Gene Expression Profile Assessment in Zebrafish (Danio rerio)

The Zebrafish Danio rerio, in the last decade, has been recognized as the best model among the vertebrate model organisms. The exquisite features and the high homology with the mammalian system have facilitated its application in many field e.g. eco-toxicology, biomedicine, genetics and physiology. The complete sequencing of the genome provided enormous value giving the possibility to develop a DNA microarray for gene expression analysis. Gene expression represents a unique way of characterizing how cells and organisms adapt to changes in the external environment (Lettieri, EHP, 2006). The measurements of gene expression levels, upon exposure to a chemical, can be used both to provide information about the mechanism of action of the toxicant, and also to form a sort of ¿genetic signature¿ for the identification of toxic products. Benzo[a]pyrene (BaP) is a well-known chemical pollutant, which belongs to the family of polycyclic aromatic hydrocarbons (PAHs) which are formed during combustion processes. Extensive studies have been published on the metabolism and the toxicity of PAHs, as well as epidemiologic studies which suggest a role of the PAHs in carcinogenetic and mutagenic effects in many species. BaP as well as other PAHs is included in the Priority Hazardous Substances adopted in November 2001 by the new Water Framework Directive (2000). Our studies focused on the analysis of gene expression changes in a liver cell line of Zebrafish upon exposure to BaP concentrations close to environmental level using DNA Microarrays. In our gene expression analysis, we identified nine genes, among them the cyctochrome P450 CYP1A involved in the xenobiotic response and the cyb5 whose role in the activation of human CYP1A2 was recently reported in the literature.JRC.H.5-Rural, water and ecosystem resource

Microbial Biodiversity and Molecular Approach

Biodiversity is given by the variety of species on Earth resulting from billions of years of evolution. Molecular-phylogenetic studies have revealed that the main diversity of life is microbial and it is distributed among three domains: Achaea, Bacteria, and Eukarya. The functioning of whole biosphere depends absolutely on the activities of the microbial world. Due to their versatility microbes are the major natural providers of ecological services as well play major role in semi-artificial systems such as sewage treatment plants, landfills, and in toxic waste bioremediation. As for other organisms many pressures and drivers are causing decrease of microbial biodiversity. Several publications document the effect of chemical pollutants e.g. Polycyclic Aromatic Hydrocarbons (PAHs), of atmospheric pollution, of temperature change and of fertilization on microbial community structure. These studies are now possible because sequencing technologies are in ongoing revolution allowing massive de novo sequencing producing millions of bases in a single day. Metagenomics, metatranscriptomics, metaproteomics and single-cell sequencing are approaches providing a view not only of the community structure (species phylogeny, richness, and distribution) but also of the functional (metabolic) potential of a community because virtually about all genes are captured and sequenced. Unfortunately, although microrganisms are very important for the functioning of whole biosphere public knowledge, awareness and political actions did not yet deal with microbes when biodiversity and its decrease are highlighted.JRC.DDG.H.5-Rural, water and ecosystem resource

Cyanotoxins: methods and approaches for their analysis and detection

Cyanotoxins are secondary metabolites produced by cyanobacteria, a group of photosynthetic prokaryota especially found in freshwater. In favourable conditions (i.e. high nutrient levels, light intensity, water temperature), cyanobacteria can form blooms, a natural phenomenon characterised by an algal biomass accumulation and the possible release of cyanotoxins in water ecosystems. Toxins represent an emerging threats for the aquatic organisms which can bioaccumulate these compounds and transfer them throughout the food chain to wildlife and humans. Other ways of exposure for humans include the oral, dermal and inhalation route. The consumption of contaminated drinking water, skin contact and swallowing water during recreational activities are among the most frequently reasons for human poisonings caused by cyanotoxins. The associated symptoms usually range from severe headache to fever, respiratory paralysis and in rare case, death. The World Health Organization (WHO) has issued a provisional guideline value of 1 µg/L in drinking water for Microcystin-LR (MC-LR), the most toxic, widespread and common toxin in water supplies. Due to the lack of complete toxicological data for a range of cyanotoxins, their concentration in drinking water is not yet well regulated even in countries belonging to the European Union (EU). In this report, attention is focused on the methodologies commonly used to detect cyanotoxins in water environments. These applications can be grouped in: I) microscopy analysis II) physicochemical methods III) molecular-based methods IV) biochemical-based methods V) chemical methods. Each technique shows specific limitations in terms of sensitivity, reliability and limit of detection. The choice of the best one to use is determined in accordance with the information they provide, the availability of facilities and the technical expertise of the operators. Most of the research about cyanotoxins has been mainly focused on microcystins (MCs). The other cyanotoxins have been much less investigated and more tools need to be developed to overcome this problem. Notwithstanding there is no a single analytical application able to detect all cyanotoxin variants in an environmental sample. Some current methods described in this report show great promise in terms of being simple, cost-effective, specific and sensitive for the analysis of a defined toxin.JRC.D.2-Water and Marine Resource

Review of the 1st Watch List under the Water Framework Directive and recommendations for the 2nd Watch List

The surface water Watch List (WL) under the Water Framework Directive (WFD) is a mechanism for obtaining high-quality Union-wide monitoring data on potential water pollutants for the purpose of determining the risk they pose and thus whether Environmental Quality Standards (EQS) should be set for them at EU level. According to the EQS Directive (article 8b) , this list should be updated every 2 years. The main objectives of this report are: • To present an overview of the data gathered during the 1st year of monitoring of the 1st WL (also called WL dataset in this report), • To assess whether this WL dataset is sufficient to determine the risk posed by the WL substances, and consequently to determine whether any of these substances can be taken out of the WL, • To propose new substance(s) to be included in the second WL, using the information and results from the latest review of the list of priority substances, as well as any other relevant information available at the time of this report. The executive summary first explains the context for the assessment. Then, mirroring the report itself, it presents an overview of the WL dataset for the different WL substances, it specifies the criteria for taking substances out of the WL and the substances proposed on the basis of these criteria, and finally it presents the criteria for including new substances in the WL and the new proposed WL candidates.JRC.D.2-Water and Marine Resource

Transcriptomics Responses in Marine Diatom Thalassiosira pseudonana Exposed to the Polycyclic Aromatic Hydrocarbon Benzo[a]pyrene

Diatoms are unicellular, photosynthetic, eukaryotic algae with a ubiquitous distribution in water environments and they play an important role in the carbon cycle. Molecular or morphological changes in these species under ecological stress conditions are expected to serve as early indicators of toxicity and can point to a global impact on the entire ecosystem. Thalassiosira pseudonana, a marine diatom and the first with a fully sequenced genome has been selected as an aquatic model organism for ecotoxicological studies using molecular tools. A customized DNA microarray containing probes for the available gene sequences has been developed and tested to analyze the effects of a common pollutant, benzo(a)pyrene (BaP), at a sub-lethal concentration. This approach in diatoms has helped to elucidate pathway/metabolic processes involved in the mode of action of this pollutant, including lipid metabolism, silicon metabolism and stress response. A doseresponse of BaP on diatoms has been made and the effect of this compound on the expression of selected genes was assessed by quantitative real time-PCR. Up-regulation of the long-chain acyl-CoA synthetase and the anti-apoptotic transmembrane Bax inhibitor, as well as down-regulation of silicon transporter 1 and a heat shock factor was confirmed at lower concentrations of BaP, but not the heat-shock protein 20. The study has allowed the identification of molecular biomarkers to BaP to be later on integrated into environmental monitoring for water quality assessment.JRC.H.1-Water Resource

Development of the First Watch List under the Environmental Quality Standards Directive

According to Directive 2008/105/EC (the Environmental Quality Standards Directive, EQSD), a new mechanism is needed to provide high-quality monitoring information on the concentrations of polluting substances in the aquatic environment across the EU. The aim of this mechanism is to support the identification of priority substances for regulation under the Water Framework Directive. A restricted number of substances (up to 10) are to be included in a dynamic Watch List, remaining there for limited time. Three compounds, i.e. diclofenac, 17-beta-estradiol (E2), and 17-alpha-ethinylestradiol (EE2) have already been identified for inclusion in the first Watch List, for the specific purpose of better informing the determination of suitable risk reduction measures. Therefore, up to seven additional substances should be identified for inclusion. This report describes the procedure to identify a short-list of substances, based on the suspected risk to or via the aquatic environment, as well as on the unavailability of sufficient monitoring data or data of sufficient quality to identify the risk posed by those substances, and to prioritise them at EU level. From the short-list, seven additional substances are proposed for inclusion in the first Watch List.JRC.H.1-Water Resource

Algal bloom and its economic impact

Harmful algal blooms (HABs) represent a natural phenomena caused by a mass proliferation of phytoplankton (cyanobacteria, diatoms, dinoflagellates) in waterbodies. Blooms can be harmful for the environment, human health and aquatic life due to the production of nocive toxins and the consequences of accumulated biomass (oxygen depletion). These blooms are occurring with increased regularity in marine and freshwater ecosystems and the reasons for their substantial intensification can be associated with a set of physical, chemical and biological factors including climate changes and anthropogenic impacts. Many bloom episodes have significant impacts on socio-economic systems. Fish mortality, illnesses caused by the consumption of contaminated seafood and the reluctance of consumers to purchase fish during HABs episodes represent only some of the economic impacts of HABs. The aim of this report is to evaluate the economic losses caused by HABs in different sectors. This was achieved by collecting data that exist in the technical literature and group them into four categories: (1) human health impacts; (2) fishery impacts; (3) tourism and recreation impacts; (4) monitoring and management costs. The data analysed refer to both marine and freshwater HABs. Among the sectors examined in this study, human health impacts appear less investigated than the other three categories. This is probably caused by the difficulty to assess the direct effects of toxins on human health because of the wide range of symptoms they can induce. Looking at the data, the interest in mitigating the economic losses associated with blooms is particularly demonstrated by studies aimed to develop monitoring and management strategies to reduce HABs episodes. Indeed, the water monitoring, when accompanied by appropriate management actions, can assure the mitigation of ongoing HABs and the reduction of negative impacts. During data collection, it has been more difficult to find economic data about blooms in Europe than in United States of America (USA). A reason may be the lack of European reports or publically available data about HABs and their socio-economic impacts. Much studies still have to be performed in this field, but the reported increase in HABs frequency will surely increase not only scientific analysis about HABs but also economic studies to report whether safeguards taken have succeeded in mitigating the economic impact associated with blooms.JRC.H.1-Water Resource

Phylogenetic and Functional Metagenomic Profiling for Assessing Microbial Biodiversity in Environmental Monitoring

Environmental management decisions have to base on holistic understanding of biodiversity and functional capability in ecosystems. Environmental metagenomics is an emerging and powerful approach allowing rapidly and reliably determine and compare microbial biodiversity and functional profiles. Advances in next generation sequencing technologies and bioinformatic tools allow set up analysis pipelines which are useful for well designed and targeted monitoring exercises already today. In the paper we demonstrate how direct sequencing of the total community DNA and analysis of the data are applicable to assess anthropogenic impact on the coastal marine ecosystems.JRC.H.1-Water Resource

Rational modification of estrogen receptor by combination of computational and experimental analysis

In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally modifying the amino acid composition of its ligand binding domain. By combining sequence alignment and structural analysis of known ER-ligand complexes with computational analysis, we were able to predict ER mutants with altered binding properties. These predictions were experimentally confirmed by producing single point variants with up to an order of magnitude increased binding affinity towards some estrogen disrupting chemicals and reaching an IC50 value of 2 nM for the 17α−Ethinylestradiol ligand. Due to increased affinity and stability, utilizing such mutated ERs instead of the wild type ER as bio-recognition element would be beneficial in an assay or biosensor.JRC.I-Institute for Health and Consumer Protection (Ispra

Modes of action of the current Priority Substances list under the Water Framework Directive and other substances of interest

The Water Framework Directive 2000/60/EC (WFD) has established a strategy for water protection that includes specific measures for pollution control to achieve good chemical and ecological status at European level. There is a need to review the approach to the current listing of priority substances (PS) under the WFD and to the current assessment of the chemical status, and consider eventually a wider range of chemical substances that could be covered in future monitoring programmes. Overall, the aim is to assess the water status more holistically and understand which the real effects are caused by the sum of the chemical substances present in the aquatic environment (including emerging pollutants /other substances of interest, metabolites and transformation products). The assessment of chemical status should be improved and linked with ecological status where relevant. Hundreds of different substances may co-occur, and even if most are present at very small concentrations they could exert a toxic effect on aquatic organisms (Carvalho et al. 2014) exposed for their entire life cycle and indirectly on human health (via food and drinking water consumption). Some of the substances in the current list of Priority Substances and in the first Watch List are considered in groups (e.g. brominated diphenylethers, neonicotinoid insecticides), but the overall approach to chemical pollution is otherwise based on the regulation of single substances. It has become increasingly clear that the risks from the vast number of chemical substances present in the environment cannot be adequately controlled on this basis. The Commission acknowledges the need to consider the potential toxic effects of mixtures of chemicals (EC COM(2012)252, 7th EAP). The challenge is to find a way of capturing a true picture of the chemical status of water bodies based on standards and methods that assess the presence of an adequate range of representative chemical effect types or modes of action (MoA), for example. The knowledge on the MoA is an important driver for linking exposure to chemicals to their effects in the aquatic environment, and therefore for development and application of the scientific methodologies for the assessment of combined effects of chemicals - the effect-based methods (EBM). The EBM, including biomarkers and bioassays, can target different levels of biological organisation in the aquatic environment, such as individual and/or sub-organism, community, and population levels (Carvalho et al. 2014, Ann-Sofie Wernersson et al. 2014). It is however much less clear how these EBM can be used to capture (predictively) the indirect effects that might occur in humans following long-term chronic exposure to pollutants via the aquatic environment. The use of effect-based monitoring approaches, complementary to chemical analysis, could allow assessing chemical status more holistically (rather than with a limited but ever-growing list of individual substances). The use of the EBM offers also the advantage of overcoming analytical difficulties (Kunz et al. 2015) and reducing monitoring costs by screening. To become a credible complement to chemical monitoring information, however, a better understanding of the capabilities and gaps of available EBM is needed. This report, based on a comprehensive literature study, reviews the current PS list and other substances of interest, considering their MoA(s). The review of data from the open sources clearly identified few groups of toxicological endpoints, with the majority driven by non-specific mechanisms (e.g. oxidative stress, activation of metabolizing / detoxifying pathways, histopathology, and others), and few groups with more specific biochemical / physiological pathways (photosynthesis inhibition, acetylcholinesterase inhibition, presence of PAHs metabolites, expression of metallothioneins). The majority of current PS and other substances of interest can be grouped, based on few common toxicological endpoints, and biomarkers are available for determining the concentrations and/or effects of some groups of substances. The identified biomarkers of effect seem to be however in general not very specific. There is clearly no “one size fits all” bioassay / EBM that could provide the toxicological potency of every PS and other substances of interest and their mixture toward all aquatic organisms in all water bodies, but rather a battery of bioassays that should be selected as “fit for purpose”. In addition, the present report allowed identification of uncertainty and inconsistency in observations, and thus identified areas where future investigations can be best directed. The present knowledge about MoA(s) remains limited, especially for the emerging substances of concern, such as pyrethroids and neonicotinoides.JRC.D.2-Water and Marine Resource