynthetic apparatus and intrinsic properties of the cells. These results indicate that during fragmentation and degradation face masks a significant chemical print can be observed in the marine environment., Marta Sendra wishes to acknowledge her contract Juan de la Cierva Incorporación (IJC2020-043162-I) funded by MCIN/AEI/10.13039/ 501100011033 and European Union NextGenerationEU/PRTR. Dr. Araceli Rodríguez-Romero is supported by the Spanish grant Juan de la Cierva Incorporación referenced as IJC2018–037545-I.

The high consumption and subsequent input of antibacterial compounds in marine ecosystems has become a worldwide problem. Their continuous presence in these ecosystems allows a direct interaction with aquatic organisms and can cause negative effects over time. The objective of the present study was to evaluate the effects of exposure to three antibacterial compounds of high consumption and presence in marine ecosystems (Ciprofloxacin CIP, Sulfadiazine SULF and Trimethoprim TRIM) on the physiology of the gilthead sea bream, Sparus aurata. Plasma parameters, enzymatic biomarkers of oxidative stress and damage and expression of genes related to stress and growth were assessed in exposed S. aurata specimens. For this purpose, sea bream specimens were exposed to individual compounds at concentrations of 5.2 ± 2.1 μg L-1 for CIP, 3.8 ± 2.7 μg L-1 for SULF and 25.7 ± 10.8 μg L-1 for TRIM during 21 days. Exposure to CIP up-regulated transcription of genes associated with the hypothalamic-pituitary-thyroid (HPT) (thyrotropin-releasing hormone, trh) and hypothalamic-pituitary-interrenal (HPI) axes (corticotropin-releasing hormone-binding protein, crhbp) in the brain, as well as altering several hepatic stress biomarkers (catalase, CAT; glutathione reductase, GR; and lipid peroxidation, LPO). Similar alterations at the hepatic level were observed after exposure to TRIM. Overall, our study indicates that S. aurata is vulnerable to environmentally relevant concentrations of CIP and TRIM and that their exposure could lead to a stress situation, altering the activity of antioxidant defense mechanisms as well as the activity of HPT and HPI axes., This study was funded by the Spanish Ramón y Cajal funding scheme supportingMH (contract reference RYC-2012-12217) and the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) within the framework of the project: Integration of Omics Tools for the Environmental Risk Assessment of Emerging Pollutants in Marine Species of Commercial Interest, HORACIO (CTM2015-70731-R).

7 pages, 3 figures.-- Under a Creative Commons license, Environmental contamination is a problem that reduces the quality of ecosystems and may make them unsuitable to accommodate life. As many ecosystems are connected, some organisms avoid the stress from continuous exposure to contaminants by moving towards less disturbed areas. However, the landscapes in which organisms move might vary regarding the concentrations of contaminants, in the form of gradients or patches of contamination. Therefore, although it is expected that organisms prefer clean areas, their sporadic contact with contamination should not be ignored, as the greater the probability of being in contact with contaminated areas, the higher the stress. The aim of this study was to assess how the stress (cortisol levels) of zebrafish (Danio rerio) varies as a consequence of heterogeneity in the chemical composition of the habitats and the presence of uncontaminated areas in this heterogeneous landscape. Zebrafish were exposed to heterogeneous contamination scenarios containing different concentrations of copper along a free-choice multi-compartmented system, in which they were able to flee from the most contaminated areas. Fish escaped from the most contaminated areas with an avoidance by 50% of population (AC50) at concentrations of 41 (copper gradient scenario), 25 (spatially limited contamination scenario) and 69 (highly contaminated scenario) μg/L. Higher cortisol levels were observed in the populations exposed to homogeneously contaminated and highly contaminated (by copper) scenarios (both with no acceptable clean area to flee to). In summary, the uncontaminated areas might be crucial for the spatial dynamics of fish populations in a chemically heterogeneous landscape due to their role as escape zones to alleviate stress, This study was funded by the Spanish Ministry of Science and Innovation [MCIN/AEI/10.13039/501100011033 (BrEStress project: #PID2019-105868RA-I00)] and BrEStress project: #CGL2017-92160-EXP

As the exposure of organisms to contaminants can provoke harmful effects, some organisms try to avoid a continuous exposure by using different strategies. The aim of the current study was to assess the ability of the shrimp Palaemon varians to detect a triclosan gradient and escape to less contaminated areas. Two multi-compartmented exposure systems (the linear system and the HeMHAS-Heterogeneous Multi-Habitat Assay System) were used and then results were compared. Finally, it was aimed how sensitive the avoidance response is by comparing it with other endpoints through a sensitivity profile by biological groups and the species sensitive distribution. The distribution of the shrimps along the triclosan gradient was dependent on the concentrations, not exceeding 3% for 54 μg/L in the linear system and 7% for 81 μg/L in the HeMHAS; 25% of organisms preferred the compartment with the lowest concentrations in both systems. Half of the population seems to avoid concentrations around 40–50 μg/L. The triclosan concentration that might start (threshold) to trigger an important avoidance (around 20%) was estimated to be of 18 μg/L. The profile of sensitivity to triclosan showed that avoidance by shrimps was less sensitive than microalgae growth and avoidance by guppy; however, it might occur even at concentrations considered safe for more than 95% of the species. In summary, (i) the HeMHAS proved to be a suitable system to simulate heterogeneous contamination scenarios, (ii) triclosan triggered the avoidance response in P. varians, and (iii) the avoidance was very sensitive compared to other ecotoxicological responses., C.V.M. Araújo is grateful to the Spanish Ministry of Science, Innovation and Universities for the Juan de la Cierva (IJCI-2014-19318) and Ramón and Cajal (RYC-2017-22324) contracts. M. Hampel benefits from a Ramón and Cajal contract (RYC-2012-12217) from the Spanish Ministry for Economy and Competitiveness. This study was funded by the Spanish Ministry of Science, Innovation and Universities, Spain (Explora Project: #CGL2017-92160-EXP) and the Ministry for Economy and Competitiveness in the framework of the project HORACIO (CTM2015-70731-R).

Cerium nanoparticles (nCeO2) are increasingly utilized in a wide variety of industrial, environmental and biomedical applications, and are therefore expected to be released in the aquatic environment. Due to its peculiar redox properties, nCeO2 may present unique hazards to environmental and human health. Previous data showed that in the hemocytes of the marine bivalve Mytilus galloprovincialis, in vitro exposure to a particular type of nCeO2 (9 nm, characterized by negative ζ-potential, high H2O2 scavenging capacity and Ce3+/Ce4+ surface ratio) reduced basal ROS production, lysosomal membrane stability and phagocytic activity in the presence of hemolymph serum; the effects observed were partly ascribed to the formation of a SOD-protein corona in the hemolymph. In this work, the in vivo effects of this type of nCeO2 were investigated in mussels exposed to 100 μg/L nCeO2 for 96 h; several lysosomal, immune, inflammatory and antioxidant biomarkers were measured at cellular (hemocytes) and tissue (gills, digestive gland) level. Molecular responses were evaluated in hemocytes and digestive gland by determining expression of 11 selected genes related to known biological functions. The results show specific immunomodulatory and antioxidant effects of nCeO2 at different levels of biological organization in the absence of Cerium tissue accumulation. These data further support the redox mechanisms at the basis of the physiological effects of nCeO2. Finally, in order to evaluate the possible impact at the whole organism level, the effects of nCeO2 were evaluated in the 48 h embryotoxicity assay in a wide concentration range. However, nCeO2 exposure resulted in a small reduction in normal embryo development. Overall, the results demonstrate that in mussels nCeO2 can selectively modulate different physiological processes at different levels of biological organization., This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement PANDORA N° 671881.

The use of nanoparticles (NPs), such as Ag and CeO2 NPs, has increased considerably in the last decade due to their importance for the production of engineered nanomaterials applied to new consumer products. This generalized use in everyday products has made the presence of NPs in aquatic systems more frequent and makes them potential environmental disturbers. Marine phytoplankton is at the bottom of the food web and, therefore, microalgae are potentially susceptible to NPs at different levels: reproductive (population growth), structural and metabolic. One of the first mechanisms of toxicity is caused by the adsorption of NPs onto the cell wall later leading to their internalization. As the cell wall may be a barrier against the intake of NPs, species lacking a cell wall would be expected to show a higher sensitivity. In the present study, two microalgae species with a marked structural difference, D. salina, lacking a cell wall, and Chlorella autotrophica, with a typical cellulosic cell wall, were exposed to ionic and NP forms of the metals Ag and Ce for 72 h. The biomarkers used as indicative of toxicity were: cell density, cell viability, cell size, cell complexity, autofluorescence of chlorophyll a, active chlorophyll, effective quantum yield of PII (photosystem II) and reactive oxygen species (ROS). Exposure to Ag and Ce in both ion and NP forms affected the reproductive, structural and physiological mechanisms of D. salina and C. autotrophica. In general, toxicity resulted in a decrease in active chlorophyll, effective quantum yield of PII, and cell density and an increase in cell complexity and ROS. For both species, treatments with Ag were more toxic than those of Ce and, for both metals, the ionic form was more toxic than the NPs. Although D. salina lacks a cell wall, it was more tolerant than C. autotrophica, indicating that the absence of a cell wall does not make it a more sensitive species. The higher tolerance of D. salina might be related to different processes to manage metal exposure that prevent toxicity such as biosorption, by producing extracellular polymeric substances, and the elimination of the compounds to the external environment.

Lines of evidence used in ecological risk assessment (ERA) are essentially three: chemistry, biology and ecotoxicology. Until now, the fundamental assumption made when measuring ecological risks is that organisms are forcedly exposed to stressors. However, when organisms can avoid disturbed habitats by escaping to less stressful areas, the assumption that exposure is mandatory to pose risk may not match field disturbance scenarios. A non-forced exposure approach using a linear free-choice multi-compartmented system has been proposed previously as a complementary tool to assess the effects on organisms' spatial avoidance/preference responses. Yet, the linearity of the latter system limits avoidance measurements to one spatial dimension. A novel, heterogeneous multi-habitat assay system (HeMHAS) consisting of 18 connected circular compartments (3 compartments on a vertical axis in each one of 6 zones on a longitudinal axis; a 2D system) is put forward here to be used in heterogeneous-habitat selection studies, as it makes it possible to assess the ability of organisms to detect contamination and other stressors and select more favorable habitats. In the present study, the avoidance to copper by zebrafish (Danio rerio) was tested after exposing organisms to a copper gradient in the HeMHAS and compared with that in the linear system. Avoidance occurred for all copper concentrations: 43% in the lowest (21 μg·L-1) to 72% in the highest (221 μg·L-1). Results obtained within the HeMHAS (AC50: 60 μg·L-1) were statistically (p = 0.72) similar to avoidance of copper by D. rerio in the linear non-forced system (AC50: 89 μg·L-1). In summary, the simulation of a copper gradient in the HeMHAS (2D system) allowed to assess the potential repellency of copper to zebrafish and to corroborate the ability of organisms to detect and avoid potentially toxic concentrations.

The knowledge about the effects of pharmaceuticals on aquatic organisms has been increasing in the last decade. However, due to the variety of compounds presents in the aquatic medium, exposure scenarios and exposed organisms, there are still many gaps in the knowledge on how mixtures of such bioactive compounds affect exposed non target organisms. The crayfish Procambarus clarkii was used to analyze the toxicity effects of mixtures of ciprofloxacin, flumequine and ibuprofen at low and high concentrations (10 and 100 μg/L) over 21 days of exposure and to assess the recovery capacity of the organism after a depuration phase following exposure during additional 7 days in clean water. The crayfish accumulated the three compounds throughout the entire exposure in the hepatopancreas. The exposure to the mixture altered the abundance of proteins associated with different cells functions such as biotransformation and detoxification processes (i.e. catalase and glutathione transferase), carbohydrate metabolism and immune responses. Additionally changes in expression of genes encoding antioxidant enzymes and in activity of the corresponding enzymes (i.e. superoxide dismutase, glutathione peroxidase and glutathione transferase) were reported. Alterations at different levels of biological organization did not run in parallel under all circumstances and can be related to changes in the redox status of the target tissue. No differences were observed between control and exposed organisms for most of selected endpoints after a week of depuration, indicating that exposure to the drug mixture did not produce permanent damage in the hepatopancreas of P. clarkii., This work was supported by the Spanish Ministry of Economy and Competitiveness (reference of Projects: CTM2012-38720-C03-03 and CTM2016-75908-R; grant number BES-2013-063426) and ERDF.

Summary Aquatic environments are the recipients of many sources of environmental stress that trigger both local and global changes. To evaluate the associated risks to organisms and ecosystems more sensitive and accurate strategies are required. The analysis of the microbiome is one of the most promising candidates for environmental diagnosis of aquatic systems. Culture‐independent interconnected meta‐omic approaches are being increasing used to fill the gaps that classical microbial approaches cannot resolve. Here, we provide a prospective view of the increasing application of these high‐throughput molecular technologies to evaluate the structure and functional activity of microbial communities in response to changes and disturbances in the environment, mostly of anthropogenic origin. Some relevant topics are reviewed, such as: (i) the use of microorganisms for water quality assessment, highlighting the incidence of antimicrobial resistance as an increasingly serious threat to global public health; (ii) the crucial role of microorganisms and their complex relationships with the ongoing climate change, and other stress threats; (iii) the responses of the environmental microbiome to extreme pollution conditions, such as acid mine drainage or oil spills. Moreover, protists and viruses, due to their huge impacts on the structure of microbial communities, are emerging candidates for the assessment of aquatic environmental health., Aquatic environments are the recipients of many sources of environmental stress that trigger both local and global changes. We provide a prospective view of the increasing application of high‐throughput molecular technologies to evaluate the structure and functional activity of microbial communities in response to changes and disturbances in the environment.

To date, the occurrence, fate and toxicity of metal-based NPs in the environment is under investigated. Their unique physicochemical, biological and optical properties, responsible for their advantageous application, make them intrinsically different from their bulk counterpart, raising the issue of their potential toxic specificity or “nanosize effect”. The aim of this study was to investigate copper bioaccumulation, subcellular distribution and toxic effect in the marine benthic species Scrobicularia plana exposed to two forms of sediment-associated copper, as nanoparticles (CuO NPs) and as soluble ions (CuCl2). Results showed that the exposure to different copper forms activated specific organism's metal handling strategies. Clams bioaccumulated soluble copper at higher concentrations than those exposed to sediment spiked with CuO NPs. Moreover, CuO NPs exposure elicited a stronger detoxification response mediated by a prompt mobilization of CuO NPs to metal-containing granules as well as a delayed induction of MT-like proteins, which conversely, sequestered soluble copper since the beginning of the exposure at levels significantly different from the control. Eventually, exposure to high concentrations of either copper form led to the same acute toxic effect (100% mortality) but the outcome was delayed in bivalves exposed to CuO NPs suggesting that the mechanisms underlying toxicity were copper form-specific. Indeed, while most of soluble copper was associated to the mitochondrial fraction suggesting an impairment of the ATP synthesis capacity at mitochondrial level, CuO NPs toxicity was most likely caused by the oxidative stress mediated by their bioaccumulation in the enzymatic and mitochondrial metabolically available fractions., This research has been funded by the Spanish National Research Plan MINECO (CTM2016-75908-R) and European Regional Development Fund.

Contamination is likely to affect the composition of an ecological landscape, leading to the rupture of ecological connectivity among habitats (ecological fragmentation), which may impact on the distribution, persistence and abundance of populations. In the current study, different scenarios within a spatially heterogeneous landscape were simulated in the Heterogeneous Multi-Habitat Assay System (HeMHAS) to evaluate the potential effect that contamination (copper at 0.5 and 25 μg/L) might have on habitat selection by the estuarine shrimp Palaemon varians in combination with two other ecological factors: predator presence and food availability. As a result, P. varians detected and avoided copper; however, in the presence of the predation signal, shrimps shifted their response by moving to previously avoided regions, even if this resulted in a higher exposure to contamination. When encouraged to move towards environments with a high availability of food, a lower connectivity among the shrimp populations isolated by both contamination and predation risk simultaneously was evidenced, when compared to populations isolated only by the risk of predation. These results indicate that contamination might: (i) trigger avoidance in shrimps, (ii) prevent colonization of attractive foraging areas, (iii) enhance populations' isolation and (iv), make populations more susceptible to local extinction., D. Salvatierra thanks the Erasmus Mundus Joint Master Degree Program for the Master's fellowship. C.V.M. Araújo is grateful to the grant (Ramón y Cajal contract: RYC-2017-22324) funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. A. Cordero-de-Castro and Á. Rodríguez-Ruiz thank the Spanish National Research Council (CSIC) for the JAE-Intro fellowship. The authors thank Carmen Navarro, Gonzalo Martínez, Manolo Yúfera, Meli Agullo and Neda Gilannejad for their laboratory assistance and Jon Nesbit for his revision of the English text. This research was funded by MCIN/AEI/10.13039/501100011033 (BrEStress project: PID2019-105868RA-I00).

Lead is a priority pollutant introduced in the aquatic environment by different sources commonly located in estuarine regions, such as ports, marinas and industries. Environmental agencies around the world set the maximum allowable concentration of lead in effluents, surface water and sediment, but few studies reported its accumulation and chronic toxicity in mangrove benthic invertebrates using concentrations believed to be safe. In the case of Brazilian mangrove environments, Ucides cordatus is a crab species of choice to be used in bioaccumulation studies. We have assessed biomarkers’ responses (DNA strand breaks, micronucleated cells, metallothioneins, enzymatic activity of aminolevulinic acid dehydratase and neutral red retention time) and the total bioaccumulation in six tissues of U. cordatus crabs resident to mangrove areas under different conservation status during a 28-day period bioassay. We also investigated Pb subcellular partition and biomarkers’ responses using a supposedly safe concentration (10 μg L−1). During the Pb exposure, the highest concentration of Pb was observed in crab gills. Crabs also showed a high ability to allocate Pb in detoxified forms. Multivariate analysis pointed out that bioaccumulation (total, active and detoxified) is linked to biomarkers. Even in supposedly safe dosage, U. cordatus triggered its defense mechanisms expressing more metallothioneins and presented relevant cyto-genotoxic damage. Our data suggest the development of biological tolerance to Pb in crabs from polluted areas. Our results provided a new insight about lead toxicity even at concentrations considered environmentally safe, which could support new strategies to manage estuarine areas considering their respective conservation status., Authors acknowledge the São Paulo State Research Foundation (FAPESP) for financial support to LFAM (Grants # 2010/01552-9, 2014/13448-2 and 2016/05414-6) and The Brazilian National Council for Scientific and Technological Development (CNPq) for a productivity fellowship to CDSP.

Coastal transitional waters are exposed to many anthropogenic threats. This study aims to assess the trace metals' pollution status of transitional waters by evaluating its biological effects in the clam Venerupis decussata. Among the studied sites along the Tunisian littoral, South Tunis and Boughrara were the most impacted, since clams from these two lagoons presented significant differences in: (i) trace metal contents, (ii) in-cell hydrogen peroxide, (iii) enzymatic and non-enzymatic defenses, (iv) damage to lipids and proteins, and (v) protein post-translational modifications. These changes related to evident histopathological traits. PCA showed a clear separation between the digestive gland and gills tissues and illustrated an impact gradient in Tunisian coastal lagoons. Water temperature was revealed as an added natural stressor that, when concurring with high pollution, may jeopardize an ecosystem's health and contribute to the accumulation of hazardous metals in organisms., This study was supported by the Spanish Ministry of Economy and Competitiveness -CTM2016-75908-R-, by the Andalusian Plan of Research, Development, and Innovation, and by the University of Cordoba “Plan Propio” program to group BIO187 (Spain). Safa Bejaoui stay in Cordoba was funded by the Tunisian government, Ministry of Higher Education and Scientific Research, University of Tunis EL Manar, Laboratory of Ecology, Biology and Physiology of Aquatic Organisms -EBPAO, LR18ES41- (Tunisia). Pedro M. Costa acknowledges Fundação para a Ciência e Tecnologia -FCT-MCES- for the grant IF/00265/2015 and for the support to UCIBIO, Applied Molecular Biosciences Unit -UID/Multi/04378/2019- (Portugal).

From the issue entitled "Special Issue: Understanding effects of global change on water quantity and quality in river basins", INTRODUCTION: The Consolider-Ingenio 2010 project SCARCE, with the full title "Assessing and predicting effects on water quantity and quality in Iberian Rivers caused by global change" aims to examine and predict the relevance of global change on water availability, water quality, and ecosystem services in Mediterranean river basins of the Iberian Peninsula, as well as their socio-economic impacts. Starting in December 2009, it brought together a multidisciplinary team of 11 partner Spanish institutions, as well as the active involvement of water authorities, river basin managers, and other relevant agents as stakeholders. METHODS: The study areas are the Llobregat, Ebro, Jucar, and Guadalquivir river basins. These basins have been included in previous studies and projects, the majority of whom considered some of the aspects included in SCARCE but individually. Historical data will be used as a starting point of the project but also to obtain longer time series. The main added value of SCARCE project is the inclusion of scientific disciplines ranging from hydrology, geomorphology, ecology, chemistry, and ecotoxicology, to engineering, modeling, and economy, in an unprecedented effort in the Mediterranean area. The project performs data mining, field, and lab research as well as modeling and upscaling of the findings to apply them to the entire river basin. RESULTS: Scales ranging from the laboratory to river basins are addressed with the potential to help improve river basin management. The project emphasizes, thus, linking basic research and management practices in a single framework. In fact, one of the main objectives of SCARCE is to act as a bridge between the scientific and the management and to transform research results on management keys and tools for improving the River Basin Management Plans. Here, we outline the general structure of the project and the activities conducted within the ten Work Packages of SCARCE., This work has been supported by the Spanish Ministry of Science and Innovation through the project Consolider-Ingenio 2010 CSD2009-00065. Special thanks are due to all partners of the SCARCE consortium and the peer review panel for ensuring quality results and fruitful collaboration within the project.

Pharmaceuticals are included in the group of emergent pollutants due to their characteristics and potential negative effects. They remain mostly unregulated or are undergoing currently some sort of regularization process. Diclofenac, for instance, has been included in a watch list of substances for European Union-wide monitoring and the priority list of the Water Framework Directive (WFD) of the European Union. Nonsteroidal anti-inflammatory drugs (NSAIDs) are a varied and chemically heterogeneous group of mainly anti-inflammatory, analgesic, and antipyretic drugs, reducing symptoms of inflammation, pain, and fever, respectively. They are widely employed and have been detected in freshwater, seawater, and sediment. Nevertheless, they are found as mixture instead of single compounds. In this chapter, we have tried to summarize how to assess the risk due to the occurrence of pharmaceuticals in aquatic ecosystems. We have focused on the mixture of diclofenac and ibuprofen using acute and sublethal toxicity data for different aquatic species. It has been presented new strategies as adverse outcome pathway to improve the understanding of the toxicity of these compounds. Although gaps of the information are pointed out, the risk levels associated with the occurrence of these compounds in aquatic ecosystems will range between no risk or high risk, depending on concentrations and environmental conditions., We would like to thank the projects CTM2016-75908-R and CTM2015-70731-R and the Junta de Andalucía PAIDI Excellence Research Group RNM306 for their support. MH is supported by the Spanish Ramón y Cajal funding scheme (contract reference RYC-2012-12217) of the Spanish Ministry of Economy, Industry and Competitiveness (MINECO).

22 pages, The sunscreens are complex products for protecting the skin of UV radiation. These products contain active ingredients organic and inorganic UV filters. The release of some of these components can provoke negative effects to aquatic ecosystems. The UV filters have shown to be present in environmental compartments (freshwater, wastewater, groundwater, seawater, sediment, and sand) and to be ubiquitous, motivated by the use in other applications. To assess the environmental risk of these products implies to know exposure conditions and toxic effects in order to establish the risk quotient. This is calculated as the ratio between predicted environmental concentration (PEC) or measured environmental concentration (MEC) and predicted no-effect concentration (PNEC). The organic compounds that presented higher risk were benzophenone-3, ethylhexyl methoxycinnamate, and 4-methylbenzylidene camphor. Nevertheless, this risk is depending on the location and environmental compartment. The lack of a database concentration of inorganic nanoparticles (TiO2 and ZnO) makes difficult to carry out a realistic assessment of environmental risk, although using modeled data an approach was carried out. The results evidenced that certain risk can be related to the release of these nanomaterials from sunscreens, although a refinement will be necessary to reduce the uncertainties. Finally, some gaps of information have been identified in order to get a more realistic environmental risk assessment. Thus, the toxicity of the mixture of sunscreens compounds under realistic conditions and the improvement of the knowledge of their mode of actions could be the next steps, We would like to thank to the projects CTM2016-75908-R funding by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and FEDER funds and the Junta de Andalucía PAIDI, Excellence Research Group RNM306 for their support

Metals have important roles in biological systems due to their essential or toxic character, the participation in controlling numerous cellular and biological processes, and their effects on the metabolism that regulates numerous cellular processes. The study of metal-induced changes on metabolic pathways is fundamental to decipher the biological response related to environmental problems. In this context, the omics, such as metallomics and metabolomics, are powerful methodological tools to reach this aim by searching for biomarkers that act as a warning of harmful environmental situations. Generally, different bioindicator organisms (mice, clams, and crabs) have been used to evaluate the biological response associated with the presence of contaminants in the laboratory, which can be extrapolated to actual environmental issues in terrestrial and aquatic systems. Suitable exposure experiments and the application of multidimensional analytical techniques based on the coupling of chromatography with inductively coupled plasma mass spectrometry for very specific analysis of metal-containing biomolecules (metallomics), as well as the use of organic mass spectrometry, are considered for the nontargeted evaluation of metabolite changes caused by the contamination (metabolomics). Translational application of this methodology to natural spaces affected by contamination allows establishing, in a closer evaluation, the processes involved in the environment degradation and its assessment.

Editors: Antonio Tovar-Sanchez, David Sanchez-Quiles, Julian Blasco. The Handbook of Environmental Chemistry book series (HEC, volume 94)

The ability of aquatic organisms to sense the surrounding environment chemically and interpret such signals correctly is crucial for their ecological niche and survival. Although it is an oversimplification of the ecological interactions, we could consider that a significant part of the decisions taken by organisms are, to some extent, chemically driven. Accordingly, chemical contamination might interfere in the way organisms behave and interact with the environment. Just as any environmental factor, contamination can make a habitat less attractive or even unsuitable to accommodate life, conditioning to some degree the decision of organisms to stay in, or move from, an ecosystem. If we consider that contamination is not always spatially homogeneous and that many organisms can avoid it, the ability of contaminants to repel organisms should also be of concern. Thus, in this critical review, we have discussed the dual role of contamination: toxicity (disruption of the physiological and behavioral homeostasis) vs. repellency (contamination-driven changes in spatial distribution/habitat selection). The discussion is centered on methodologies (forced exposure against non-forced multi-compartmented exposure systems) and conceptual improvements (individual stress due to the toxic effects caused by a continuous exposure against contamination-driven spatial distribution). Finally, we propose an approach in which Stress and Landscape Ecology could be integrated with each other to improve our understanding of the threat contaminants represent to aquatic ecosystems., SI

Since ancient times, humans have felt the need to protect their skin from the harmful effects of the sun: first with the use of vegetable oils or mud that were applied on the skin and then with the wearing of clothes, hats, or umbrellas. Today, the use of sunscreens around the world has become widespread. It has been shown that the use of these cosmetics can release large quantities of chemicals into coastal waters, either directly through bathing or indirectly through waste water treatment plants and atmospheric depositions. Due to the nature of the active ingredients of sunscreens, organic and inorganic UV filters, it has been proven that they can bioaccumulate and bioconcentrate in sediments and biota and can enter the food chain, being a problem whose true magnitude is still unknown.