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Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2021.151783, Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring., The work is the result of the DNAqua-Net workshop held in Rome, on June 10–12, 2019, organised by IRSA-CNR and supported by European Cooperation in Science and Technology (COST) grant CA15219 (DNAqua-Net). It largely benefited from the discussions with DNAqua-Net members during this event as well as other DNAqua-Net conferences.This study was carried out within the UNIVPM VIRIDE Project (n. 2017EKFA98) funded by the Ministry of University and Research of Italy (MUR) and the Italian National Project on the use of environmental DNA for the identification of marine non-indigenous species funded by the Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA). JP, TC, and LAPG were supported by the Swiss National Science Foundation (grant 31003A_179125) as well as FL with an Early Postdoc.Mobility grant (P2GEP3_171829). MS-M was supported by the Ministry of Education, Youth and Sports of the Czech Republic, European Regional Development Fund-Project CZ.02.1.01/0.0/0.0/16_019/0000845 and LTC 20063. KP was supported by the European Regional Development Fund within National Programme for Addressing Socio-Economic Challenges through R&D (L190003VLVI RITA1/02-60-05) and base financed project P190250PKKH of Estonian University of Life Sciences, IM was supported by the Slovenian Research Agency (ARRS) programme P4-0085. SD was supported by the Portuguese Foundation for Science and Technology (FCT) (CEECIND/00667/2017). LC was supported by the PON AIM 1854833 – PON Ricerca e Innovazione 2014–2020 – Azione I.2 – D.D. n. 407, 27/02/2018 – Attraction and International Mobility.VK was supported by grants PUT1389 (ETAg) and Institute of Technology, University of Tartu basic funding grant.

This paper explores the diversity and taxonomy of species within Fragilaria sensu stricto, an abundant and ecologically important diatom genus, taking advantage of cultured and DNA ‐barcoded material. The goal is to facilitate the identification of European taxa within this complex, providing a unified view on morphological and molecular diversity. There is a general agreement that the separation of species within the group of Fragilaria is difficult because morphological descriptions of species are not consistent between authorities, ongoing taxonomic revisions have resulted in species described with standards of the late 20th and 21st centuries alongside descriptions based on 19th century (light microscopical) criteria, and because not all diagnostic characters can be seen in all specimens encountered in routine analyses. Consequent confusion could blur potentially important ecological distinctions between species. Our study demonstrated that some species defined on morphological criteria could be confirmed using the rbc L chloroplast gene as a genetic marker, for example, Fragilaria gracilis , Fragilaria tenera , Fragilaria perminuta , and Fragilaria subconstricta . However, even for those species, preliminary identifications based on morphology often differed from identifications based on phylogenetic clustering combined with detailed morphological study. Clades were well‐defined by rbc L, but based on morphology, the terminal taxa of these clades did not match the currently described Fragilaria species. To clarify recognition of these taxa, we describe three new species: Fragilaria agnesiae , Fragilaria heatherae , and Fragilaria joachimii . info:eu-repo/semantics/acceptedVersion

International audience; Littoral benthic diatom communities are pivotal indicators which react to coastline sources of pollution. Littoral communities, however, may be also strongly influenced by other environmental factors: we assumed that even in lakes without anthropogenic pressure, communities could be heterogeneous. To investigate the natural causes of community heterogeneity along shoreline, we sampled 58 pristine high-altitude lakes in the French Alps. Inside each lake, three different littoral stations were sampled. Water chemical composition was measured with major environmental settings. Lakes with homogeneous communities along their shoreline were dominated by pioneer species adapted to strong physical disturbances. The water renewal time in these lakes was short (a few days) and this conveyed strong turbulences. Lakes with heterogeneous communities along their shore had longer water renewal time and were dominated by high-profile diatoms that were able to compete for light and nutrients. The within-lake heterogeneity could be explained by the variability in coastline terrestrial habitats. This study illustrates how water turbulences can limit the expression of some ecological processes like interspecific competition and reduce heterogeneity when it is an overriding stressor. Finally, implications for lake managers are given in terms of monitoring efforts.

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data, (undefined)

Diatoms (Bacillariophyta) are ubiquitous microalgae which produce a siliceous exoskeleton and which make a major contribution to the productivity of oceans and freshwaters. They display a huge diversity, which makes them excellent ecological indicators of aquatic ecosystems. Usually, diatoms are identified using characteristics of their exoskeleton morphology. DNA-barcoding is an alternative to this and the use of High-Throughput-Sequencing enables the rapid analysis of many environmental samples at a lower cost than analyses under microscope. However, to identify environmental sequences correctly, an expertly curated reference library is needed. Several curated libraries for protists exists; none, however are dedicated to diatoms. Diat.barcode is an open-access library dedicated to diatoms which has been maintained since 2012. Data come from two sources (1) the NCBI nucleotide database and (2) unpublished sequencing data of culture collections. Since 2017, several experts have collaborated to curate this library for rbcL, a chloroplast marker suitable for species-level identification of diatoms. For the latest version of the database (version 7), 605 of the 3482 taxonomical names originally assigned by the authors of the rbcL sequences were modified after curation. The database is accessible at https://www6.inra.fr/carrtel-collection_eng/Barcoding-database.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring., This paper is a deliverable of the European Cooperation in Science and Technology (COST) Action DNAqua-Net (CA15219) Working Group 1, led by Torbjørn Ekrem and Fedor Čiampor. Thanks to the University of Minho and University of Pécs for hosting workshops and working group meetings. We also thank staff at National Environment Agencies and others that provided national checklists of taxa used in biomonitoring, and otherwise assisted with checklist proof-reading: Jarmila Makovinská and Emília Mišíková Elexová (Slovakia); Steinar Sandøy and Dag Rosland (Norway); Mišel Jelič (Croatia); Marlen Vasquez (Cyprus); Adam Petrusek (Czech Republic); Kristel Panksep (Estonia); Panagiotis Kaspiditis (Greece); Matteo Montagna (Italy); Marija Katarzyte (Lithuania); Ana Rotter (Slovenia); Rosa Trabajo (Spain); Florian Altermatt (Switzerland); Kristian Meissner (Finland), Rigers Bakiu (Albania), Valentina Stamenkovic and Jelena Hinic (Macedonia); Patricia Mergen (Belgium); Gael Denys & the French Biodiversity Agency (France); Mary Kelly-Quinn (Ireland); Piotr Panek and Andrzej Zawal (Poland); Cesare Mario Puzzi (Italy); Carole Fitzpatrick (United Kingdom); Simon Vitecek (Austria); Ana Filipa Filipe (Portugal); Peter Anton Stæhr & Anne Winding (Denmark); Michael Monaghan (Germany); Alain Dohet, Lionel L'Hoste, Nora Welschbillig & Luc Ector (Luxembourg), Lujza Keresztes, (Romania). The authors also want to thank Dirk Steinke for providing the original European ERMS list for marine taxa and Florian Malard for comments on the manuscript. The preparation of the AMBI checklist was carried out in the scope of a Short-term Scientific Mission (ECOST-STSM-CA15219-150217- 082111) granted to SD visiting AZTI, Spain. ZC was supported by grants EFOP-3.6.1.-16-2016-00004 and 20765-3/2018/FEKUTSTRAT. TE was supported by the NorBOL-grant (226134/F50) from the Research Coun cil of Norway. BR, FL and MFG contributed through support from the GBOL project, which is generously funded by the German Federal Min istry of Education and Research (FKZ 01LI1101 and 01LI1501). MG contributed through support of the Polish National Science Centre, grants N N303 5794 39 and 2014/15/B/NZ8/00266. SF was funded by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER).

International audience; Climate change is predicted to increase drought occurrence and severity in small continental watercourses. Here, we studied the structure and the functional diversity of benthic diatom assemblages in lowland intermittent and permanent watercourses of the Carpathian Basin. We assumed that the community structure of intermittent and permanent watercourses would be markedly different, and the functional diversity in both would be strongly influenced by autumn drought. We found that intermittent streams were primarily characterized by small-sized generalists and aerophilic taxa, while permanent watercourses were inhabited by large-sized planktic or fast moving groups. The functional richness was significantly lower in intermittent than in permanent streams. This decrease in the functional richness of benthic algal communities may negatively affect the functioning of lotic algal communities. We conclude that diatom assemblages in lowland intermittent watercourses are sensitive indicators of changes in ecosystem properties, and should be considered in appropriate evaluation and management of extreme climatic events on aquatic ecosystems.

International audience; Extensive studies on the taxonomic resolution required for bioassessment purposes have determined that resolution above species level (genus, family) is sufficient for their use as indicators of relevant environmental pressures. The high‐throughput se - quencing (HTS) and meta‐barcoding methods now used for bioassessment tradition - ally employ an arbitrary sequence similarity threshold (SST) around 95% or 97% to cluster sequences into operational taxonomic units, which is considered descriptive of species‐level resolution. In this study, we analyzed the effect of the SST on the resulting diatom‐based ecological quality index, which is based on OTU abundance distribution along a defined environmental gradient, ideally avoiding taxonomic as - signments that could result in high rates of unclassified OTUs and biased final values. A total of 90 biofilm samples were collected in 2014 and 2015 from 51 stream sites on Mayotte Island in parallel with measures of relevant physical and chemical param - eters. HTS sequencing was performed on the biofilms using the rbcL region as the genetic marker and diatom‐specific primers. Hierarchical clustering was used to group sequences into OTUs using 20 experimental SST levels (80%–99%). An OTU‐ based quality index (Idx OTU ) was developed based on a weighted average equation using the abundance profiles of the OTUs. The developed Idx OTU revealed significant correlations between the Idx OTU values and the reference pressure gradient, which reached maximal performance using an SST of 90% (well above species level delimi - tation). We observed an interesting and important trade‐off with the power to dis - criminate between sampling sites and index stability that will greatly inform future applications of the index. Taken together, the results from this study detail a thor - oughly optimized and validated approach to generating robust, reproducible, and complete indexes that will greatly facilitate effective and efficient environmental monitoring

International audience; The effectiveness of environmental protection measures is based on the early identification and diagnosis of anthropogenic pressures. Similarly, restoration actions require precise monitoring of changes in the ecological quality of ecosystems, in order to highlight their effectiveness. Monitoring the ecological quality relies on bioindicators, which are organisms revealing the pressures exerted on the environment through the composition of their communities. Their implementation, based on the morphological identification of species, is expensive because it requires time and experts in taxonomy. Recent genomic tools should provide access to reliable and high-throughput environmental monitoring by directly inferring the composition of bioindicators' communities from their DNA (metabarcoding). The French-Swiss program SYNAQUA (INTERREG France-Switzerland 2017-2019) proposes to use and validate the tools of environmental genomic for biomonitoring and aims ultimately at their implementation in the regulatory bio-surveillance. SYNAQUA will test the metabarcoding approach focusing on two bioindicators, diatoms, and aquatic oligochaetes, which are used in freshwater biomonitoring in France and Switzerland. To go towards the renewal of current biomonitoring practices, SYNAQUA will (1) bring together different actors: scientists, environmental managers, consulting firms, and biotechnological companies, (2) apply this approach on a large scale to demonstrate its relevance, (3) propose robust and reliable tools, and (4) raise public awareness and train the various actors likely to use these new tools. Biomonitoring approaches based on such environmental genomic tools should address the European need for reliable, higher-throughput monitoring to improve the protection of aquatic environments under multiple pressures, guide their restoration , and follow their evolution.

International audience; Benthic diatoms inhabiting littoral zones are regarded as promising bioindicators in lakes. Some pre-standard protocols were proposed and suggested to collect a single site per lake to assess its overall quality. In large lakes, such as Lake Geneva, a spatial heterogeneity of diatom assemblages was suspected, because of differing human pressures. Thirty-five sites were sampled along the shoreline in locations showing various population densities and crossed by rivers flowing into the lake. Different assemblages were found; one corresponded to more oligotrophic areas and was mostly situated on the shoreline next to low population densities; it was mostly composed of low-profile diatoms. A second one corresponded to more eutrophic areas and was mostly situated near densely inhabited areas; it was composed of many high-profile diatoms. A third one was in close vicinity to river inlets, with high abundances of motile diatoms. This last assemblage was spatially restricted to the river plume. After testing several diatom indices, one showed better capacities to assess nutrient level (EPI-L). If we avoid areas near river inlets, the ecological quality assessed with diatom indices ranged from moderate to high ecological status depending on the vicinity situated more or less near densely populated areas.

Increasing release of pharmaceuticals and personal care products (PPCPs) into aquatic ecosystems is a growing environmental concern. Triclosan and fluoxetine are two widely used PPCPs and frequently detected in aquatic ecosystems. In this study, the sensitivities of 7 algal species from 4 genera to triclosan, fluoxetine and their mixture were evaluated. The results showed that the inhibitory effect on algal growth (EC50-96h) of triclosan varied with 50 times differences among the 7 algal species. Chlorella ellipsoidea was the least susceptible species and Dunaliella parva was the most sensitive species to triclosan. The inhibitory effect of fluoxetine was less variable than triclosan. Slightly higher toxicity of fluoxetine than triclosan was shown in the 7 tested algal species. No consistent pattern of the effects from mixture of triclosan and fluoxetine was observed among the 7 algal species and among the 4 genera. Additive effects of the mixture occured in 4 species and antagonistic effects in the other 3 species but no synergistic effect was detected. The algal species might show some sign of phylogenetic response to triclosan, as evidenced by the wide range of differences in their sensitivity at the genus level. This study provides important data which could be beneficial for biomonitoring programs on the ecological risk (algal species diversity) of these two chemicals.

Aquatic biomonitoring has become an essential task in Europe and many other regions as a consequence of strong anthropogenic pressures affecting the health of lakes, rivers, oceans and groundwater. A typical assessment of the environmental quality status, such as it is required by European but also North American and other legislation, relies on matching the composition of assemblages of organisms identified using morphological criteria present in aquatic ecosystems to those expected in the absence of anthropogenic pressures. Through decade-long and difficult intercalibration exercises among networks of regulators and scientists in European countries, a pragmatic biomonitoring approach was developed and adopted, which now produces invaluable information. Nonetheless, this approach is based on several hundred different protocols, making it susceptible to issues with comparability, scale and resolution. Furthermore, data acquisition is often slow due to a lack of taxonomic experts for many taxa and regions and time-consuming morphological identification of organisms. High-throughput genetic screening methods such as (e)DNA metabarcoding have been proposed as a possible solution to these shortcomings. Such "next-generation biomonitoring", also termed "biomonitoring 2.0", has many advantages over the traditional approach in terms of speed, comparability and costs. It also creates the potential to include new bioindicators and thereby further improves the assessment of aquatic ecosystem health. However, several major conceptual and technological challenges still hinder its implementation into legal and regulatory frameworks. Academic scientists sometimes tend to overlook legal or socioeconomic constraints, which regulators have to consider on a regular basis. Moreover, quantification of species abundance or biomass remains a significant bottleneck to releasing the full potential of these approaches. Here, we highlight the main challenges for next-generation aquatic biomonitoring and outline principles and good practic, COST - European Cooperation in Science and Technology(CA15219). COST Action DNAqua-Net (CA15219), supported by the COST (European Cooperation in Science and Technology) programme

International audience; Hospital wastewater (HWW) receives increasing attention because of its specific composition and higher concentrations of some micropollutants. Better knowledge of HWW is needed in order to improve management strategies and to ensure the preservation of wastewater treatment efficiency and freshwater ecosystems. This context pushed forward the development of a pilot study site named Site Pilote de Bellecombe (SIPIBEL), which collects and treats HWW separately from urban wastewater, applying the same conventional treatment process. This particular configuration offers the opportunity for various scientific investigations. It enables to compare hospital and urban wastewater, the efficiency of the two parallel treatment lines, and the composition of the resulting hospital and urban treated effluents, as well as the evaluation of their effects on the environment. The study site takes into account environmental, economic, and social issues and promotes scientific and technical multidisciplinary actions. ᅟ.

Diatoms are used routinely to assess pollution level in rivers and lakes. Current methods are based on identification by light microscopy, which is laborious. An alternative is to identify species based on short DNA fragments and High-Throughput Sequencing (HTS). However a potential limitation is the incomplete coverage of species in reference barcode libraries. Usually these libraries are compiled by isolating cells, before culturing and sequencing them, which is tedious and often unsuccessful. Here we propose the use of rbcL sequences from environmental samples analysed by HTS. We set several criteria to ensure good sequence quality and correspondence with the target species observed in microscopy: the sequence needed to be abundant in the sample, and with no insertions nor deletions or stop codon, phylogenetic neighbour taxa had to correspond to neighbour taxonomic taxa expected from morphological observations. Four species from tropical rivers are given as examples, including one that is new to science. info:eu-repo/semantics/publishedVersion

ECOTOX, new questions for terrestrial and aquatic ecotoxicology

International audience; Assessment of ecological status for the European Water Framework Directive (WFD) is based on "Biological Quality Elements" (BQEs), namely phytoplankton, benthic flora, benthic invertebrates and fish. Morphological identification of these organisms is a time-consuming and expensive procedure. Here, we assess the options for complementing and, perhaps, replacing morphological identification with procedures using eDNA, metabarcoding or similar approaches. We rate the applicability of DNA-based identification for the individual BQEs and water categories (rivers, lakes, transitional and coastal waters) against eleven criteria, summarised under the headlines representativeness (for example suitability of current sampling methods for DNA-based identification, errors from DNA-based species detection), sensitivity (for example capability to detect sensitive taxa, unassigned reads), precision of DNA-based identification (knowledge about uncertainty), comparability with conventional approaches (for example sensitivity of metrics to differences in DNA-based identification), cost effectiveness and environmental impact. Overall, suitability of DNA-based identification is particularly high for fish, as eDNA is a well-suited sampling approach which can replace expensive and potentially harmful methods such as gill-netting, trawling or electrofishing. Furthermore, there are attempts to replace absolute by relative abundance in metric calculations. For invertebrates and phytobenthos, the main challenges include the modification of indices and completing barcode libraries. For phytoplankton, the barcode libraries are even more problematic, due to the high taxonomic diversity in plankton samples. If current assessment concepts are kept, DNA-based identification is least appropriate for macrophytes (rivers, lakes) and angiosperms/macroalgae (transitional and coastal waters), which are surveyed rather than sampled. We discuss general implications of implementing DNA-based identification into standard ecological assessment, in particular considering any adaptations to the WFD that may be required to facilitate the transition to molecular data.

International audience; Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior.

Special Issue « ECOTOX, new questions for terrestrial and aquatic ecotoxicology »

National audience

Epub 2017 Jul 06; Diatoms are used as indicators of freshwater ecosystems integrity. Developing diatom-based tools to assess impact of herbicide pollution is expected by water managers. But, defining sensitivities of all species to multiple herbicides would be unattainable. The existence of a phylogenetic signal of herbicide sensitivity was shown among diatoms and should enable prediction of new species sensitivity. However, diatoms present a cryptic diversity that may lead to variation in their sensitivity to herbicides that would need to be taken into account. Using bioassays, the sensitivity to four herbicides (Atrazine, Terbutryn, Diuron, Isoproturon) was evaluated for 11 freshwater diatom taxa and intraspecific variability was assessed for two of them (Nitzschia palea and Achnanthidium spp.). Intraspecific variability of herbicide sensitivity was always smaller than interspecific variability, but intraspecific variability was more important in N. palea than in Achnanthidium spp. Indeed, one species showed no intraspecific phylogenetic signal (N. palea) whereas the other did (Achnanthidium spp.). On one hand, species boundaries are not set properly for Achnanthidium spp. which encompass several taxa. On the other hand, there is a higher phenotypic plasticity for N. palea. Finally, a phylogenetic signal of herbicide sensitivity was measured at the interspecific level, opening up prospects for setting up reliable biomonitoring tools based on sensitivity prediction, insofar as species boundaries are correctly defined.

Current freshwater biomonitoring with diatoms is based on microscopic examination of the morphology of their silica skeleton. This standardized approach is time consuming and requires a high degree of taxonomic expertise. Metabarcoding combined with high-throughput sequencing (HTS) has great potential for next-generation biomonitoring applications but requires standardization. Molecular inventories are strongly influenced by the DNA extraction method used, but the effect of extraction protocols has not been tested to enable selection of the best DNA extraction method for HTS metabarcoding. We used 5 DNA extraction methods combining various types of cell lysis and DNA purification to extract DNA from 8 pure diatom cultures and 8 samples from streams and lakes with differing water quality. We compared the methods based on: 1) quality and purity of the extracted DNA, 2) community inventories obtained from HTS targeting the ribulose-1, 5-bisphosphate carboxylase (rbcL) barcode, and 3) similarity between molecular and microscopy-based inventories of community composition and the Specific Pollution sensitivity Index [SPI]. A method based on GenEluteTM-LPA had higher extraction efficiency than the 4 commercial kits but had the highest polymerase chain reaction inhibition level. All 5 methods were efficient for HTS, and method did not affect operational taxonomic unit richness. We observed variations in the relative abundance of some taxa within Nitzschia, Amphora, Encyonerna, Gomphonema, and Navicula between 2 of the 5 methods, but method did not affect global diatom community composition or SPI values. SPI values calculated from microscopy-based inventories and molecular inventories based on all 5 extraction methods were strongly correlated. For convenience purposes (high DNA quantity and low cost), we encourage standardization of FITS diatom biomonitoring based on the SA-Gen method.

Diatom-based ecological quality assessment methods have been implemented and used regularly in the Water Framework Directive. These indices use the species' abundance profiles along a specific environmental gradient, which they aim to assess. However, this approach has several problematic issues including the unstable and fast-changing diatom taxonomy. The use of traits can be a solution if their responses to the environmental pressure are well-defined. Here, we developed taxonomy-based and trait-based diatom assemblage indices to assess the ecological status of riverine sites on a tropical island. The two indices are based on two sub-indices that measure the diatom assemblage response to a nutrient and organic matter/turbidity gradient. Both taxonomy- and trait-based indices correlated significantly with the selected environmental gradients of the test database, which was not used during index development. We showed that traits could be used for quality assessment of the Mayotte rivers and require much less effort than taxonomy-based indices. There were differences between the two types of indices, which are discussed in this paper. As a perspective for further studies, tests of trait-based indices among different eco-regions would be challenging.

International audience; Phylogeny has not yet been fully accepted in the field of ecotoxicology, despite studies demonstrating its potential for developing environmental biomonitoring tools, as it can provide an a priori assessment of the sensitivity of several indicator organisms. We therefore investigated the relationship between phylogeny and sensitivity to herbicides in freshwater diatom species. This study was performed on four photosystem 11 inhibitor herbicides (atrazine, terbutryn, diuron, and isoproturon) and 14 diatom species representative of Lake Geneva biofilm diversity. Using recent statistical tools provided by phylogenetics, we observed a strong phylogenetic signal for diatom sensitivity to herbicides. There was a major division in sensitivity to herbicides within the phylogenetic tree. The most sensitive species were mainly centrics and araphid diatoms (in this study, Thalassiosirales and Fragilariales), whereas the most resistant species were mainly pennates (in this study, Cymbellales, Naviculales, and Bacillariales). However, there was considerable variability in diatom sensitivity within the raphid clade, which could be explained by differences in trophic preferences (autotrophy or heterotrophy). These traits appeared to be complementary in explaining the differences in sensitivity observed at a refined phylogenetic level. Using phylogeny together with complementary traits, as trophic preferences, may help to predict the sensitivity of communities with a view to protecting their ecosystem.

International audience; Benthic diatom communities are used in the ecological assessment of Portuguese rivers through the calculation of an autecological index, the IPS (Indice de Polluosensibilite Specifique), officially adopted for Portugal. This index requires a high level of taxonomic expertise for morphological identification of individuals at species level. Advances in genomics, such as the DNA metabarcoding combined with high-throughput sequencing techniques offer a promising alternative to this classical approach, decreasing expertise requirement and therefore reducing time and costs. The aim of this study was to test the potential of DNA metabarcoding of diatoms in the bioassessment of Portuguese rivers, by comparing the classifications of the IPS obtained with morphological and molecular approaches. A total of 88 samples were collected in four river types and a quality gradient in central Portugal in the spring of 2017, following the Water Framework Directive (WFD) standards. The morphological approach comprised taxonomic identification of at least 400 valves, under the light microscope. Molecular approach included eDNA extraction followed by PCR amplification and DNA high-throughput sequencing (Illumina MiSeq) using a diatom-specific 312 bp rbcL DNA barcode. Sequences were analysed with Mothur software, producing Operational Taxonomic Units (OTUs) that were taxonomically assigned using the R-Syst::diatom reference library. We also tested the effect of a correction factor (CF) based on biovolume for the quantification of molecular data. When comparing the number of species identified, morphological, molecular without and with CF inventories revealed 306, 125 and 111 species, respectively. Molecular data showed 67% of unassigned OTUs. These results were mainly due to the incompleteness of the reference library, which currently represents the major difficulty in taxonomic assignment of DNA sequences in metabarcoding. Regarding IPS values, the results indicated a good correlation between morphological and molecular methods, particularly when applying the CF. Furthermore, Principal Coordinates analysis plot based on species abundances revealed a quality gradient in all 3 methodologies (morphological, molecular without and with CF). These results support the hypothesis that DNA metabarcoding is a valid approach for ecological quality assessment of Portuguese rivers. Yet, there is still work to be done on this new methodology to enable a smooth transition between the traditional and this new approach without misspend accumulated knowledge from the last decades on water quality assessment.

International audience; Benthic diatoms evolved in a biofilm structure, at the interface between water and substrata. Biofilms can adsorb toxicants, such as herbicides, but little is known about the exposure of biofilm organisms, such as benthic diatoms, to these adsorbed herbicides. We assessed the sensitivity of 11 benthic diatoms species to 6 herbicides under both planktonic and benthic conditions using single-species bioassays. The concentration that reduced the growth rate of the population by 10% (EC10) and 50% (EC50), respectively, varied depending on the species, the herbicides, and the growth forms involved. As a general trend, the more hydrophobic the herbicide, the more species were found to be sensitive under benthic growth conditions. Statistical differences (alpha < 5%) were observed between the sensitivities under planktonic and benthic growth conditions for many hydrophobic herbicides. A protective effect of the biofilm against herbicides was observed, and this tended to decrease (at both the EC10 and EC50 levels) with increasing hydrophobicity. The biofilm matrix appeared to control exposure to herbicides, and consequently their toxicity towards benthic diatoms. For metolachlor, terbutryn and irgarol, benthic thresholds derived from species sensitivity distributions were more protective than planktonic thresholds. For hydrophobic herbicides, deriving sensitivity thresholds from data obtained under benthic growth seems to offer a promising alternative. (C) 2013 Elsevier B.V. All rights reserved.

ECOTOX, le Réseau des écotoxicologues de l'INRA et de ses partenaires. Forum ECO-TOX, Ecoparc Rovaltain

Special Issue « ECOTOX, the INRA's network of ecotoxicologists »

The fifth meeting of INRA’s national network of ecotoxicologists took place on 25 to 27 November 2014 in Biarritz, France. The main aim of the meeting was to bring together ecotoxicologists from INRA and associated partners, providing them ample opportunity to share and discuss their latest scientific results as well as the national policy of research in ecotoxicology and to precise perspectives for the network.

Persist-Env est un projet de recherche finance par l’Anses pour evaluer la persistance environnementale des medicaments et des bacteries pathogenes en sortie de station d’epuration. Le projet s’interesse notamment a l’impact de la mise en place de filiere individuelle d’epuration des effluents hospitaliers. Sipibel (pour « site pilote de Bellecombe », situe en Haute-Savoie) teste actuellement cette strategie de traitement sur les effluents du centre hospitalier Alpes-Leman. L’objectif est de developper des connaissances scientifiques et des connaissances operationnelles autour de la question de l’interet du traitement separatif.

International audience; A high number of species often represents a relevant redundancy in terms of ecological adaptation strategies. Collecting species to groups based on their functional adaptations can handle this redundancy and obtain the "real" functional complexity of ecosystems. Functional traits are proxies of adaptation strategies under particular environmental conditions, and a set of functional traits are interpreted as life-strategies. Organisms with life-strategies occupying a similar niche can be collected in ecological groups (functional group/guild). In this study, we review the latest trait-based approaches and existing attempts at functional classifications in phytobenthos studies. Advantages and shortcomings of these classifications are discussed with perspectives of their utility in ecological status assessment.

A great number of chemicals (metallic and synthetic compounds) are used by human activities. Most of them enter our environment and lead to its contamination, as well as to adverse effects on wildlife and humans. Some compounds of natural origin (toxins, biopesticides...) are also of concern. One challenge for ecotoxicologists remains to understand and predict the long-term impact of these mixtures of chemicals, often on trace concentrations, on living organisms. This implies to develop tools and assessment strategies at different biological levels: individuals, populations, and communities. In that context, this special issue of Environmental Science and Pollution Research highlights selected papers from the members of ECOTOX, the network of ecotoxicologists of the National Institute for Agricultural Research (INRA) (http://www6.inra.fr/ecotox_eng/). Set up in 2009 to stimulate research on ecotoxicology within INRA and to unify the teams involved in the field, the network now brought together academics of other French research institutes and universities, as well as professionals. A total of 150 scientists over 50 laboratories or companies subscribes to the mailing list of the network. The ECOTOX network is sponsored by the INRA, and particularly the scientific divisions BForest, Grassland and Freshwater Ecology^ and BEnvironment and Agronomy , as well as the BSustainable Management of Crop Health^ metaprogram (SMaCH). The network has three main priority objectives: (i) to encourage scientific thinking within the community of ecotoxicologists, whether they are dealing with aquatic or terrestrial ecosystems, (ii) to contribute to defining a national research strategy in this field, and (iii) to favor scientific production. For many years, the network offers opportunities for exchanges within the French community of ecotoxicologists, especially through regular scientific seminars They were held in Thonon les Bains (1999), Antibes (2002), Dinard (2006), St-Lager (2011), and Biarritz (2014); see the conference report on the last seminar in the present special issue. The network is also involved in the coordination and structuring of the national research in ecotoxicology, by interacting with other French networks and structures. In addition, the network has also set up a thematic watch system (http://www6.inra.fr/ecotox_eng/ Watch), providing to numerous partners a bimonthly bulletin. Finally, the network provides means to present and publish the results and highlights from the research developed by its scientists. That special issue is one illustration of that objective. Twenty-three papers were included in this special issue. These papers highlighted current research on the fate, mode of action, and adverse outcomes for a variety of environmental contaminants, on a variety of biological targets. Some of these results were presented during the last seminar in Biarritz. They aimed at establishing the Responsible editor: Philippe Garrigues

International audience; Mixture risk assessment predictions have rarely been confronted with biological changes observed in the environment. In this study, long-term monitoring of a European great lake, Lake Geneva, provides the opportunity to assess to what extent the predicted toxicity of herbicide mixtures explains the changes in the composition of the phytoplankton community next to other classical limnology parameters such as nutrients. To reach this goal, the gradient of the mixture toxicity of 14 herbicides regularly detected in the lake was calculated using concentration addition and response addition models. A temporal gradient of toxicity was observed which decreased from 2004 to 2009. Redundancy analysis and partial redundancy analysis showed that this gradient explains a significant portion of the variation in phytoplankton community composition with and without having removed the effect of all other covariables. Moreover, species that are significantly influenced, positively or negatively, by the decrease of toxicity in the lake over time are highlighted. It can be concluded that the herbicide mixture toxicity is one of the key parameters to explain phytoplankton changes in Lake Geneva.

International audience; The European Union’s Water Framework Directive has set a target of achieving good ecological status for all aquatic environments in Europe by 2015. In order to determine the quality of aquatic environments, biological indicators such as diatoms are often used. However, biotic diatom indices can be difficult and time consuming to use because of complexity of species determination. We investigated whether the biological traits of diatoms in rivers (life-forms, size classes and ecological guilds) could be used to assess organic pollution and trophic level. We worked on a data set comprising 315 diatom species, determined at 328 river stations of south-east France and a variety of parameters. The abundances of some biological traits differed significantly between the different organic pollution and trophic levels, particularly stalked diatoms, and the motile and low-profile guilds.

International audience; The effects of chemical and physical factors on periphyton structure, diversity and functioning were investigated in an outdoor mesocosm experiment. Stream biofilms were subjected to a pesticide mix (diuron and azoxystrobin) under two different hydraulic regimes. The hydraulic regimes differed by spatial variations of flow conditions (turbulent with high variations vs. laminar with low variations). The effects of the hydraulic regime and pesticides were assessed at the level of the periphytic communities. We focused on the change in the biodiversity of these communities under the two hydraulic regimes, and on the role of these biodiversity changes in case of pesticide contamination. Changes in structural (biomass, cell density), diversity (community composition assessed by PCR-DGGE and microscopic analysis) and functional (bacterial and algal production, sensitivity to the herbicide) parameters were monitored throughout a 2-month experiment. The results showed that exposure to pesticides affected the phytobenthic community targeted by the herbicide, impacting on both its growth dynamics and its primary production. Conversely, the impact of the flow regime was greater than that of pesticides on the non-target bacterial community with higher bacterial density and production in laminar mesocosms (uniform regime). An interaction between flow and pollution effects was also observed. Communities that developed in turbulent mesocosms (heterogeneous regime) were more diversified, as a result of increased microhabitat heterogeneity due to high spatial variations. However, this higher biodiversity did not increase the ability of these biofilms to tolerate pesticides, as expected. On the contrary, the sensitivity of these communities to pesticide contamination was, in fact, increased.

[Departement_IRSTEA]Eaux [TR1_IRSTEA]BELCA; International audience; The pollution of lakes and rivers by pesticides is a growing problem worldwide. However, the impacts of these substances on microbial communities are still poorly understood, partly because next-generation sequencing (NGS) has rarely been used in an ecotoxicology context to study bacterial communities despite its interest for accessing rare taxa. Microcosm experiments were carried out to evaluate the effects of tebuconazole (TBZ) on the structure and composition of bacterial communities from two types of freshwater ecosystem (lakes and rivers) with differing histories of pollutant contamination (pristine vs. previously exposed sites). Pyrosequencing revealed that bacterial diversity was higher in the river than in the lakes and in previously exposed sites than in pristine sites. Lakes and river stations shared very few OTUs, and differences at the phylum level were identified between these ecosystems (i.e. the relative importance of Actinobacteria and Gammaproteobacteria). Despite differences between these ecosystems and their contamination history, no significant effect of TBZ on bacterial community structure or composition was observed. Compared to functional parameters that displayed variable responses, we demonstrated that a combination of classical methods and NGS is necessary to investigate the ecotoxicological responses of microbial communities to pollutants.

International audience; Seasonal changes in the structure and composition of a benthic microalgal community may lead to different responses to herbicide contamination during different seasons. Consequently, the thresholds derived from risk assessment tools such as species sensitivity distributions (SSDs) must allow for these changes. We built a single-substance SSD for each of four herbicides (atrazine, terbutryn, diuron and isoproturon), which was specific to the sensitivity of the benthic diatoms found in Lake Geneva, in order to derive protective thresholds for a mixture of these four herbicides using the concentration addition model. We then investigated (1) the structural parameters of a Lake Geneva benthic microalgal community during two contrasting seasons (summer 2012 and winter 2013), (2) the response of these communities to a herbicide mixture, and (3) the protective levels of the thresholds derived. The winter community was characterized by having greater biomass, diatom species richness, and diversity metrics, and lower non-diatom species richness than the summer community. The differences in the diatom communities composition in these seasons appeared to be primarily driven by the environmental nitrate concentrations and the temperature. Moreover, the species in the winter community were more resistant to herbicides than those found in the summer community. Consequently, the protective threshold for this herbicide mixture obtained in this study was in fact protective for the winter community, but not for the summer community based on their structural parameters. Thus, the protective level against herbicides of the threshold for the benthic microalgal community should take into account changes in the environmental physico-chemical conditions that strongly influence the structure and composition of the community. The fact that the succession of species over time (i.e., over the seasons) is difficult to predict introduces uncertainties into the estimation of protective thresholds and questions their applicability year round.

International audience; Diatoms are main bioindicators used to assess the ecological quality of rivers, but their identification is difficult and time-consuming. Next Generation Sequencing (NGS) can be used to study communities of microorganisms, so we carried out a test of the reliability of 454 pyrosequencing for estimating diatom inventories in environmental samples. We used small subunit ribosomal deoxyribonucleic acid (SSU rDNA), ribulose-1, 5-bisphosphate carboxylase (rbcL), and cytochrome oxidase I (COI) markers and examined reference libraries to define thresholds between the intra- and interspecific and intra- and intergeneric genetic distances. Based on tests of 1 mock community, we used a threshold of 99% identity for SSU rDNA and rbcL sequences to study freshwater diatoms at the species level. We applied 454 pyrosequencing to 4 contrasting environmental samples (with one in duplicate), assigned taxon names to environmental sequences, and compared the qualitative and quantitative molecular inventories to those obtained by microscopy. Species richness detected by microscopy was always higher than that detected by pyrosequencing. Some morphologically detected taxa may have been persistent frustules from dead cells. Some taxa detected by molecular analysis were not detected by morphology and vice versa. The main source of divergence appears to be inadequate taxonomic coverage in DNA reference libraries. Only a small percentage of species (but almost all genera) in morphological inventories were included in DNA reference libraries. DNA reference libraries contained a smaller percentage of species from tropical (27.1–38.1%) than from temperate samples (53.7–77.8%). Agreement between morphological and molecular inventories was better for species with relative abundance >1% than for rare species. The rbcL marker appeared to provide more reproducible results (94.9% species similarity between the 2 duplicates) and was very useful for molecular identification, but procedural standardization is needed. The water-quality ranking assigned to a site via the Pollution Sensitivity diatom index was the same whether calculated with molecular or morphological data. Pyrosequencing is a promising approach for detecting all species, even rare ones, once reference libraries have been developed.

International audience; DNA barcoding, being developed for biomonitoring, requires a database of reference sequences and knowledge of how much sequences can deviate before they are assigned to separate species. The molecular hunt for hidden species also raises the question of species definitions. We examined whether there are objective criteria for sequence-based species delimitation in diatoms, using Nitzschia palea, an important monophyletic indicator species already known to contain cryptic diversity. Strains from a wide geographical range were sequenced for 28S rRNA, COI and rbcL. Homogeneity indices and the Chao index failed to objectively select a precise number of species existing in N. palea as well as an evolutionary method based on coalescence theory. COI always gave higher diversity estimations than 28S rRNA or rbcL. Mating data did not provide a precise calibration of molecular species thresholds. Rarefaction curves indicated that further MOTUs would be detected with more isolates than we sampled (81 clones, 42 localities). Although some genotypes had intercontinental distributions, there was a positive relationship between genetic and geographical distance, suggesting even higher richness than we assessed, given that many regions were not sampled. Overall, no objective criteria were found for species separation; instead barcoding will need a consensual approach to molecular species limits.

International audience; The recent emergence of barcoding approaches coupled to those of next-generation sequencing (NGS) has raised new perspectives for studying environmental communities. In this framework, we tested the possibility to derive accurate inventories of diatom communities from pyrosequencing outputs with an available DNA reference library. We used three molecular markers targeting the nuclear, chloroplast and mitochondrial genomes (SSU rDNA, rbcL and cox1) and three samples of a mock community composed of 30 known diatom strains belonging to 21 species. In the goal to detect methodological biases, one sample was constituted directly from pooled cultures, whereas the others consisted of pooled PCR products. The NGS reads obtained by pyrosequencing (Roche 454) were compared first to a DNA reference library including the sequences of all the species used to constitute the mock community, and second to a complete DNA reference library with a larger taxonomic coverage. A stringent taxonomic assignation gave inventories that were compared to the real one. We detected biases due to DNA extraction and PCR amplification that resulted in false-negative detection. Conversely, pyrosequencing errors appeared to generate false positives, especially in case of closely allied species. The taxonomic coverage of DNA reference libraries appears to be the most crucial factor, together with marker polymorphism which is essential to identify taxa at the species level. RbcL offers a high resolving power together with a large DNA reference library. Although needing further optimization, pyrosequencing is suitable for identifying diatom assemblages and may find applications in the field of freshwater biomonitoring.

International audience; Tools that may be used to characterize the state of aquatic ecosystems and the impacts associated to pesticides are currently requested for the monitoring of ecosystems. Chemical analysis alone is not sufficient for such purposes and biomonitoring is essential. Many bioindicators based on diatoms are commonly used to assess the quality of aquatic environments but have not been developed to specifically address the impacts of pesticides. It is therefore crucial to develop dedicated tools. Our research aimed at performing a field study (analysis of monitoring network data) in order to evaluate the responses of different metrics towards pesticide pressure. For diatom communities, toxic pressure associated to pesticides, especially herbicides, had a weak effect on species composition and on the various tested metrics. Nutrient and organic matter concentrations (and also typology) are the main influencing factors. When trophic and saprobic levels are controlled, it is possible to identify the effects of herbicides on some metrics. The only responding metric is the abundance of the "high profile" guild, which corresponds to taxa presenting an important contact surface with water flow. Its abundance decreases when herbicide toxic pressure increases. It could therefore be used to assess the impact of herbicides on diatom communities, but in a framework where confounding factors variability (e.g., nutrients, organic matter, river order) is reduced.

International audience; The Gomphonema parvulum complex includes species displaying considerable morphological variability and a wide geographical distribution. These characteristics make them difficult to identify by microscopy and raise the question of their taxonomic validity and of the possibility of biogeographical differentiation between them. In this context, we isolated 39 G. parvulum s.l. strains from rivers located in a tropical island (Mayotte) and in mainland Europe. By sequencing three DNA fragments (ITS, rbcL and cox1), four clades (A, B, C and D) were clearly identified, and an additional one (B') was distinguishable only on the rbcL sequence. The main four clades can be separated by their morphological criteria, in particular by the shape of the central area, but some overlaps were found between them. We therefore consider that the G. parvulum complex contains at least four semi-cryptic species corresponding to the four main clades. One of them (A) was found only on Mayotte, while two others (C and D) were found only in Europe. The last clade (B) contained strains from both Europe and Mayotte. Pyrosequencing data confirmed the geographical differences in the distribution of these species, suggesting that the G. parvulum complex displays biogeographic structure. (c) 2013 Elsevier GmbH. All rights reserved.

International audience

International audience

Key-words: Bacillariophyta, biomonitoring, check-list, metric, river Diatoms are a very diverse group of microalgae that encompass about 100000 taxa. This makes an excellent bioindicator especially of rivers. The majority of biotic indices based on diatoms are constructed on species pollution-sensitivities. Nevertheless, recent studies showed the interest to use complementary metrics for biomonitoring: life-forms, cell-sizes and ecological guilds. Unfortunately, taxa memberships to such metrics are scattered in numerous floras and papers. Therefore, studies using such metrics often referred to grey literature or home-made checklists. The objective of this paper is to make available to the scientific community an important check-list of taxa frequently observed in European rivers with their memberships to these metrics. After defining these metrics, 1115 taxa were codified using Biofresh and Omnidia codes in an Excel file. Metrics memberships were then explored in a taxonomical framework. It clearly appears that metrics memberships are determined by taxonomy. Lifeforms and cell sizes taxa memberships and their study in the framework of evolutionary history of diatoms is a field that is worth studying for a better understanding of diatoms ecology. Such inputs should be beneficial for the future improvement of diatom-based monitoring tools.

International audience

International audience; Although benthic diatoms are widely used in ecological studies of aquatic systems, there is still a dearth of data concerning species sensitivities towards several contaminants. Within the same community, different species may respond differently depending on their physiological and ecological characteristics. This lack of knowledge makes specific appropriate risk assessment impossible. To find out whether species sensitivity distribution (SSD) could be used to estimate the risk of herbicide toxicity for diatoms, we need to know whether their sensitivity depends on their physiological and ecological characteristics. We carried out single-species bioassays on 11 diatom species exposed to 8 herbicides. Dose-responses relationships were used to extrapolate the Effective Concentration 5 (EC5) and the Effective Concentration 50 (EC50) for each exposure. These data were used to fit a SSD curve for each herbicide, and to determine the Hazardous concentration 5 (HC5) and 50 (HC50). Our results revealed a high level of variability of the sensitivity in the set of species tested. For photosystem-II inhibitor (PSII) herbicides, diatoms species displayed a typical grouping of sensitivity levels consistent with their trophic mode and their ecological guild. N-heterotroph and "motile" guild species were more tolerant of PSII inhibitors, while N-autotroph and "low profile" guild species were more sensitive. Comprehensive SSD curves were obtained for 5 herbicides, but not for sulfonylurea herbicides or for dimetachlor, which had toxicity levels that were below the range of concentration tested. The SSD curves provided the following ranking of toxicity: diuron > terbutryn > isoproturon > atrazine > metolachlor. The HC that affected 5% of the species revealed that, even at the usual environmental concentrations of herbicides, diatom assemblages could be affected, especially by isoproturon, terbutryn, and diuron.

International audience; The pollution of lakes and rivers by the agricultural use of pesticides is a growing problem worldwide. As these substances have complex effects on ecosystems, we need to improve our understanding of their fate and their impact on biological communities. With this in mind, we carried out experimental study of the responses of planktonic bacterial communities from lakes and benthic bacterial communities from streams to the fungicide tebuconazol, and of its degradation by these different communities. For each type of ecosystem, communities were collected from pristine environments (Lake Aiguebelette and the upstream segment of the river Morcille, France) and pre-exposed environments (Lake Léman and the downstream segment of the river Morcille, France in which tebuconazol was found). The experiments were carried out in 20L microcosms in which communities from each of the four environments were either not Exposed (control), or exposed to a low (2μg/L) or high (20μg/L) concentration of the fungicide, in order to simulate the contamination levels encountered in aquatic ecosystems. Results obtained after three weeks of incubation showed that 60 to 70% of the initial concentration of tebuconazol had been degraded in the stream microcosms, whereas only minor degradation had occurred in the lake microcosms (15-20%). The fungicide had no significant impact on benthic bacterial abundance, although the proportion of dead bacteria increased. The changes observed in planktonic bacterial abundance and dead bacteria were transient and depended on previous exposure. To complete these findings, the bacterial diversity is characterizing by pyrosequencing of the gene coding for 16S rRNA. This high-throughput sequencing technology provides a deep inventory of the diversity in microbial communities. All the resulting findings will be discussed with regard to the fungicide history of the different communities studied, and to the rate at which these communities degrade the fungicide

International audience; Pollution of aquatic ecosystems by pesticide contamination is a major environmental concern. Numerous authors have addressed the frequent occurrence of chronic or acute herbicide contamination of freshwater ecosystems in both agricultural and urban areas of the world (Devault et al. 2007; Gilliom 2007; Schuler and Rand 2008; Woudneh et al. 2009). The physiological characteristics of photosynthetic microorganisms make them attractive as targets for herbicides in aquatic ecosystems. Since these primary producers form the basis of trophic structure in many aquatic environments, herbicides may threaten the entire equilibrium of the ecosystems they contaminate.

National audience